Androgen-androgen receptor system improves chronic inflammatory conditions by suppressing monocyte chemoattractant protein-1 gene expression in adipocytes via transcriptional regulation

International Nuclear Information System (INIS)

Morooka, Nobukatsu; Ueguri, Kei; Yee, Karen Kar Lye; Yanase, Toshihiko; Sato, Takashi

2016-01-01

Age-related decreases in sex hormones are closely related to chronic inflammation in obesity and metabolic diseases. Particularly, the molecular basis of androgen activity in regulating inflammation and controlling metabolism remains largely unknown. Obese adipocytes secrete monocyte chemoattractant protein-1 (MCP-1), a key chemokine that promotes the infiltration of monocytes/macrophages into adipose tissue, thereby leading to metabolic disorders. Here, we studied the role of androgen-androgen receptor (AR) action in regulating MCP-1 expression in adipose tissue. We observed the induction of Mcp-1 expression in 3T3-L1 adipocytes co-cultured with RAW264.7 macrophages. Additionally, Mcp-1 expression was upregulated by culturing in conditioned medium derived from inflammatory macrophages (M1-Mφ) containing tumor necrosis factor-alpha (TNF-α). We found that sex hormones downregulated TNF-α-induced Mcp-1 and interleukin (Il)-6 expression in 3T3-L1 adipocytes. Furthermore, luciferase-reporter analysis indicated that MCP-1 promoter activity was predominantly suppressed by dihydrotestosterone (DHT)-AR interactions through functional canonical nuclear factor-kappa B (NF-κB) sites, whereas non-canonical NF-κB site containing important flanking sequences exhibited minor contributions to DHT-AR transcriptional repression. These findings suggested that androgen-AR suppressed obesity-induced chronic inflammation in adipose tissue. - Highlights: • DHT, non-aromatizable androgen suppresses Mcp-1 expression in adipocytes. • Mcp-1 transcription was negatively regulated by DHT-AR action. • DHT-AR selectively regulates Mcp-1 transcription through distinct NF-κB sites.

Monocyte chemoattractant protein 1, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 in exudative age-related macular degeneration.

Science.gov (United States)

Jonas, Jost B; Tao, Yong; Neumaier, Michael; Findeisen, Peter

2010-10-01

To examine intraocular concentrations of monocyte chemoattractant protein 1 (MCP-1), soluble intercellular adhesion molecule 1 (sICAM-1), soluble vascular cell adhesion molecule 1 (sVCAM-1), and vascular endothelial growth factor (VEGF) in eyes with exudative age-related macular degeneration (AMD). The investigation included a study group of 28 patients (28 eyes) with exudative AMD and a control group of 25 patients (25 eyes) with cataract. The concentrations of MCP-1, sICAM-1, sVCAM-1, and VEGF in aqueous humor samples obtained during surgery were measured using a solid-phase chemiluminescence immunoassay. The study group as compared with the control group had higher aqueous concentrations of sICAM-1 (mean [SD], 844 [2073] vs 246 [206] pg/mL, respectively; P < .001), sVCAM-1 (mean [SD], 7978 [7120] vs 2999 [1426] pg/mL, respectively; P < .001), and MCP-1 (mean [SD], 587 [338] vs 435 [221] pg/mL, respectively; P = .07). The concentration of VEGF did not vary significantly between the groups (P = .76). The MCP-1 concentration was significantly associated with macular thickness (r = 0.40; P = .004). It decreased significantly with the type of subfoveal neovascular membrane (classic membrane type, occult membrane, retinal pigment epithelium detachment) (P = .009). The concentrations of sICAM-1, sVCAM-1, and VEGF were not significantly associated with membrane type and macular thickness (P ≥ .18). Concentrations of MCP-1, sICAM-1, and sVCAM-1 are significantly associated with exudative AMD, even in the presence of normal VEGF concentrations. Intraocular MCP-1 concentrations are correlated with the subfoveal neovascular membrane type and the amount of macular edema. One may infer that MCP-1, sICAM-1, and sVCAM-1 could potentially be additional target molecules in therapy for exudative AMD.

Magnetic Nanoparticles Conjugated with Peptides Derived from Monocyte Chemoattractant Protein-1 as a Tool for Targeting Atherosclerosis

Directory of Open Access Journals (Sweden)

Chung-Wei Kao

2018-05-01

Full Text Available Atherosclerosis is a multifactorial inflammatory disease that may progress silently for long period, and it is also widely accepted as the main cause of cardiovascular diseases. To prevent atherosclerotic plaques from generating, imaging early molecular markers and quantifying the extent of disease progression are desired. During inflammation, circulating monocytes leave the bloodstream and migrate into incipient lipid accumulation in the artery wall, following conditioning by local growth factors and proinflammatory cytokines; therefore, monocyte accumulation in the arterial wall can be observed in fatty streaks, rupture-prone plaques, and experimental atherosclerosis. In this work, we synthesized monocyte-targeting iron oxide magnetic nanoparticles (MNPs, which were incorporated with the peptides derived from the chemokine receptor C-C chemokine receptor type 2 (CCR2-binding motif of monocytes chemoattractant protein-1 (MCP-1 as a diagnostic tool for potential atherosclerosis. MCP-1-motif MNPs co-localized with monocytes in in vitro fluorescence imaging. In addition, with MNPs injection in ApoE knockout mice (ApoE KO mice, the well-characterized animal model of atherosclerosis, MNPs were found in specific organs or regions which had monocytes accumulation, especially the aorta of atherosclerosis model mice, through in vivo imaging system (IVIS imaging and magnetic resonance imaging (MRI. We also performed Oil Red O staining and Prussian Blue staining to confirm the co-localization of MCP-1-motif MNPs and atherosclerosis. The results showed the promising potential of MCP-1-motif MNPs as a diagnostic agent of atherosclerosis.

Platelet-derived growth factor (PDGF-BB-mediated induction of monocyte chemoattractant protein 1 in human astrocytes: implications for HIV-associated neuroinflammation

Directory of Open Access Journals (Sweden)

Bethel-Brown Crystal

2012-12-01

Full Text Available Abstract Chemokine (C-C motif ligand 2, also known as monocyte chemoattractant protein 1 (MCP-1 is an important factor for the pathogenesis of HIV-associated neurocognitive disorders (HAND. The mechanisms of MCP-1-mediated neuropathogenesis, in part, revolve around its neuroinflammatory role and the recruitment of monocytes into the central nervous system (CNS via the disrupted blood-brain barrier (BBB. We have previously demonstrated that HIV-1/HIV-1 Tat upregulate platelet-derived growth factor (PDGF-BB, a known cerebrovascular permeant; subsequently, the present study was aimed at exploring the regulation of MCP-1 by PDGF-BB in astrocytes with implications in HAND. Specifically, the data herein demonstrate that exposure of human astrocytes to HIV-1 LAI elevated PDGF-B and MCP-1 levels. Furthermore, treating astrocytes with the human recombinant PDGF-BB protein significantly increased the production and release of MCP-1 at both the RNA and protein levels. MCP-1 induction was regulated by activation of extracellular-signal-regulated kinase (ERK1/2, c-Jun N-terminal kinase (JNK and p38 mitogen-activated protein (MAP kinases and phosphatidylinositol 3-kinase (PI3K/Akt pathways and the downstream transcription factor, nuclear factor κB (NFκB. Chromatin immunoprecipitation (ChIP assays demonstrated increased binding of NFκB to the human MCP-1 promoter following PDGF-BB exposure. Conditioned media from PDGF-BB-treated astrocytes increased monocyte transmigration through human brain microvascular endothelial cells (HBMECs, an effect that was blocked by STI-571, a tyrosine kinase inhibitor (PDGF receptor (PDGF-R blocker. PDGF-BB-mediated release of MCP-1 was critical for increased permeability in an in vitro BBB model as evidenced by blocking antibody assays. Since MCP-1 is linked to disease severity, understanding its modulation by PDGF-BB could aid in understanding the proinflammatory responses in HAND. These results suggest that astrocyte

Chemoattraction of Ichthyophthirius multifiliis (Ciliophora) theronts to host molecules

DEFF Research Database (Denmark)

Buchmann, Kurt; Nielsen, Michael Engelbrecht

1999-01-01

not attract theronts. In contrast, sera and mucus from a range of teleosts (including marine fish) were effective attractants. Fractionation by gel filtration of fish serum allowed determination of the molecular size of the attracting proteins. Further biochemical studies suggested the chemoattractants...

Loss of monocyte chemoattractant protein-1 alters macrophage polarization and reduces NFκB activation in the foreign body response.

Science.gov (United States)

Moore, Laura Beth; Sawyer, Andrew J; Charokopos, Antonios; Skokos, Eleni A; Kyriakides, Themis R

2015-01-01

Implantation of biomaterials elicits a foreign body response characterized by fusion of macrophages to form foreign body giant cells and fibrotic encapsulation. Studies of the macrophage polarization involved in this response have suggested that alternative (M2) activation is associated with more favorable outcomes. Here we investigated this process in vivo by implanting mixed cellulose ester filters or polydimethylsiloxane disks in the peritoneal cavity of wild-type (WT) and monocyte chemoattractant protein-1 (MCP-1) knockout mice. We analyzed classical (M1) and alternative (M2) gene expression via quantitative polymerase chain reaction, immunohistochemistry and enzyme-linked immunosorbent assay in both non-adherent cells isolated by lavage and implant-adherent cells. Our results show that macrophages undergo unique activation that displays features of both M1 and M2 polarization including induction of tumor necrosis factor α (TNF), which induces the expression and nuclear translocation of p50 and RelA determined by immunofluorescence and Western blot. Both processes were compromised in fusion-deficient MCP-1 KO macrophages in vitro and in vivo. Furthermore, inclusion of BAY 11-7028, an inhibitor of NFκB activation, reduced nuclear translocation of RelA and fusion in WT macrophages. Our studies suggest that peritoneal implants elicit a unique macrophage polarization phenotype leading to induction of TNF and activation of the NFκB pathway. Published by Elsevier Ltd.

Complement 5a Enhances Hepatic Metastases of Colon Cancer via Monocyte Chemoattractant Protein-1-mediated Inflammatory Cell Infiltration*

Science.gov (United States)

Piao, Chunmei; Cai, Lun; Qiu, Shulan; Jia, Lixin; Song, Wenchao; Du, Jie

2015-01-01

Complement 5a (C5a), a potent immune mediator generated by complement activation, promotes tumor growth; however, its role in tumor metastasis remains unclear. We demonstrate that C5a contributes to tumor metastases by modulating tumor inflammation in hepatic metastases of colon cancer. Colon cancer cell lines generate C5a under serum-free conditions, and C5a levels increase over time in a murine syngeneic colon cancer hepatic metastasis model. Furthermore, in the absence of C5a receptor or upon pharmacological inhibition of C5a production with an anti-C5 monoclonal antibody, tumor metastasis is severely impaired. A lack of C5a receptor in colon cancer metastatic foci reduces the infiltration of macrophages, neutrophils, and dendritic cells, and the role for C5a receptor on these cells were further verified by bone marrow transplantation experiments. Moreover, C5a signaling increases the expression of the chemokine monocyte chemoattractant protein-1 and the anti-inflammatory molecules arginase-1, interleukin 10, and transforming growth factor β, but is inversely correlated with the expression of pro-inflammatory molecules, which suggests a mechanism for the role of C5a in the inflammatory microenvironment required for tumor metastasis. Our results indicate a new and potentially promising therapeutic application of complement C5a inhibitor for the treatment of malignant tumors. PMID:25739439

Biglycan- and Sphingosine Kinase-1 Signaling Crosstalk Regulates the Synthesis of Macrophage Chemoattractants

Directory of Open Access Journals (Sweden)

Louise Tzung-Harn Hsieh

2017-03-01

Full Text Available In its soluble form, the extracellular matrix proteoglycan biglycan triggers the synthesis of the macrophage chemoattractants, chemokine (C-C motif ligand CCL2 and CCL5 through selective utilization of Toll-like receptors (TLRs and their adaptor molecules. However, the respective downstream signaling events resulting in biglycan-induced CCL2 and CCL5 production have not yet been defined. Here, we show that biglycan stimulates the production and activation of sphingosine kinase 1 (SphK1 in a TLR4- and Toll/interleukin (IL-1R domain-containing adaptor inducing interferon (IFN-β (TRIF-dependent manner in murine primary macrophages. We provide genetic and pharmacological proof that SphK1 is a crucial downstream mediator of biglycan-triggered CCL2 and CCL5 mRNA and protein expression. This is selectively driven by biglycan/SphK1-dependent phosphorylation of the nuclear factor NF-κB p65 subunit, extracellular signal-regulated kinase (Erk1/2 and p38 mitogen-activated protein kinases. Importantly, in vivo overexpression of soluble biglycan causes Sphk1-dependent enhancement of renal CCL2 and CCL5 and macrophage recruitment into the kidney. Our findings describe the crosstalk between biglycan- and SphK1-driven extracellular matrix- and lipid-signaling. Thus, SphK1 may represent a new target for therapeutic intervention in biglycan-evoked inflammatory conditions.

Adiponectin, interleukin-6, monocyte chemoattractant protein-1, and regional fat mass during 12-month randomized treatment with metformin and/or oral contraceptives in polycystic ovary syndrome

DEFF Research Database (Denmark)

Glintborg, Dorte; Mumm, Hanne; Altinok, Magda Lambaa

2014-01-01

CONTEXT: Central obesity in polycystic ovary syndrome (PCOS) is associated with increased inflammatory markers and increased risk for type 2 diabetes. OBJECTIVE: To evaluate if improved body composition during treatment with metformin (M) vs. oral contraceptive pills (OCP) was associated...... with changes in circulating adiponectin, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1. PATIENTS AND INTERVENTIONS: Ninety patients with PCOS were randomized to 12-month treatment with M (2 g/day), M + OCP (150 mg desogestrel + 30 microgram ethinylestradiol) or OCP. Adiponectin, IL-6, MCP-1...... during the three types of medical intervention. Treatment with M and M + OCP was superior to OCP regarding decreased regional fat mass. Baseline adiponectin and IL-6 were associated with BMI, waist, and trunk fat mass. Changes in trunk fat were significantly associated with changes in IL-6 and MCP-1...

Complement 5a Enhances Hepatic Metastases of Colon Cancer via Monocyte Chemoattractant Protein-1-mediated Inflammatory Cell Infiltration.

Science.gov (United States)

Piao, Chunmei; Cai, Lun; Qiu, Shulan; Jia, Lixin; Song, Wenchao; Du, Jie

2015-04-24

Complement 5a (C5a), a potent immune mediator generated by complement activation, promotes tumor growth; however, its role in tumor metastasis remains unclear. We demonstrate that C5a contributes to tumor metastases by modulating tumor inflammation in hepatic metastases of colon cancer. Colon cancer cell lines generate C5a under serum-free conditions, and C5a levels increase over time in a murine syngeneic colon cancer hepatic metastasis model. Furthermore, in the absence of C5a receptor or upon pharmacological inhibition of C5a production with an anti-C5 monoclonal antibody, tumor metastasis is severely impaired. A lack of C5a receptor in colon cancer metastatic foci reduces the infiltration of macrophages, neutrophils, and dendritic cells, and the role for C5a receptor on these cells were further verified by bone marrow transplantation experiments. Moreover, C5a signaling increases the expression of the chemokine monocyte chemoattractant protein-1 and the anti-inflammatory molecules arginase-1, interleukin 10, and transforming growth factor β, but is inversely correlated with the expression of pro-inflammatory molecules, which suggests a mechanism for the role of C5a in the inflammatory microenvironment required for tumor metastasis. Our results indicate a new and potentially promising therapeutic application of complement C5a inhibitor for the treatment of malignant tumors. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Monocyte chemoattractant protein-1 and interleukin-8 levels in urine and serum of patents with hemolytic uremic syndrome.

Science.gov (United States)

van Setten, P A; van Hinsbergh, V W; van den Heuvel, L P; Preyers, F; Dijkman, H B; Assmann, K J; van der Velden, T J; Monnens, L A

1998-06-01

The epidemic form of the hemolytic uremic syndrome (HUS) in children is hallmarked by endothelial cell damage, most predominantly displayed by the glomerular capillaries. The influx of mononuclear (MO) and polymorphonuclear cells (PMNs) into the glomeruli may be an important event in the initiation, prolongation, and progression of glomerular endothelial cell damage in HUS patients. The molecular mechanisms for the recruitment of these leukocytes into the kidney are unclear, but monocyte chemoattractant protein-1 (MCP-1) and IL-8 are suggested to be prime candidates. In this study, we analyzed the presence of both chemokines in 24-h urinary (n = 15) and serum (n = 14) samples of HUS children by specific ELISAs. Furthermore, kidney biopsies of three different HUS children were examined for MO and PMN cell infiltration by histochemical techniques and electron microscopy. Whereas the chemokines MCP-1 and IL-8 were present in only very limited amounts in urine of 17 normal control subjects, serial samples of HUS patients demonstrated significantly elevated levels of both chemokines. HUS children with anuria showed higher initial and maximum chemokine levels than their counterparts without anuria. A strong positive correlation was observed between urinary MCP-1 and IL-8 levels. Whereas initial serum IL-8 levels were significantly increased in HUS children, serum MCP-1 levels were only slightly elevated compared with serum MCP-1 in control children. No correlation was found between urinary and serum chemokine concentrations. Histologic and EM studies of HUS biopsy specimens clearly showed the presence of MOs and to a lesser extent of PMNs in the glomeruli. The present data suggest an important local role for MOs and PMNs in the process of glomerular endothelial-cell damage. The chemokines MCP-1 and IL-8 may possibly be implicated in the pathogenesis of HUS through the recruitment and activation of MOs and PMNs, respectively.

Radiation-Induced Thymidine Phosphorylase Upregulation in Rectal Cancer Is Mediated by Tumor-Associated Macrophages by Monocyte Chemoattractant Protein-1 From Cancer Cells

International Nuclear Information System (INIS)

Kim, Tae-Dong; Li Ge; Song, Kyoung-Sub; Kim, Jin-Man; Kim, Jun-Sang; Kim, Jong-Seok; Yun, Eun-Jin; Park, Jong-Il; Park, Hae-Duck; Hwang, Byung-Doo; Lim, Kyu; Yoon, Wan-Hee

2009-01-01

Purpose: The mechanisms of thymidine phosphorylase (TP) regulation induced by radiation therapy (XRT) in various tumors are poorly understood. We investigated the effect and mechanisms of preoperative XRT on TP expression in rectal cancer tissues. Methods and Materials: TP expression and CD68 and monocyte chemoattractant protein-1 (MCP-1) levels in rectal cancer tissues and cancer cell lines were evaluated before and after XRT in Western blotting, immunohistochemistry, enzyme-linked immunoassay, and reverse transcription-polymerase chain reaction studies. Isolated peripheral blood monocytes were used in the study of chemotaxis under the influence of MCP-1 released by irradiated colon cancer cells. Results: Expression of TP was significantly elevated by 9 Gy of XRT in most rectal cancer tissues but not by higher doses of XRT. In keeping with the close correlation of the increase in both TP expression and the number of tumor-associated macrophages (TAMs), anti-TP immunoreactivity was found in the CD68-positive TAMs and not the neoplastic cells. Expression of MCP-1 was increased in most cases after XRT, and this increase was strongly correlated with TP expression. However, this increase in MCP-1 expression occurred in tumor cells and not stromal cells. The XRT upregulated MCP-1 mRNA and also triggered the release of MCP-1 protein from cultured colon cancer cells. The supernatant of irradiated colon cancer cells showed strong chemotactic activity for monocyte migration, but this activity was completely abolished by neutralizing antibody. Conclusions: Use of XRT induces MCP-1 expression in cancer cells, which causes circulating monocytes to be recruited into TAMs, which then upregulate TP expression in rectal cancer tissues

Monocyte chemoattractant protein-1 promoter -2518 polymorphism and susceptibility to vasculitis, rheumatoid arthritis, and multiple sclerosis: A meta-analysis.

Science.gov (United States)

Lee, Y H; Bae, S-C

2016-03-20

The purpose of this study was to examine whether the monocyte chemoattractant protein-1 (MCP-1) promoter -2518 A/G polymorphism (rs1024611) is associated with susceptibility to vasculitis, rheumatoid arthritis (RA), or multiple sclerosis (MS). A meta-analysis was conducted on the association between the MCP-1 -2518 A/G polymorphism and vasculitis, RA, and MS. Fourteen studies from 13 articles, including six on vasculitis, five on RA, and three on MS, consisting of 3,038 patients and 3,545 controls were available for the meta-analysis. The meta-analysis revealed no association between the MCP-1 -2518 G allele and vasculitis (odds ratio [OR] = 0.990, 95% confidence interval [CI] = 0.749-1.309, p = 0.943). Stratification by ethnicity indicated no association between the G allele of the MCP-1 -2518 A/G polymorphism and vasculitis in Asians and Caucasians. Meta-analysis by vasculitis type revealed an association between the GG+GA genotype of the MCP-1 -2518 A/G polymorphism and Behçet's disease (BD; OR = 1.349, 95% CI = 1.013-1.796, p = 0.040). However, sensitivity analysis showed that the association was not statistically significant after removing a study that was conducted in China (OR = 1.030, 95% CI = 0.667-1.590, p = 0.895), which indicated that the association was not statistically robust. The meta-analysis revealed no association between the MCP-1 -2518 G allele and RA (OR = 0.986, 95% CI = 0.890-1.093, p = 0.793) or MS (OR = 1.281, 95% CI = 0.802-2.046, p = 0.301). Our meta-analysis demonstrates that the MCP-1 -2518 A/G polymorphism is not associated with susceptibility to vasculitis, RA, or MS.

Chondroitin Sulfate Inhibits Monocyte Chemoattractant Protein-1 Release From 3T3-L1 Adipocytes: A New Treatment Opportunity for Obesity-Related Inflammation?

Directory of Open Access Journals (Sweden)

Thomas V Stabler

2017-08-01

Full Text Available Monocyte chemoattractant protein-1 (MCP-1 overproduction from inflamed adipose tissue is a major contributor to obesity-related metabolic syndromes. 3T3-L1 embryonic fibroblasts were cultured and differentiated into adipocytes using an established protocol. Adipocytes were treated with lipopolysaccharide (LPS to induce inflammation and thus MCP-1 release. At the same time, varying concentrations of chondroitin sulfate (CS were added in a physiologically relevant range (10-200 µg/mL to determine its impact on MCP-1 release. Chondroitin sulfate, a natural glycosaminoglycan of connective tissue including the cartilage extracellular matrix, was chosen on the basis of our previous studies demonstrating its anti-inflammatory effect on macrophages. Because the main action of MCP-1 is to induce monocyte migration, cultured THP-1 monocytes were used to test whether CS at the highest physiologically relevant concentration could inhibit cell migration induced by human recombinant MCP-1. Chondroitin sulfate (100-200 µg/mL inhibited MCP-1 release from inflamed adipocytes in a dose-dependent manner ( P  < .01, 95% confidence interval [CI]: −5.89 to −3.858 at 100 µg/mL and P  < .001, 95% CI: −6.028 to −3.996 at 200 µg/mL but had no effect on MCP-1–driven chemotaxis of THP-1 monocytes. In summary, CS could be expected to reduce macrophage infiltration into adipose tissue by reduction in adipocyte expression and release of MCP-1 and as such might reduce adipose tissue inflammation in response to pro-inflammatory stimuli such as LPS, now increasingly recognized to be relevant in vivo.

Monocyte Chemoattractant Protein-1 in the choroid plexus: a potential link between vascular pro-inflammatory mediators and the CNS during peripheral tissue inflammation

Science.gov (United States)

Mitchell, K.; Yang, H.-Y. T.; Berk, J. D.; Tran, J. H.; Iadarola, M. J.

2009-01-01

During peripheral tissue inflammation, inflammatory processes in the CNS can be initiated by blood-borne pro-inflammatory mediators. The choroid plexus, the site of CSF production, is a highly specialized interface between the vascular system and CNS, and thus, this structure may be an important element in communication between the vascular compartment and the CNS during peripheral tissue inflammation. We investigated the potential participation of the choroid plexus in this process during peripheral tissue inflammation by examining expression of the SCYA2 gene which codes for monocyte chemoattractant protein-1 (MCP-1). MCP-1 protein was previously reported to be induced in a variety of cells during peripheral tissue inflammation. In the basal state, SCYA2 is highly expressed in the choroid plexus as compared to other CNS tissues. During hind paw inflammation, SCYA2 expression was significantly elevated in choroid plexus, whereas it remained unchanged in a variety of brain regions. The SCYA2-expressing cells were strongly associated with the choroid plexus as vascular depletion of blood cells by whole-body saline flush did not significantly alter SCYA2 expression in the choroid plexus. In situ hybridization suggested that the SCYA2-expressing cells were localized to the choroid plexus stroma. To elucidate potential molecular mechanisms of SCYA2 increase, we examined genes in the NF-κβ signaling cascade including TNF-α, IL-1β and IκBα in choroid tissue. Given that we also detected increased levels of MCP-1 protein by ELISA, we sought to identify potential downstream targets of MCP-1 and observed altered expression levels of mRNAs encoding tight junction proteins TJP2 and claudin 5. Finally, we detected a substantial up-regulation of the transcript encoding E-selectin, a molecule which could participate in leukocyte recruitment to the choroid plexus along with MCP-1. Together, these results suggest that profound changes occur in the choroid plexus during

Correlation of urinary monocyte chemo-attractant protein-1 with other parameters of renal injury in type-II diabetes mellitus

Directory of Open Access Journals (Sweden)

Ibrahim Salwa

2008-01-01

Full Text Available Diabetic nephropathy (DN is the leading cause of end-stage renal disease in the western world. Increased number of interstitial macrophages has been observed in biopsies from patients with DN. Monocyte chemo-attractant protein-1 (MCP-1 is the strongest known chemo-tactic factor for monocytes and is upregulated in DN. We examined urinary levels of MCP-1 in patients with type-2 diabetes mellitus (DM to assess its possible correlation with other para-meters of renal injury. The urinary MCP-1 level was assessed in 75 patients with type-2 DM (25 patients each with no microalbuminuria, with macroalbuminuria and, with renal impairment and compared them with matched healthy control subjects. The HbA1c and estimated glomerular fil-tration rate (eGFR derived from the abbreviated Modification of Diet in Renal Disease (MDRD equation were examined in the study groups in relation to the urinary MCP-1. The urinary MCP-1 level was significantly higher in patients with micro and macroalbuminuria (167.41 ± 50.23 and 630.87 ± 318.10 ng/gm creatinine respectively as compared with normoalbuminuric patients and healthy controls (63.85 ± 21.15 and 61.50 ± 24.81 ng/gm creatinine, p< 0.001. MCP-1 correlated positively with urine albumin/creatinine ratio (ACR (r= 0.75, p< 0.001, HbA1c (r= 0.55, p< 0.001 and inversely with eGFR (r=-0.60, p< 0.001. Our findings suggest that hyperglycemia is associated with increased urinary levels of MCP-1 that is closely linked to renal damage as reflected by proteinuria and eGFR levels. Collectively, these findings suggest that MCP-1 is in-volved in the pathogenesis of diabetic nephropathy through its various stages.

Urine Epidermal Growth Factor, Monocyte Chemoattractant Protein-1 or Their Ratio as Biomarkers for Interstitial Fibrosis and Tubular Atrophy in Primary Glomerulonephritis

Directory of Open Access Journals (Sweden)

Supanat Worawichawong

2016-12-01

Full Text Available Background/Aims: The degree of tubular atrophy and interstitial fibrosis (IFTA is an important prognostic factor in glomerulonephritis. Imbalance between pro-inflammatory cytokines such as monocyte chemoattractant protein- 1 (MCP-1 and protective cytokines such as epidermal growth factor (EGF likely determine IFTA severity. In separate studies, elevated MCP-1 and decreased EGF have been shown to be associated with IFTA severity. In this study, we aim to evaluate the predictive value of urinary EGF/MCP-1 ratio compared to each biomarker individually for moderate to severe IFTA in primary glomerulonephritis (GN. Methods: Urine samples were collected at biopsy from primary GN (IgA nephropathy, focal and segmental glomerulosclerosis, minimal change disease, membranous nephropathy. MCP-1 and EGF were analyzed by enzyme-linked immunosorbent assay. Results: EGF, MCP-1 and EGF/MCP-1 ratio from primary GN, all correlated with IFTA (n=58. By univariate analysis, glomerular filtration rate, EGF, and EGF/MCP-1 ratio were associated with IFTA. By multivariate analysis, only EGF/MCP-1 ratio was independently associated with IFTA. EGF/MCP-1 ratio had a sensitivity of 88% and specificity of 74 % for IFTA. EGF/MCP-1 had good discrimination for IFTA (AUC=0.85, but the improvement over EGF alone was not significant. Conclusion: EGF/MCP-1 ratio is independently associated IFTA severity in primary glomerulonephritis, but the ability of EGF/MCP-1 ratio to discriminate moderate to severe IFTA may not be much better than EGF alone.

Mechanism of sphingosine 1-phosphate- and lysophosphatidic Acid-induced up-regulation of adhesion molecules and eosinophil chemoattractant in nerve cells.

LENUS (Irish Health Repository)

Costello, Richard W

2012-02-01

The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via G-protein coupled receptors S1P(1-5) and LPA(1-3) respectively, and are implicated in allergy. Eosinophils accumulate at innervating cholinergic nerves in asthma and adhere to nerve cells via intercellular adhesion molecule-1 (ICAM-1). IMR-32 neuroblastoma cells were used as an in vitro cholinergic nerve cell model. The G(i) coupled receptors S1P(1), S1P(3), LPA(1), LPA(2) and LPA(3) were expressed on IMR-32 cells. Both S1P and LPA induced ERK phosphorylation and ERK- and G(i)-dependent up-regulation of ICAM-1 expression, with differing time courses. LPA also induced ERK- and G(i)-dependent up-regulation of the eosinophil chemoattractant, CCL-26. The eosinophil granule protein eosinophil peroxidase (EPO) induced ERK-dependent up-regulation of transcription of S1P(1), LPA(1), LPA(2) and LPA(3), providing the situation whereby eosinophil granule proteins may enhance S1P- and\\/or LPA- induced eosinophil accumulation at nerve cells in allergic conditions.

Mechanism of sphingosine 1-phosphate- and lysophosphatidic Acid-induced up-regulation of adhesion molecules and eosinophil chemoattractant in nerve cells.

LENUS (Irish Health Repository)

Costello, Richard W

2011-05-01

The lysophospholipids sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) act via G-protein coupled receptors S1P(1-5) and LPA(1-3) respectively, and are implicated in allergy. Eosinophils accumulate at innervating cholinergic nerves in asthma and adhere to nerve cells via intercellular adhesion molecule-1 (ICAM-1). IMR-32 neuroblastoma cells were used as an in vitro cholinergic nerve cell model. The G(i) coupled receptors S1P(1), S1P(3), LPA(1), LPA(2) and LPA(3) were expressed on IMR-32 cells. Both S1P and LPA induced ERK phosphorylation and ERK- and G(i)-dependent up-regulation of ICAM-1 expression, with differing time courses. LPA also induced ERK- and G(i)-dependent up-regulation of the eosinophil chemoattractant, CCL-26. The eosinophil granule protein eosinophil peroxidase (EPO) induced ERK-dependent up-regulation of transcription of S1P(1), LPA(1), LPA(2) and LPA(3), providing the situation whereby eosinophil granule proteins may enhance S1P- and\\/or LPA- induced eosinophil accumulation at nerve cells in allergic conditions.

Folic acid is a potent chemoattractant of free-living amoebae in a new and amazing species of protist, Vahlkampfia sp.

Science.gov (United States)

Maeda, Yasuo; Mayanagi, Taira; Amagai, Aiko

2009-03-01

Folic acid (folate; vitamin Bc) is well recognized as essential for the proper metabolism of the essential amino acid methionine as well as for the synthesis of adenine and thymine. A folate deficiency has been Implicated in a wide variety of disorders from Alzheimer's disease to depression and neural tube defects. In the cellular slime molds, including Dictyostelium, vegetative growth-phase cells are known to chemotactically move toward folate that is secreted by bacterial food sources such as Escherichia coli. Intracellular folate signal transductlon, including G proteins, Ca(2+)channels, and the PIP3 pathway, has been reported in D. discoideum. To our surprise, the genuine chemoattractant(s) of free-living protozoan amoebae have remained to be determined, possibly because of lack of a pertinent method for assaying chemotaxis. We recently isolated a primitive free-living amoeba from the soil of Costa Rica and identified it as a new species of the genus Vahlkampfia belonging to Subclass Gymnamoebia, which includes Entamoeba and Acanthamoeba. The amoebae can grow and multiply quite rapidly, engulfing nearby bacteria such as E. coli. Importantly, we have demonstrated here using a quite simple but finely designed chemotaxis assay that the Vahlkampfia amoebae exhibit chemotaxis toward higher folate concentrations. Riboflavin and cyanocobalamin were also found to serve as positive chemoattractants. Among these chemoattractants, folate is of particular importance because its function seems to be evolutionarily conserved as a potent chemoattractant of amoeboid cells in a wide range of organisms as well as in the Protista and cellular slime molds.

Requirement for C-X-C chemokines (macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant) in IgG immune complex-induced lung injury

DEFF Research Database (Denmark)

Shanley, T P; Schmal, H; Warner, R L

1997-01-01

chemokines, macrophage inflammatory protein-2 (MIP-2) and cytokine-induced neutrophil chemoattractant (CINC). Both mRNA and protein for MIP-2 and CINC appeared in a time-dependent manner after initiation of IgG immune complex deposition in lung. There exists a 69% homology between the amino acid sequences...... for these proteins, and we found cross-reactivity between polyclonal Abs raised to these chemokines. By purifying the blocking Abs using double affinity methods (with Ag-immobilized beads), this cross-reactivity was removed. Individually, anti-MIP-2 and anti-CINC Ab significantly reduced lung injury (as measured...... activity in BAL fluids collected 2 h after injury from animals undergoing immune complex deposition could be shown to be chiefly due to the combined contributions of MIP-2 (39%), CINC (28%), and C5a (21%). When either MIP-2 or CINC was blocked in vivo, up-regulation of Mac-1 expression on neutrophils...

Stromal cell derived factor-1α (SDF-1α) directed chemoattraction of transiently CXCR4 overexpressing mesenchymal stem cells into functionalized three-dimensional biomimetic scaffolds

DEFF Research Database (Denmark)

Thieme, S; Ryser, Martin; Gentsch, Marcus

2009-01-01

Three-dimensional (3D) bone substitute material should not only serve as scaffold in large bone defects but also attract mesenchymal stem cells, a subset of bone marrow stromal cells (BMSCs) that are able to form new bone tissue. An additional crucial step is to attract BMSCs from the surface int...... invaded up to 250 mum into SDF-1alpha-releasing 3D scaffolds, whereas CXCR4-overexpressing BMSC invaded up to 500 mum within 5 days. Thus, the SDF-1alpha/CXCR4 chemoattraction system can be used to efficiently recruit BMSCs into SDF-1alpha-releasing 3D scaffolds in vitro and in vivo....

Loss of PPAR gamma in immune cells impairs the ability of abscisic acid to improve insulin sensitivity by suppressing monocyte chemoattractant protein-1 expression and macrophage infiltration into white adipose tissue.

Science.gov (United States)

Guri, Amir J; Hontecillas, Raquel; Ferrer, Gerardo; Casagran, Oriol; Wankhade, Umesh; Noble, Alexis M; Eizirik, Decio L; Ortis, Fernanda; Cnop, Miriam; Liu, Dongmin; Si, Hongwei; Bassaganya-Riera, Josep

2008-04-01

Abscisic acid (ABA) is a natural phytohormone and peroxisome proliferator-activated receptor gamma (PPARgamma) agonist that significantly improves insulin sensitivity in db/db mice. Although it has become clear that obesity is associated with macrophage infiltration into white adipose tissue (WAT), the phenotype of adipose tissue macrophages (ATMs) and the mechanisms by which insulin-sensitizing compounds modulate their infiltration remain unknown. We used a loss-of-function approach to investigate whether ABA ameliorates insulin resistance through a mechanism dependent on immune cell PPARgamma. We characterized two phenotypically distinct ATM subsets in db/db mice based on their surface expression of F4/80. F4/80(hi) ATMs were more abundant and expressed greater concentrations of chemokine receptor (CCR) 2 and CCR5 when compared to F4/80(lo) ATMs. ABA significantly decreased CCR2(+) F4/80(hi) infiltration into WAT and suppressed monocyte chemoattractant protein-1 (MCP-1) expression in WAT and plasma. Furthermore, the deficiency of PPARgamma in immune cells, including macrophages, impaired the ability of ABA to suppress the infiltration of F4/80(hi) ATMs into WAT, to repress WAT MCP-1 expression and to improve glucose tolerance. We provide molecular evidence in vivo demonstrating that ABA improves insulin sensitivity and obesity-related inflammation by inhibiting MCP-1 expression and F4/80(hi) ATM infiltration through a PPARgamma-dependent mechanism.

Purification and characterization of a chemoattractant from electric shock-induced earthworm secretion, its receptor binding, and signal transduction through the vomeronasal system of garter snakes.

Science.gov (United States)

Jiang, X C; Inouchi, J; Wang, D; Halpern, M

1990-05-25

Following shocks with low voltage electric current, earthworms, Lumbricus terrestris, secrete a yellow mucus that has alarm properties for conspecifics and chemoattractive properties for garter snakes, Thamnophis sirtalis. A proteinaceous chemoattractant for garter snakes has been isolated and purified to homogeneity from such secretions by means of permeation chromatography and semipreparative nondenaturing polyacrylamide gel electrophoresis. The purified protein is highly attractive to garter snakes; it loses its activity after proteolytic digestion. It is a glycoprotein consisting of a single polypeptide chain with an NH2-terminal alanine. This chemoattractant has a minimum molecular mass of 15.4 kDa calculated from its amino acid and carbohydrate contents and an apparent molecular mass of about 20 kDa as estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It has a pI of about 4.0, and it binds wheat germ agglutinin but not concanavalin A. This chemoattractant shows a protein to carbohydrate ratio of 2.0 +/- 0.08 (n = 5) and a ratio of total sugar to amino sugar of 1.9 +/- 0.08 (n = 3). The sequence of its NH2-terminal 15 amino acid residues has been determined. Studies were also conducted on the chemosignal transduction through the vomeronasal sensory system of the garter snake. Dot blot analysis showed that the purified chemoattractant bound to snake vomeronasal sensory epithelial membrane fractions. It did not bind to membrane extracts of the nonsensory epithelium of the vomeronasal mushroom body. The chemoattractant also bound specifically to vomeronasal sensory epithelial membrane in a reversible and saturable fashion with Kd and Bmax values of about 0.3 microM and 0.4 nmol/mg of protein, respectively. In electrophysiological studies, the chemoattractant applied to the vomeronasal epithelium caused an increase in firing rate of individual neurons in the accessory olfactory bulb of garter snakes, the projection site for vomeronasal

Role of CC chemokines (macrophage inflammatory protein-1 beta, monocyte chemoattractant protein-1, RANTES) in acute lung injury in rats

DEFF Research Database (Denmark)

Bless, N M; Huber-Lang, M; Guo, R F

2000-01-01

The role of the CC chemokines, macrophage inflammatory protein-1 beta (MIP-1 beta), monocyte chemotactic peptide-1 (MCP-1), and RANTES, in acute lung inflammatory injury induced by intrapulmonary deposition of IgG immune complexes injury in rats was determined. Rat MIP-1 beta, MCP-1, and RANTES...... were cloned, the proteins were expressed, and neutralizing Abs were developed. mRNA and protein expression for MIP-1 beta and MCP-1 were up-regulated during the inflammatory response, while mRNA and protein expression for RANTES were constitutive and unchanged during the inflammatory response....... Treatment of rats with anti-MIP-1 beta Ab significantly decreased vascular permeability by 37% (p = 0.012), reduced neutrophil recruitment into lung by 65% (p = 0.047), and suppressed levels of TNF-alpha in bronchoalveolar lavage fluids by 61% (p = 0.008). Treatment of rats with anti-rat MCP-1 or anti...

Progranulin Is a Chemoattractant for Microglia and Stimulates Their Endocytic Activity

Science.gov (United States)

Pickford, Fiona; Marcus, Jacob; Camargo, Luiz Miguel; Xiao, Qiurong; Graham, Danielle; Mo, Jan-Rung; Burkhardt, Matthew; Kulkarni, Vinayak; Crispino, Jamie; Hering, Heike; Hutton, Michael

2011-01-01

Mutations resulting in progranulin haploinsufficiency cause disease in patients with a subset of frontotemporal lobar degeneration; however, the biological functions of progranulin in the brain remain unknown. To address this subject, the present study initially assessed changes in gene expression and cytokine secretion in rat primary cortical neurons treated with progranulin. Molecular pathways enriched in the progranulin gene set included cell adhesion and cell motility pathways and pathways involved in growth and development. Secretion of cytokines and several chemokines linked to chemoattraction but not inflammation were also increased from progranulin-treated primary neurons. Therefore, whether progranulin is involved in recruitment of immune cells in the brain was investigated. Localized lentiviral expression of progranulin in C57BL/6 mice resulted in an increase of Iba1-positive microglia around the injection site. Moreover, progranulin alone was sufficient to promote migration of primary mouse microglia in vitro. Primary microglia and C4B8 cells demonstrated more endocytosis of amyloid β1-42 when treated with progranulin. These data demonstrate that progranulin acts as a chemoattractant in the brain to recruit or activate microglia and can increase endocytosis of extracellular peptides such as amyloid β. PMID:21224065

The biophysical model for accuracy of cellular sensing spatial gradients of multiple chemoattractants

International Nuclear Information System (INIS)

Chang, Qiang; Zuo, Li

2013-01-01

Spatial gradients of surrounding chemoattractants are the key factors in determining the directionality of eukaryotic cell movement. Thus, it is important for cells to accurately measure the spatial gradients of surrounding chemoattractants. Here, we study the precision of sensing the spatial gradients of multiple chemoattractants using cooperative receptor clusters. Cooperative receptors on cells are modeled as an Ising chain of Monod–Wyman–Changeux clusters subject to multiple chemical-gradient fields to study the physical limits of multiple chemoattractants spatial gradients sensing. We found that eukaryotic cells cannot sense each chemoattractant gradient individually. Instead, cells can only sense a weighted sum of surrounding chemical gradients. Moreover, the precision of sensing one chemical gradient is signicantly affected by coexisting chemoattractant concentrations. These findings can provide a further insight into the role of chemoattractants in immune response and help develop novel treatments for inflammatory diseases. (paper)

Monocyte chemoattractant protein-1 (MCP-1 regulates macrophage cytotoxicity in abdominal aortic aneurysm.

Directory of Open Access Journals (Sweden)

Qiwei Wang

Full Text Available AIMS: In abdominal aortic aneurysm (AAA, macrophages are detected in the proximity of aortic smooth muscle cells (SMCs. We have previously demonstrated in a murine model of AAA that apoptotic SMCs attract monocytes and other leukocytes by producing MCP-1. Here we tested whether infiltrating macrophages also directly contribute to SMC apoptosis. METHODS AND RESULTS: Using a SMC/RAW264.7 macrophage co-culture system, we demonstrated that MCP-1-primed RAWs caused a significantly higher level of apoptosis in SMCs as compared to control macrophages. Next, we detected an enhanced Fas ligand (FasL mRNA level and membrane FasL protein expression in MCP-1-primed RAWs. Neutralizing FasL blocked SMC apoptosis in the co-culture. In situ proximity ligation assay showed that SMCs exposed to primed macrophages contained higher levels of receptor interacting protein-1 (RIP1/Caspase 8 containing cell death complexes. Silencing RIP1 conferred apoptosis resistance to SMCs. In the mouse elastase injury model of aneurysm, aneurysm induction increased the level of RIP1/Caspase 8 containing complexes in medial SMCs. Moreover, TUNEL-positive SMCs in aneurysmal tissues were frequently surrounded by CD68(+/FasL(+ macrophages. Conversely, elastase-treated arteries from MCP-1 knockout mice display a reduction of both macrophage infiltration and FasL expression, which was accompanied by diminished apoptosis of SMCs. CONCLUSION: Our data suggest that MCP-1-primed macrophages are more cytotoxic. MCP-1 appears to modulate macrophage cytotoxicity by increasing the level of membrane bound FasL. Thus, we showed that MCP-1-primed macrophages kill SMCs through a FasL/Fas-Caspase8-RIP1 mediated mechanism.

The G-protein coupled chemoattractant receptor FPR2 promotes malignant phenotype of human colon cancer cells

Science.gov (United States)

Xiang, Yi; Yao, Xiaohong; Chen, Keqiang; Wang, Xiafei; Zhou, Jiamin; Gong, Wanghua; Yoshimura, Teizo; Huang, Jiaqiang; Wang, Rongquan; Wu, Yuzhang; Shi, Guochao; Bian, Xiuwu; Wang, Jiming

2016-01-01

The G-protein coupled chemoattractant receptor formylpeptide receptor-2 (FPR2 in human, Fpr2 in mice) is expressed by mouse colon epithelial cells and plays a critical role in mediating mucosal homeostasis and inflammatory responses. However, the biological role of FPR2 in human colon is unclear. Our investigation revealed that a considerable number of human colon cancer cell lines expressed FPR2 and its ligands promoted cell migration and proliferation. Human colon cancer cell lines expressing high levels of FPR2 also formed more rapidly growing tumors in immunocompromised mice as compared with cell lines expressing lower levels of FPR2. Knocking down of FPR2 from colon cancer cell lines highly expressing FPR2 reduced their tumorigenicity. Clinically, FPR2 is more highly expressed in progressive colon cancer, associated with poorer patient prognosis. These results suggest that FPR2 can be high-jacked by colon cancer cells for their growth advantage, thus becoming a potential target for therapeutic development. PMID:27904774

Adiponectin, interleukin-6, monocyte chemoattractant protein-1, and regional fat mass during 12-month randomized treatment with metformin and/or oral contraceptives in polycystic ovary syndrome.

Science.gov (United States)

Glintborg, Dorte; Mumm, Hanne; Altinok, Magda Lambaa; Richelsen, Bjørn; Bruun, Jens Meldgaard; Andersen, Marianne

2014-08-01

Central obesity in polycystic ovary syndrome (PCOS) is associated with increased inflammatory markers and increased risk for type 2 diabetes. To evaluate if improved body composition during treatment with metformin (M) vs. oral contraceptive pills (OCP) was associated with changes in circulating adiponectin, interleukin (IL)-6, and monocyte chemoattractant protein (MCP)-1. Ninety patients with PCOS were randomized to 12-month treatment with M (2 g/day), M + OCP (150 mg desogestrel + 30 microgram ethinylestradiol) or OCP. Adiponectin, IL-6, MCP-1, whole body DXA scans, and clinical evaluations were performed before and after the intervention period in the 65 study completers. Changes in inflammatory markers and changes in total and regional fat mass estimates. Adiponectin, IL-6, and MCP-1 levels were unchanged during the three types of medical intervention. Treatment with M and M + OCP was superior to OCP regarding decreased regional fat mass. Baseline adiponectin and IL-6 were associated with BMI, waist, and trunk fat mass. Changes in trunk fat were significantly associated with changes in IL-6 and MCP-1 during M + OCP. Long-term treatment with M alone or in combination with OCP was associated with improved body composition compared to OCP, whereas inflammatory markers were unchanged. OCP was not associated with increased inflammatory markers despite a small but significant weight gain.

Active spice-derived components can inhibit inflammatory responses of adipose tissue in obesity by suppressing inflammatory actions of macrophages and release of monocyte chemoattractant protein-1 from adipocytes.

Science.gov (United States)

Woo, Hae-Mi; Kang, Ji-Hye; Kawada, Teruo; Yoo, Hoon; Sung, Mi-Kyung; Yu, Rina

2007-02-13

Inflammation plays a key role in obesity-related pathologies such as cardiovascular disease, type II diabetes, and several types of cancer. Obesity-induced inflammation entails the enhancement of the recruitment of macrophages into adipose tissue and the release of various proinflammatory proteins from fat tissue. Therefore, the modulation of inflammatory responses in obesity may be useful for preventing or ameliorating obesity-related pathologies. Some spice-derived components, which are naturally occurring phytochemicals, elicit antiobesity and antiinflammatory properties. In this study, we investigated whether active spice-derived components can be applied to the suppression of obesity-induced inflammatory responses. Mesenteric adipose tissue was isolated from obese mice fed a high-fat diet and cultured to prepare an adipose tissue-conditioned medium. Raw 264.7 macrophages were treated with the adipose tissue-conditioned medium with or without active spice-derived components (i.e., diallyl disulfide, allyl isothiocyanate, piperine, zingerone and curcumin). Chemotaxis assay was performed to measure the degree of macrophage migration. Macrophage activation was estimated by measuring tumor necrosis factor-alpha (TNF-alpha), nitric oxide, and monocyte chemoattractant protein-1 (MCP-1) concentrations. The active spice-derived components markedly suppressed the migration of macrophages induced by the mesenteric adipose tissue-conditioned medium in a dose-dependent manner. Among the active spice-derived components studied, allyl isothiocyanate, zingerone, and curcumin significantly inhibited the cellular production of proinflammatory mediators such as TNF-alpha and nitric oxide, and significantly inhibited the release of MCP-1 from 3T3-L1 adipocytes. Our findings suggest that the spice-derived components can suppress obesity-induced inflammatory responses by suppressing adipose tissue macrophage accumulation or activation and inhibiting MCP-1 release from adipocytes

RACK1, A Multifaceted Scaffolding Protein: Structure and Function

LENUS (Irish Health Repository)

Adams, David R

2011-10-06

Abstract The Receptor for Activated C Kinase 1 (RACK1) is a member of the tryptophan-aspartate repeat (WD-repeat) family of proteins and shares significant homology to the β subunit of G-proteins (Gβ). RACK1 adopts a seven-bladed β-propeller structure which facilitates protein binding. RACK1 has a significant role to play in shuttling proteins around the cell, anchoring proteins at particular locations and in stabilising protein activity. It interacts with the ribosomal machinery, with several cell surface receptors and with proteins in the nucleus. As a result, RACK1 is a key mediator of various pathways and contributes to numerous aspects of cellular function. Here, we discuss RACK1 gene and structure and its role in specific signaling pathways, and address how posttranslational modifications facilitate subcellular location and translocation of RACK1. This review condenses several recent studies suggesting a role for RACK1 in physiological processes such as development, cell migration, central nervous system (CN) function and circadian rhythm as well as reviewing the role of RACK1 in disease.

In situ tissue regeneration: chemoattractants for endogenous stem cell recruitment.

Science.gov (United States)

Vanden Berg-Foels, Wendy S

2014-02-01

Tissue engineering uses cells, signaling molecules, and/or biomaterials to regenerate injured or diseased tissues. Ex vivo expanded mesenchymal stem cells (MSC) have long been a cornerstone of regeneration therapies; however, drawbacks that include altered signaling responses and reduced homing capacity have prompted investigation of regeneration based on endogenous MSC recruitment. Recent successful proof-of-concept studies have further motivated endogenous MSC recruitment-based approaches. Stem cell migration is required for morphogenesis and organogenesis during development and for tissue maintenance and injury repair in adults. A biomimetic approach to in situ tissue regeneration by endogenous MSC requires the orchestration of three main stages: MSC recruitment, MSC differentiation, and neotissue maturation. The first stage must result in recruitment of a sufficient number of MSC, capable of effecting regeneration, to the injured or diseased tissue. One of the challenges for engineering endogenous MSC recruitment is the selection of effective chemoattractant(s). The objective of this review is to synthesize and evaluate evidence of recruitment efficacy by reported chemoattractants, including growth factors, chemokines, and other more recently appreciated MSC chemoattractants. The influence of MSC tissue sources, cell culture methods, and the in vitro and in vivo environments is discussed. This growing body of knowledge will serve as a basis for the rational design of regenerative therapies based on endogenous MSC recruitment. Successful endogenous MSC recruitment is the first step of successful tissue regeneration.

Interleukin-17A and Toll-Like Receptor 3 Ligand Poly(I:C Synergistically Induced Neutrophil Chemoattractant Production by Bronchial Epithelial Cells.

Directory of Open Access Journals (Sweden)

Hirotaka Matsuzaki

Full Text Available Chronic inflammatory airway diseases, such as bronchial asthma and chronic obstructive pulmonary disease, are common respiratory disorders worldwide. Exacerbations of these diseases are frequent and worsen patients' respiratory condition and overall health. However, the mechanisms of exacerbation have not been fully elucidated. Recently, it was reported that interleukin (IL-17A might play an important role in neutrophilic inflammation, which is characteristic of such exacerbations, through increased production of neutrophil chemoattractants. Therefore, we hypothesized that IL-17A was involved in the pathogenesis of acute exacerbation, due to viral infection in chronic inflammatory airway diseases. In this study, we assessed chemokine production by bronchial epithelial cells and investigated the underlying mechanisms. Comprehensive chemokine analysis showed that, compared with poly(I:C alone, co-stimulation of BEAS-2B cells with IL-17A and poly(I:C strongly induced production of such neutrophil chemoattractants as CXC chemokine ligand (CXCL8, growth-related oncogene (GRO, and CXCL1. Co-stimulation synergistically induced CXCL8 and CXCL1 mRNA and protein production by BEAS-2B cells and normal human bronchial epithelial cells. Poly(I:C induced chemokine expression by BEAS-2B cells mainly via Toll-like receptor 3/TIR-domain-containing adapter-inducing interferon-β-mediated signals. The co-stimulation with IL-17A and poly(I:C markedly activated the p38 and extracellular-signal-regulated kinase 1/2 pathway, compared with poly(I:C, although there was little change in nuclear factor-κB translocation into the nucleus or the transcriptional activities of nuclear factor-κB and activator protein 1. IL-17A promoted stabilization of CXCL8 mRNA in BEAS-2B cells treated with poly(I:C. In conclusion, IL-17A appears to be involved in the pathogenesis of chronic inflammatory airway disease exacerbation, due to viral infection by promoting release of neutrophil

New insights into potential functions for the protein 4.1superfamily of proteins in kidney epithelium

Energy Technology Data Exchange (ETDEWEB)

Calinisan, Venice; Gravem, Dana; Chen, Ray Ping-Hsu; Brittin,Sachi; Mohandas, Narla; Lecomte, Marie-Christine; Gascard, Philippe

2005-06-17

Members of the protein 4.1 family of adapter proteins are expressed in a broad panel of tissues including various epithelia where they likely play an important role in maintenance of cell architecture and polarity and in control of cell proliferation. We have recently characterized the structure and distribution of three members of the protein 4.1 family, 4.1B, 4.1R and 4.1N, in mouse kidney. We describe here binding partners for renal 4.1 proteins, identified through the screening of a rat kidney yeast two-hybrid system cDNA library. The identification of putative protein 4.1-based complexes enables us to envision potential functions for 4.1 proteins in kidney: organization of signaling complexes, response to osmotic stress, protein trafficking, and control of cell proliferation. We discuss the relevance of these protein 4.1-based interactions in kidney physio-pathology in the context of their previously identified functions in other cells and tissues. Specifically, we will focus on renal 4.1 protein interactions with beta amyloid precursor protein (beta-APP), 14-3-3 proteins, and the cell swelling-activated chloride channel pICln. We also discuss the functional relevance of another member of the protein 4.1 superfamily, ezrin, in kidney physiopathology.

The roles of MCP-1 and protein kinase C delta activation in human eosinophilic leukemia EoL-1 cells.

Science.gov (United States)

Lee, Ji-Sook; Yang, Eun Ju; Kim, In Sik

2009-12-01

Idiopathic hypereosinophilc syndrome is a disorder associated with clonally eosinophilic proliferation. The importance of FIP1-like-1-platelet-derived growth factor receptor-alpha (FIP1L1-PDGFRA) in the pathogenesis and classification of HES has been recently reported. In this study, we investigated the contribution of monocyte chemoattractant protein-1 (MCP-1)/CCL2 to chemotactic activity and protein kinase C delta (PKC delta in the human eosinophilic leukemia cell line EoL-1. These cells express CCR2 protein among the CC chemokine receptors (CCR1-5). MCP-1 induces strong migration of EoL-1 cells and the chemotaxis signal in response to MCP-1 involves a G(i)/G(o) protein, phospholipase C (PLC), PKC delta, p38 MAPK and NF-kappaB. MCP-1 activates p38 MAPK via G(i)/G(o) protein, PLC and PKC delta cascade. MCP-1 also induces NF-kappaB translocation and the activation is inhibited by PKC delta activation. The increase in the basal expression and activity of PKC delta in EoL-1 cells, compared to normal eosinophils, inhibits apoptosis in EoL-1 cells. Anti-apoptotic mechanism of PKC delta is related to inhibition of caspase 3 and caspase 9, but not to FIP1L1-PDGFRA. PKC delta functions as an anti-apoptotic molecule, and is involved in EoL-1 cell movement stimulated by MCP-1. This study contributes to an understanding of MCP-1 in eosinophil biology and pathogenic mechanism of eosinophilic disorders.

Ascorbic acid deficiency stimulates hepatic expression of inflammatory chemokine, cytokine-induced neutrophil chemoattractant-1, in scurvy-prone ODS rats.

Science.gov (United States)

Horio, Fumihiko; Kiyama, Keiichiro; Kobayashi, Misato; Kawai, Kaori; Tsuda, Takanori

2006-02-01

ODS rat has a hereditary defect in ascorbic acid biosynthesis and is a useful animal model for elucidating the physiological role of ascorbic acid. We previously demonstrated by using ODS rats that ascorbic acid deficiency changes the hepatic gene expression of acute phase proteins, as seen in acute inflammation. In this study, we investigated the effects of ascorbic acid deficiency on the production of inflammatory chemokine, cytokine-induced neutrophil chemoattractant-1 (CINC-1), in ODS rats. Male ODS rats (6 wk of age) were fed a basal diet containing ascorbic acid (300 mg/kg diet) or a diet without ascorbic acid for 14 d. Obvious symptoms of scurvy were not observed in the ascorbic acid-deficient rats. Ascorbic acid deficiency significantly elevated the serum concentration of CINC-1 on d 14. The liver and spleen CINC-1 concentrations in the ascorbic acid-deficient rats were significantly elevated to 600% and 180% of the respective values in the control rats. However, the lung concentration of CINC-1 was not affected by ascorbic acid deficiency. Ascorbic acid deficiency significantly elevated the hepatic mRNA level of CINC-1 (to 480% of the value in the control rats), but not the lung mRNA level. These results demonstrate that ascorbic acid deficiency elevates the serum, liver and spleen concentrations of CINC-1 as seen in acute inflammation, and suggest that ascorbic acid deficiency stimulate the hepatic CINC-1 gene expression.

Arachidonic acid is a chemoattractant for Dictyostelium discoideum

Indian Academy of Sciences (India)

Arachidonic acid is a chemoattractant for Dictyostelium discoideum cells ... Arachidonic acid; chemotaxis; fatty acids; iplA ... Previously, we have shown that arachidonic acid (AA) induces an increase in the cytosolic Ca2+ concentration by causing the release of Ca2+ from intracellular stores and activating influx of ...

Gut Microbiota in Type 2 Diabetes Individuals and Correlation with Monocyte Chemoattractant Protein1 and Interferon Gamma from Patients Attending a Tertiary Care Centre in Chennai, India

Directory of Open Access Journals (Sweden)

Premalatha Pushpanathan

2016-01-01

Full Text Available Background: Type 2 diabetes mellitus (T2DM and obesity are associated with changes in gut microbiota and characterized by chronic low-grade inflammation. Monocyte chemoattractant protein-1 (MCP-1 and interferon gamma (IFNγ are proinflammatory cytokines which play an important role in the development of T2DM. We undertook this study to analyze the gut microbiota of T2DM and nondiabetic subjects and to determine the profile of MCP 1 and IFNγ in the same subjects attending a tertiary care center in Chennai, Tamil Nadu, India. Methods: The study included 30 subjects with clinical details. Stool and blood samples were collected from all the subjects. DNA was extracted from fecal samples and polymerase chain reaction was done using fusion primers. Metagenomic analysis was performed using ion torrent sequencing. The reads obtained were in FASTA format and reported as operational taxonomic units. Human MCP 1 and IFNγ enzyme linked immunosorbent assay (ELISA were performed for 23 serum samples. Results: The study consisted of 30 subjects; 17 were T2DM and 13 were nondiabetics. The gut microbiota among T2DM consisted predominantly of Gram negative bacteria; Escherichia and Prevotella, when compared with the nondiabetic group with predominantly Gram positive organisms suchas Faecalibacterium, Eubacterium, and Bifidobacterium. The mean MCP-1 values in the diabetic group were 232.8 pg/ml and in the nondiabetic group 170.84 pg/ml. IFNγ (mean 385.5 pg/ml was raised in glycated hemoglobin (HbA1c group of 6.5–7.5% which was statistically significant. Association of Escherichia with T2DM and association of Bifidobacteria in the nondiabetics were also statistically significant. Conclusion: Escherichia counts were elevated in T2DM with HbA1c of 6.5–8.5% which was statistically significant suggesting that lipopolysaccharides present in the cell wall of Gram-negative bacteria may be responsible for low-grade inflammation as evidenced by elevated MCP-1 and

Prokaryotic expression and in vitro functional analysis of IL-1β and MCP-1 from guinea pig.

Science.gov (United States)

Dirisala, Vijaya R; Jeevan, Amminikutty; Ly, Lan H; McMurray, David N

2013-06-01

The Guinea pig (Cavia porcellus) is an excellent animal model for studying human tuberculosis (TB) and also for a number of other infectious and non-infectious diseases. One of the major roadblocks in effective utilization of this animal model is the lack of readily available immunological reagents. In order to address this issue, guinea pig interleukin 1 beta (IL-1β) and monocyte chemoattractant protein-1 (MCP-1) were efficiently cloned and expressed in a prokaryotic expression vector, and the expressed proteins in soluble form from both the genes were confirmed by N-terminal sequencing. The biological activity of recombinant guinea pig IL-1β was demonstrated by its ability to drive proliferation in thymocytes, and the recombinant guinea pig MCP-1 exhibited chemotactic activity for guinea pig resident peritoneal macrophages. These biologically active recombinant guinea pig proteins will facilitate an in-depth understanding of the role they play in the immune responses of the guinea pig to TB and other diseases.

Mechanisms of EHD/RME-1 Protein Function in Endocytic Transport

Science.gov (United States)

Grant, Barth D.; Caplan, Steve

2009-01-01

The evolutionarily conserved Eps15 homology domain (EHD)/receptor-mediated endocytosis (RME)-1 family of C-terminal EH domain proteins has recently come under intense scrutiny because of its importance in intracellular membrane transport, especially with regard to the recycling of receptors from endosomes to the plasma membrane. Recent studies have shed new light on the mode by which these adenosine triphosphatases function on endosomal membranes in mammals and Caenorhabditis elegans. This review highlights our current understanding of the physiological roles of these proteins in vivo, discussing conserved features as well as emerging functional differences between individual mammalian paralogs. In addition, these findings are discussed in light of the identification of novel EHD/RME-1 protein and lipid interactions and new structural data for proteins in this family, indicating intriguing similarities to the Dynamin superfamily of large guanosine triphosphatases. PMID:18801062

Progesterone from the cumulus cells is the sperm chemoattractant secreted by the rabbit oocyte cumulus complex.

Directory of Open Access Journals (Sweden)

Héctor Alejandro Guidobaldi

Full Text Available Sperm chemotaxis in mammals have been identified towards several female sources as follicular fluid (FF, oviduct fluid, and conditioned medium from the cumulus oophorus (CU and the oocyte (O. Though several substances were confirmed as sperm chemoattractant, Progesterone (P seems to be the best chemoattractant candidate, because: 1 spermatozoa express a cell surface P receptor, 2 capacitated spermatozoa are chemotactically attracted in vitro by gradients of low quantities of P; 3 the CU cells produce and secrete P after ovulation; 4 a gradient of P may be kept stable along the CU; and 5 the most probable site for sperm chemotaxis in vivo could be near and/or inside the CU. The aim of this study was to verify whether P is the sperm chemoattractant secreted by the rabbit oocyte-cumulus complex (OCC in the rabbit, as a mammalian animal model. By means of videomicroscopy and computer image analysis we observed that only the CU are a stable source of sperm attractants. The CU produce and secrete P since the hormone was localized inside these cells by immunocytochemistry and in the conditioned medium by enzyme immunoassay. In addition, rabbit spermatozoa express a cell surface P receptor detected by western blot and localized over the acrosomal region by immunocytochemistry. To confirm that P is the sperm chemoattractant secreted by the CU, the sperm chemotactic response towards the OCC conditioned medium was inhibited by three different approaches: P from the OCC conditioned medium was removed with an anti-P antibody, the attractant gradient of the OCC conditioned medium was disrupted by a P counter gradient, and the sperm P receptor was blocked with a specific antibody. We concluded that only the CU but not the oocyte secretes P, and the latter chemoattract spermatozoa by means of a cell surface receptor. Our findings may be of interest in assisted reproduction procedures in humans, animals of economic importance and endangered species.

Long-term functional impairment of hemopoietic progenitor cells engineered to express the S1 catalytic subunit of pertussis toxin.

Science.gov (United States)

Bonig, Halvard; Rohmer, Laurence; Papayannopoulou, Thalia

2005-06-01

A large body of data suggests that pertussis toxin (PTX)-sensitive G protein signals in mature and immature hemopoietic cells control their migration patterns in vitro and in vivo. These effects were derived after treatment of cells or animals with PTX. To circumvent several inherent problems of PTX holotoxin treatment, we expressed the S1 catalytic activity of PTX, thus blocking Gi protein signaling, in 32D murine myeloid progenitor cells and in primary human CD34+ cells, and studied its functional consequences. S1 was expressed using viral vectors. Effects of Gi protein blockade on proliferation, migration, adhesion, and gene expression were tested in vitro. S1 expression was nontoxic for the cells; expression and function were stable long-term and not overridden by compensatory mechanisms. S1-transduced 32D cells and primary CD34+ cells migrated poorly and did not contract their cytoskeleton upon treatment with the chemoattractant stromal cell-derived factor -1 (SDF-1), similar to the phenotype induced by PTX treatment. Gene expression studies comparing S1-transduced and control 32D cells uncovered four genes, expression of which was regulated by Gi protein blockade. Of interest, although SDF-1 signaling was inhibited, comparison between SDF-1-treated and untreated cells suggests that SDF-1 stimulation does not depend on de novo gene expression in these cells. Furthermore, when injected into nonobese diabetic/severe combined immunodeficient mice, seeding of S1-expressing 32D cells to bone marrow was largely blocked. Expression of S1 is an effective approach for studying long-term functional consequences of Gi protein blockade in hemopoietic cells in vitro and in vivo.

Tumor protein 53-induced nuclear protein 1 (TP53INP1 enhances p53 function and represses tumorigenesis

Directory of Open Access Journals (Sweden)

Jeyran eShahbazi

2013-05-01

Full Text Available Tumor protein 53-induced nuclear protein 1 (TP53INP1 is a stress-induced p53 target gene whose expression is modulated by transcription factors such as p53, p73 and E2F1. TP53INP1 gene encodes two isoforms of TP53INP1 proteins, TP53INP1α and TP53INP1β, both of which appear to be key elements in p53 function. When associated with homeodomain-interacting protein kinase-2 (HIPK2, TP53INP1 phosphorylates p53 protein at Serine 46, enhances p53 protein stability and its transcriptional activity, leading to transcriptional activation of p53 target genes such as p21, PIG-3 and MDM2, cell growth arrest and apoptosis upon DNA damage stress. The anti-proliferative and pro-apoptotic activities of TP53INP1 indicate that TP53INP1 has an important role in cellular homeostasis and DNA damage response. Deficiency in TP53INP1 expression results in increased tumorigenesis; while TP53INP1 expression is repressed during early stages of cancer by factors such as miR-155. This review aims to summarize the roles of TP53INP1 in blocking tumor progression through p53-dependant and p53-independent pathways, as well as the elements which repress TP53INP1 expression, hence highlighting its potential as a therapeutic target in cancer treatment.

Action of a diffusible target-derived chemoattractant on cortical axon branch induction and directed growth.

Science.gov (United States)

Sato, M; Lopez-Mascaraque, L; Heffner, C D; O'Leary, D D

1994-10-01

Cortical axons innervate their brainstem target, the basilar pons, by the initiation and extension of collateral branches interstitially along their length. To address whether a diffusible pons-derived chemoattractant controls these events, we used cocultures in collagen matrices and time-lapse microscopy. Pontine explants enhanced by 5-fold the de novo initiation of transient branches along cortical axons; most branches were directed toward pons. Of the branches extended toward pons, 2%-3% were stabilized; those extended away were not. Pontine explants also enhanced the stable bifurcation of growth cones and prompted directional changes by growth cone turning and collateral extension. These effects were distance dependent and mimicked by pons-conditioned medium. This evidence indicates that the pons activity promotes branch initiation interstitially along cortical axons, a novel property for a chemoattractant, and provides a directional cue for their growth. These findings suggest that the pons chemoattractant serves as a diffusible target-recognition molecule.

Multiple Degradation Pathways of Chemoattractant Mediated Cyclic GMP Accumulation in Dictyostelium

NARCIS (Netherlands)

Haastert, Peter J.M. van; Lookeren Campagne, Michiel M. van; Kesbeke, Fanja

1983-01-01

Chemoattractants induce a transient accumulation of cGMP levels in Dictyostelium. Intracellular cGMP levels reach a peak at 10 s and prestimulated cGMP levels are recovered at about 30 s. Intracellular and extracellular cGMP levels were detected simultaneously after stimulation of D. lacteum cells

Neural regeneration protein is a novel chemoattractive and neuronal survival-promoting factor

International Nuclear Information System (INIS)

Gorba, Thorsten; Bradoo, Privahini; Antonic, Ana; Marvin, Keith; Liu, Dong-Xu; Lobie, Peter E.; Reymann, Klaus G.; Gluckman, Peter D.; Sieg, Frank

2006-01-01

Neurogenesis and neuronal migration are the prerequisites for the development of the central nervous system. We have identified a novel rodent gene encoding for a neural regeneration protein (NRP) with an activity spectrum similar to the chemokine stromal-derived factor (SDF)-1, but with much greater potency. The Nrp gene is encoded as a forward frameshift to the hypothetical alkylated DNA repair protein AlkB. The predicted protein sequence of NRP contains domains with homology to survival-promoting peptide (SPP) and the trefoil protein TFF-1. The Nrp gene is first expressed in neural stem cells and expression continues in glial lineages. Recombinant NRP and NRP-derived peptides possess biological activities including induction of neural migration and proliferation, promotion of neuronal survival, enhancement of neurite outgrowth and promotion of neuronal differentiation from neural stem cells. NRP exerts its effect on neuronal survival by phosphorylation of the ERK1/2 and Akt kinases, whereas NRP stimulation of neural migration depends solely on p44/42 MAP kinase activity. Taken together, the expression profile of Nrp, the existence in its predicted protein structure of domains with similarities to known neuroprotective and migration-inducing factors and the high potency of NRP-derived synthetic peptides acting in femtomolar concentrations suggest it to be a novel gene of relevance in cellular and developmental neurobiology

KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1

International Nuclear Information System (INIS)

Goettel, Jeremy A.; Liang, Dongchun; Hilliard, Valda C.; Edelblum, Karen L.; Broadus, Matthew R.; Gould, Kathleen L.; Hanks, Steven K.; Polk, D. Brent

2011-01-01

The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway is a highly conserved signaling pathway that regulates diverse cellular processes including differentiation, proliferation, and survival. Kinase suppressor of Ras-1 (KSR1) binds each of the three ERK cascade components to facilitate pathway activation. Even though KSR1 contains a C-terminal kinase domain, evidence supporting the catalytic function of KSR1 remains controversial. In this study, we produced recombinant wild-type or kinase-inactive (D683A/D700A) KSR1 proteins in Escherichia coli to test the hypothesis that KSR1 is a functional protein kinase. Recombinant wild-type KSR1, but not recombinant kinase-inactive KSR1, underwent autophosphorylation on serine residue(s), phosphorylated myelin basic protein (MBP) as a generic substrate, and phosphorylated recombinant kinase-inactive MAPK/ERK kinase-1 (MEK1). Furthermore, FLAG immunoprecipitates from KSR1 -/- colon epithelial cells stably expressing FLAG-tagged wild-type KSR1 (+KSR1), but not vector (+vector) or FLAG-tagged kinase-inactive KSR1 (+D683A/D700A), were able to phosphorylate kinase-inactive MEK1. Since TNF activates the ERK pathway in colon epithelial cells, we tested the biological effects of KSR1 in the survival response downstream of TNF. We found that +vector and +D683A/D700A cells underwent apoptosis when treated with TNF, whereas +KSR1 cells were resistant. However, +KSR1 cells were sensitized to TNF-induced cell loss in the absence of MEK kinase activity. These data provide clear evidence that KSR1 is a functional protein kinase, MEK1 is an in vitro substrate of KSR1, and the catalytic activities of both proteins are required for eliciting cell survival responses downstream of TNF.

Monocyte chemoattractant protein 1 (MCP-1) in temporal arteritis and polymyalgia rheumatica

DEFF Research Database (Denmark)

Ellingsen, T; Elling, P; Olson, A

2000-01-01

was localised to the vessel wall in patients with TA. In TA, PMR, and healthy controls MCP-1 was quantified by enzyme linked immunosorbent assay (ELISA) in plasma. RESULTS: MCP-1 was localised to the majority of mononuclear cells, some smooth muscle cells, and giant cells in the arterial biopsy specimens from...

Isolation and functional characterization of CE1 binding proteins

Directory of Open Access Journals (Sweden)

Yu Ji-hyun

2010-12-01

Full Text Available Abstract Background Abscisic acid (ABA is a plant hormone that controls seed germination, protective responses to various abiotic stresses and seed maturation. The ABA-dependent processes entail changes in gene expression. Numerous genes are regulated by ABA, and promoter analyses of the genes revealed that cis-elements sharing the ACGTGGC consensus sequence are ubiquitous among ABA-regulated gene promoters. The importance of the core sequence, which is generally known as ABA response element (ABRE, has been demonstrated by various experiments, and its cognate transcription factors known as ABFs/AREBs have been identified. Although necessary, ABRE alone is not sufficient, and another cis-element known as "coupling element (CE" is required for full range ABA-regulation of gene expression. Several CEs are known. However, despite their importance, the cognate transcription factors mediating ABA response via CEs have not been reported to date. Here, we report the isolation of transcription factors that bind one of the coupling elements, CE1. Results To isolate CE1 binding proteins, we carried out yeast one-hybrid screens. Reporter genes containing a trimer of the CE1 element were prepared and introduced into a yeast strain. The yeast was transformed with library DNA that represents RNA isolated from ABA-treated Arabidopsis seedlings. From the screen of 3.6 million yeast transformants, we isolated 78 positive clones. Analysis of the clones revealed that a group of AP2/ERF domain proteins binds the CE1 element. We investigated their expression patterns and analyzed their overexpression lines to investigate the in vivo functions of the CE element binding factors (CEBFs. Here, we show that one of the CEBFs, AtERF13, confers ABA hypersensitivity in Arabidopsis, whereas two other CEBFs enhance sugar sensitivity. Conclusions Our results indicate that a group of AP2/ERF superfamily proteins interacts with CE1. Several CEBFs are known to mediate defense or

Isolation and functional characterization of CE1 binding proteins.

Science.gov (United States)

Lee, Sun-ji; Park, Ji Hye; Lee, Mi Hun; Yu, Ji-hyun; Kim, Soo Young

2010-12-16

Abscisic acid (ABA) is a plant hormone that controls seed germination, protective responses to various abiotic stresses and seed maturation. The ABA-dependent processes entail changes in gene expression. Numerous genes are regulated by ABA, and promoter analyses of the genes revealed that cis-elements sharing the ACGTGGC consensus sequence are ubiquitous among ABA-regulated gene promoters. The importance of the core sequence, which is generally known as ABA response element (ABRE), has been demonstrated by various experiments, and its cognate transcription factors known as ABFs/AREBs have been identified. Although necessary, ABRE alone is not sufficient, and another cis-element known as "coupling element (CE)" is required for full range ABA-regulation of gene expression. Several CEs are known. However, despite their importance, the cognate transcription factors mediating ABA response via CEs have not been reported to date. Here, we report the isolation of transcription factors that bind one of the coupling elements, CE1. To isolate CE1 binding proteins, we carried out yeast one-hybrid screens. Reporter genes containing a trimer of the CE1 element were prepared and introduced into a yeast strain. The yeast was transformed with library DNA that represents RNA isolated from ABA-treated Arabidopsis seedlings. From the screen of 3.6 million yeast transformants, we isolated 78 positive clones. Analysis of the clones revealed that a group of AP2/ERF domain proteins binds the CE1 element. We investigated their expression patterns and analyzed their overexpression lines to investigate the in vivo functions of the CE element binding factors (CEBFs). Here, we show that one of the CEBFs, AtERF13, confers ABA hypersensitivity in Arabidopsis, whereas two other CEBFs enhance sugar sensitivity. Our results indicate that a group of AP2/ERF superfamily proteins interacts with CE1. Several CEBFs are known to mediate defense or abiotic stress response, but the physiological functions

Protein-protein interaction network-based detection of functionally similar proteins within species.

Science.gov (United States)

Song, Baoxing; Wang, Fen; Guo, Yang; Sang, Qing; Liu, Min; Li, Dengyun; Fang, Wei; Zhang, Deli

2012-07-01

Although functionally similar proteins across species have been widely studied, functionally similar proteins within species showing low sequence similarity have not been examined in detail. Identification of these proteins is of significant importance for understanding biological functions, evolution of protein families, progression of co-evolution, and convergent evolution and others which cannot be obtained by detection of functionally similar proteins across species. Here, we explored a method of detecting functionally similar proteins within species based on graph theory. After denoting protein-protein interaction networks using graphs, we split the graphs into subgraphs using the 1-hop method. Proteins with functional similarities in a species were detected using a method of modified shortest path to compare these subgraphs and to find the eligible optimal results. Using seven protein-protein interaction networks and this method, some functionally similar proteins with low sequence similarity that cannot detected by sequence alignment were identified. By analyzing the results, we found that, sometimes, it is difficult to separate homologous from convergent evolution. Evaluation of the performance of our method by gene ontology term overlap showed that the precision of our method was excellent. Copyright © 2012 Wiley Periodicals, Inc.

Ex vivo expanded human regulatory T cells delay islet allograft rejection via inhibiting islet-derived monocyte chemoattractant protein-1 production in CD34+ stem cells-reconstituted NOD-scid IL2rγnull mice.

Science.gov (United States)

Xiao, Fang; Ma, Liang; Zhao, Min; Huang, Guocai; Mirenda, Vincenzo; Dorling, Anthony; Lechler, Robert; Lombardi, Giovanna

2014-01-01

Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by immune-mediated destruction of insulin-secreting β cells of the pancreas. Near complete dependence on exogenous insulin makes T1DM very difficult to control, with the result that patients are exposed to high blood glucose and risk of diabetic complications and/or intermittent low blood glucose that can cause unconsciousness, fits and even death. Allograft transplantation of pancreatic islets restores normoglycemia with a low risk of surgical complications. However, although successful immediately after transplantation, islets are progressively lost, with most of the patients requiring exogenous insulin within 2 years post-transplant. Therefore, there is an urgent requirement for the development of new strategies to prevent islet rejection. In this study, we explored the importance of human regulatory T cells in the control of islets allograft rejection. We developed a pre-clinical model of human islet transplantation by reconstituting NOD-scid IL2rγnull mice with cord blood-derived human CD34+ stem cells and demonstrated that although the engrafted human immune system mediated the rejection of human islets, their survival was significantly prolonged following adoptive transfer of ex vivo expanded human Tregs. Mechanistically, Tregs inhibited the infiltration of innate immune cells and CD4+ T cells into the graft by down-regulating the islet graft-derived monocyte chemoattractant protein-1. Our findings might contribute to the development of clinical strategies for Treg therapy to control human islet rejection. We also show for the first time that CD34+ cells-reconstituted NOD-scid IL2rγnull mouse model could be beneficial for investigating human innate immunity in vivo.

Ex Vivo Expanded Human Regulatory T Cells Delay Islet Allograft Rejection via Inhibiting Islet-Derived Monocyte Chemoattractant Protein-1 Production in CD34+ Stem Cells-Reconstituted NOD-scid IL2rγnull Mice

Science.gov (United States)

Xiao, Fang; Ma, Liang; Zhao, Min; Huang, Guocai; Mirenda, Vincenzo; Dorling, Anthony

2014-01-01

Type 1 diabetes mellitus (T1DM) is an autoimmune disease caused by immune-mediated destruction of insulin-secreting β cells of the pancreas. Near complete dependence on exogenous insulin makes T1DM very difficult to control, with the result that patients are exposed to high blood glucose and risk of diabetic complications and/or intermittent low blood glucose that can cause unconsciousness, fits and even death. Allograft transplantation of pancreatic islets restores normoglycemia with a low risk of surgical complications. However, although successful immediately after transplantation, islets are progressively lost, with most of the patients requiring exogenous insulin within 2 years post-transplant. Therefore, there is an urgent requirement for the development of new strategies to prevent islet rejection. In this study, we explored the importance of human regulatory T cells in the control of islets allograft rejection. We developed a pre-clinical model of human islet transplantation by reconstituting NOD-scid IL2rγnull mice with cord blood-derived human CD34+ stem cells and demonstrated that although the engrafted human immune system mediated the rejection of human islets, their survival was significantly prolonged following adoptive transfer of ex vivo expanded human Tregs. Mechanistically, Tregs inhibited the infiltration of innate immune cells and CD4+ T cells into the graft by down-regulating the islet graft-derived monocyte chemoattractant protein-1. Our findings might contribute to the development of clinical strategies for Treg therapy to control human islet rejection. We also show for the first time that CD34+ cells-reconstituted NOD-scid IL2rγnull mouse model could be beneficial for investigating human innate immunity in vivo. PMID:24594640

Ex vivo expanded human regulatory T cells delay islet allograft rejection via inhibiting islet-derived monocyte chemoattractant protein-1 production in CD34+ stem cells-reconstituted NOD-scid IL2rγnull mice.

Directory of Open Access Journals (Sweden)

Fang Xiao

Full Text Available Type 1 diabetes mellitus (T1DM is an autoimmune disease caused by immune-mediated destruction of insulin-secreting β cells of the pancreas. Near complete dependence on exogenous insulin makes T1DM very difficult to control, with the result that patients are exposed to high blood glucose and risk of diabetic complications and/or intermittent low blood glucose that can cause unconsciousness, fits and even death. Allograft transplantation of pancreatic islets restores normoglycemia with a low risk of surgical complications. However, although successful immediately after transplantation, islets are progressively lost, with most of the patients requiring exogenous insulin within 2 years post-transplant. Therefore, there is an urgent requirement for the development of new strategies to prevent islet rejection. In this study, we explored the importance of human regulatory T cells in the control of islets allograft rejection. We developed a pre-clinical model of human islet transplantation by reconstituting NOD-scid IL2rγnull mice with cord blood-derived human CD34+ stem cells and demonstrated that although the engrafted human immune system mediated the rejection of human islets, their survival was significantly prolonged following adoptive transfer of ex vivo expanded human Tregs. Mechanistically, Tregs inhibited the infiltration of innate immune cells and CD4+ T cells into the graft by down-regulating the islet graft-derived monocyte chemoattractant protein-1. Our findings might contribute to the development of clinical strategies for Treg therapy to control human islet rejection. We also show for the first time that CD34+ cells-reconstituted NOD-scid IL2rγnull mouse model could be beneficial for investigating human innate immunity in vivo.

The chaperone like function of the nonhistone protein HMGB1

International Nuclear Information System (INIS)

Osmanov, Taner; Ugrinova, Iva; Pasheva, Evdokia

2013-01-01

-synthetic acetylation for the chaperone function of HMGB1 protein. The presence of an acetyl groups at Lys 2 decreases strongly the stimulating effect of the protein in the stepwise salt dialysis experiment and the same tendency persisted in the dialysis free experiment

Unexpected Functional Divergence of Bat Influenza Virus NS1 Proteins.

Science.gov (United States)

Turkington, Hannah L; Juozapaitis, Mindaugas; Tsolakos, Nikos; Corrales-Aguilar, Eugenia; Schwemmle, Martin; Hale, Benjamin G

2018-03-01

Recently, two influenza A virus (FLUAV) genomes were identified in Central and South American bats. These sequences exhibit notable divergence from classical FLUAV counterparts, and functionally, bat FLUAV glycoproteins lack canonical receptor binding and destroying activity. Nevertheless, other features that distinguish these viruses from classical FLUAVs have yet to be explored. Here, we studied the viral nonstructural protein NS1, a virulence factor that modulates host signaling to promote efficient propagation. Like all FLUAV NS1 proteins, bat FLUAV NS1s bind double-stranded RNA and act as interferon antagonists. Unexpectedly, we found that bat FLUAV NS1s are unique in being unable to bind host p85β, a regulatory subunit of the cellular metabolism-regulating enzyme, phosphoinositide 3-kinase (PI3K). Furthermore, neither bat FLUAV NS1 alone nor infection with a chimeric bat FLUAV efficiently activates Akt, a PI3K effector. Structure-guided mutagenesis revealed that the bat FLUAV NS1-p85β interaction can be reengineered (in a strain-specific manner) by changing two to four NS1 residues (96L, 99M, 100I, and 145T), thereby creating a hydrophobic patch. Notably, ameliorated p85β-binding is insufficient for bat FLUAV NS1 to activate PI3K, and a chimeric bat FLUAV expressing NS1 with engineered hydrophobic patch mutations exhibits cell-type-dependent, but species-independent, propagation phenotypes. We hypothesize that bat FLUAV hijacking of PI3K in the natural bat host has been selected against, perhaps because genes in this metabolic pathway were differentially shaped by evolution to suit the unique energy use strategies of this flying mammal. These data expand our understanding of the enigmatic functional divergence between bat FLUAVs and classical mammalian and avian FLUAVs. IMPORTANCE The potential for novel influenza A viruses to establish infections in humans from animals is a source of continuous concern due to possible severe outbreaks or pandemics. The

Breast carcinoma cells modulate the chemoattractive activity of human bone marrow-derived mesenchymal stromal cells by interfering with CXCL12.

Science.gov (United States)

Wobus, Manja; List, Catrin; Dittrich, Tobias; Dhawan, Abhishek; Duryagina, Regina; Arabanian, Laleh S; Kast, Karin; Wimberger, Pauline; Stiehler, Maik; Hofbauer, Lorenz C; Jakob, Franz; Ehninger, Gerhard; Anastassiadis, Konstantinos; Bornhäuser, Martin

2015-01-01

We investigated whether breast tumor cells can modulate the function of mesenchymal stromal cells (MSCs) with a special emphasis on their chemoattractive activity towards hematopoietic stem and progenitor cells (HSPCs). Primary MSCs as well as a MSC line (SCP-1) were cocultured with primary breast cancer cells, MCF-7, MDA-MB231 breast carcinoma or MCF-10A non-malignant breast epithelial cells or their conditioned medium. In addition, the frequency of circulating clonogenic hematopoietic progenitors was determined in 78 patients with breast cancer and compared with healthy controls. Gene expression analysis of SCP-1 cells cultured with MCF-7 medium revealed CXCL12 (SDF-1) as one of the most significantly downregulated genes. Supernatant from both MCF-7 and MDA-MB231 reduced the CXCL12 promoter activity in SCP-1 cells to 77% and 47%, respectively. Moreover, the CXCL12 mRNA and protein levels were significantly reduced. As functional consequence of lower CXCL12 levels, we detected a decreased trans-well migration of HSPCs towards MSC/tumor cell cocultures or conditioned medium. The specificity of this effect was confirmed by blocking studies with the CXCR4 antagonist AMD3100. Downregulation of SP1 and increased miR-23a levels in MSCs after contact with tumor cell medium as well as enhanced TGFβ1 expression were identified as potential molecular regulators of CXCL12 activity in MSCs. Moreover, we observed a significantly higher frequency of circulating colony-forming hematopoietic progenitors in patients with breast cancer compared with healthy controls. Our in vitro results propose a potential new mechanism by which disseminated tumor cells in the bone marrow may interfere with hematopoiesis by modulating CXCL12 in protected niches. © 2014 UICC.

Dopamine attenuates the chemoattractant effect of interleukin-8: a novel role in the systemic inflammatory response syndrome.

LENUS (Irish Health Repository)

Sookhai, S

2012-02-03

Activated neutrophil (PMN) adherence to vascular endothelium comprises a key step for both transendothelial migration and initiation of potentially deleterious release of PMN products. The biogenic amine, dopamine (DA), has been used for several decades in patients to maintain hemodynamic stability. The effect of dopamine on PMN transendothelial migration and adhesion receptor expression and on the endothelial molecules, E-selectin and ICAM-1, was evaluated. PMN were isolated from healthy controls, stimulated with lipopolysaccharide (LPS), and tumor necrosis factor-alpha (TNF-alpha) and treated with dopamine. CD 11b and CD 18 PMN adhesion receptor expression were assessed flow cytometrically. In a separate experiment, the chemoattractant peptide, IL-8, was placed in the lower chamber of transwells, and PMN migration was assessed. Human umbilical vein endothelial cells (HUVEC) were stimulated with LPS\\/TNF-alpha and incubated with dopamine. ICAM-1 and E-selectin endothelial molecule expression were assessed flow cytometrically. There was a significant increase in transendothelial migration in stimulated PMN compared with normal PMN (40 vs. 14%, P < 0.001). In addition, PMN CD11b\\/CD18 was significantly upregulated in stimulated PMN compared with normal PMN (252.4\\/352.4 vs. 76.7\\/139.4, P < 0.001) as were endothelial E-selectin\\/ICAM-1 expression compared with normal EC (8.1\\/9 vs. 3.9\\/3.8, P < 0.05). After treatment with dopamine, PMN transmigration was significantly decreased compared with stimulated PMN (8% vs. 40%, P < 0.001). Furthermore, dopamine also attenuated PMN CD11b\\/CD18 and the endothelial molecules E-selectin and ICAM-1 compared with stimulated PMN\\/EC that were not treated dopamine (174\\/240 vs. 252\\/352, P < 0.05 and 4\\/4.4 vs. 8.1\\/9, P < 0.05. respectively). The chemoattractant effect of IL-8 was also attenuated. These results identify for the first time that dopamine attenuates the initial interaction between PMN and the endothelium

Protein kinase D1 (PKD1) influences androgen receptor (AR) function in prostate cancer cells

International Nuclear Information System (INIS)

Mak, Paul; Jaggi, Meena; Syed, Viqar; Chauhan, Subhash C.; Hassan, Sazzad; Biswas, Helal; Balaji, K.C.

2008-01-01

Protein kinase D1 (PKD1), founding member of PKD protein family, is down-regulated in advanced prostate cancer (PCa). We demonstrate that PKD1 and androgen receptor (AR) are present as a protein complex in PCa cells. PKD1 is associated with a transcriptional complex which contains AR and promoter sequence of the Prostate Specific Antigen (PSA) gene. Ectopic expression of wild type PKD1 and the kinase dead mutant PKD1 (K628W) attenuated the ligand-dependent transcriptional activation of AR in prostate cancer cells and yeast cells indicating that PKD1 can affect AR transcription activity, whereas knocking down PKD1 enhanced the ligand-dependent transcriptional activation of AR. Co-expression of kinase dead mutant with AR significantly inhibited androgen-mediated cell proliferation in both LNCaP and DU145 PC cells. Our data demonstrate for the first time that PKD1 can influence AR function in PCa cells

Functional anthology of intrinsic disorder. 1. Biological processes and functions of proteins with long disordered regions.

Science.gov (United States)

Xie, Hongbo; Vucetic, Slobodan; Iakoucheva, Lilia M; Oldfield, Christopher J; Dunker, A Keith; Uversky, Vladimir N; Obradovic, Zoran

2007-05-01

Identifying relationships between function, amino acid sequence, and protein structure represents a major challenge. In this study, we propose a bioinformatics approach that identifies functional keywords in the Swiss-Prot database that correlate with intrinsic disorder. A statistical evaluation is employed to rank the significance of these correlations. Protein sequence data redundancy and the relationship between protein length and protein structure were taken into consideration to ensure the quality of the statistical inferences. Over 200,000 proteins from the Swiss-Prot database were analyzed using this approach. The predictions of intrinsic disorder were carried out using PONDR VL3E predictor of long disordered regions that achieves an accuracy of above 86%. Overall, out of the 710 Swiss-Prot functional keywords that were each associated with at least 20 proteins, 238 were found to be strongly positively correlated with predicted long intrinsically disordered regions, whereas 302 were strongly negatively correlated with such regions. The remaining 170 keywords were ambiguous without strong positive or negative correlation with the disorder predictions. These functions cover a large variety of biological activities and imply that disordered regions are characterized by a wide functional repertoire. Our results agree well with literature findings, as we were able to find at least one illustrative example of functional disorder or order shown experimentally for the vast majority of keywords showing the strongest positive or negative correlation with intrinsic disorder. This work opens a series of three papers, which enriches the current view of protein structure-function relationships, especially with regards to functionalities of intrinsically disordered proteins, and provides researchers with a novel tool that could be used to improve the understanding of the relationships between protein structure and function. The first paper of the series describes our

Strong morphological defects in conditional Arabidopsis abp1 knock-down mutants generated in absence of functional ABP1 protein.

Science.gov (United States)

Michalko, Jaroslav; Glanc, Matouš; Perrot-Rechenmann, Catherine; Friml, Jiří

2016-01-01

The Auxin Binding Protein 1 (ABP1) is one of the most studied proteins in plants. Since decades ago, it has been the prime receptor candidate for the plant hormone auxin with a plethora of described functions in auxin signaling and development. The developmental importance of ABP1 has recently been questioned by identification of Arabidopsis thaliana abp1 knock-out alleles that show no obvious phenotypes under normal growth conditions. In this study, we examined the contradiction between the normal growth and development of the abp1 knock-outs and the strong morphological defects observed in three different ethanol-inducible abp1 knock-down mutants ( abp1-AS, SS12K, SS12S). By analyzing segregating populations of abp1 knock-out vs. abp1 knock-down crosses we show that the strong morphological defects that were believed to be the result of conditional down-regulation of ABP1 can be reproduced also in the absence of the functional ABP1 protein. This data suggests that the phenotypes in  abp1 knock-down lines are due to the off-target effects and asks for further reflections on the biological function of ABP1 or alternative explanations for the missing phenotypic defects in the abp1 loss-of-function alleles.

Renoprotective Effects of Low-protein Diet with the Use of Keto-analogues of Essential Amino Acids Using

Directory of Open Access Journals (Sweden)

O.I. Romadanova

2013-10-01

Full Text Available The article discusses the role of low-protein diet and keto-analogues of essential amino acids in patients with impaired renal function. On the basis of results of the study on regularities of monocyte chemoattractant protein level (MCP-1, depending on the origin of glomerular damage and stage of chronic kidney disease and the dynamics of its changes under the influence of different approaches in complex treatment, it is concluded that administration of low-protein diet with inclusion of Ketosteril is necessary already at the early stages of the diseases for renoprotective action. Use of Ketosteril should be prolonged and continuous in order to increase the predialysis period and improve the quality of life in patients with chronic kidney disease.

Association of papillomavirus E6 proteins with either MAML1 or E6AP clusters E6 proteins by structure, function, and evolutionary relatedness.

Directory of Open Access Journals (Sweden)

Nicole Brimer

2017-12-01

Full Text Available Papillomavirus E6 proteins bind to LXXLL peptide motifs displayed on targeted cellular proteins. Alpha genus HPV E6 proteins associate with the cellular ubiquitin ligase E6AP (UBE3A, by binding to an LXXLL peptide (ELTLQELLGEE displayed by E6AP, thereby stimulating E6AP ubiquitin ligase activity. Beta, Gamma, and Delta genera E6 proteins bind a similar LXXLL peptide (WMSDLDDLLGS on the cellular transcriptional co-activator MAML1 and thereby repress Notch signaling. We expressed 45 different animal and human E6 proteins from diverse papillomavirus genera to ascertain the overall preference of E6 proteins for E6AP or MAML1. E6 proteins from all HPV genera except Alpha preferentially interacted with MAML1 over E6AP. Among animal papillomaviruses, E6 proteins from certain ungulate (SsPV1 from pigs and cetacean (porpoises and dolphins hosts functionally resembled Alpha genus HPV by binding and targeting the degradation of E6AP. Beta genus HPV E6 proteins functionally clustered with Delta, Pi, Tau, Gamma, Chi, Mu, Lambda, Iota, Dyokappa, Rho, and Dyolambda E6 proteins to bind and repress MAML1. None of the tested E6 proteins physically and functionally interacted with both MAML1 and E6AP, indicating an evolutionary split. Further, interaction of an E6 protein was insufficient to activate degradation of E6AP, indicating that E6 proteins that target E6AP co-evolved to separately acquire both binding and triggering of ubiquitin ligase activation. E6 proteins with similar biological function clustered together in phylogenetic trees and shared structural features. This suggests that the divergence of E6 proteins from either MAML1 or E6AP binding preference is a major event in papillomavirus evolution.

Systemic MCP1/CCR2 blockade and leukocyte specific MCP1/CCR2 inhibition affect aortic aneurysm formation differently

NARCIS (Netherlands)

de Waard, Vivian; Bot, Ilze; de Jager, Saskia C. A.; Talib, Sara; Egashira, Kensuke; de Vries, Margreet R.; Quax, Paul H. A.; Biessen, Erik A. L.; van Berkel, Theo J. C.

2010-01-01

Objective: CCR2, the receptor for monocyte chemoattractant protein 1 (MCP1), is involved in atherosclerosis and abdominal aortic aneurysms (AAAs). Here, we explored the potential beneficial blockade of the MCP1/CCR2 pathway. Methods: We applied an AAA model in aging apolipoprotein E deficient mice

Cathepsin D Specifically Cleaves the Chemokines Macrophage Inflammatory Protein-1α, Macrophage Inflammatory Protein-1β, and SLC That Are Expressed in Human Breast Cancer

Science.gov (United States)

Wolf, Marlene; Clark-Lewis, Ian; Buri, Caroline; Langen, Hanno; Lis, Maddalena; Mazzucchelli, Luca

2003-01-01

Cathepsin D (Cath-D) expression in human primary breast cancer has been associated with a poor prognosis. In search of a better understanding of the Cath-D substrates possibly involved in cancer invasiveness and metastasis, we investigated the potential interactions between this protease and chemokines. Here we report that purified Cath-D, as well as culture supernatants from the human breast carcinoma cell lines MCF-7 and T47D, selectively degrade macrophage inflammatory protein (MIP)-1α (CCL3), MIP-1β (CCL4), and SLC (CCL21). Proteolysis was totally blocked by the protease inhibitor pepstatin A, and specificity of Cath-D cleavage was demonstrated using a large chemokine panel. Whereas MIP-1α and MIP-1β degradation was rapid and complete, cleavage of SLC was slow and not complete. Mass spectrometry analysis showed that Cath-D cleaves the Leu58 to Trp59 bond of SLC producing two functionally inactive fragments. Analysis of Cath-D proteolysis of a series of monocyte chemoattractant protein-3/MIP-1β hybrids indicated that processing of MIP-1β might start by cleaving off amino acids located in the C-terminal domain. In situ hybridization studies revealed MIP-1α, MIP-1β, and Cath-D gene expression mainly in the stromal compartment of breast cancers whereas SLC transcripts were found in endothelial cells of capillaries and venules within the neoplastic tissues. Cath-D production in the breast carcinoma cell lines MCF-7 and T47D, as assessed by enzyme-linked immunosorbent assay of culture supernatants and cell lysates, was not affected by stimulation with chemokines such as interleukin-8 (CXCL8), SDF-1 (CXCL12), and SLC. These data suggest that inactivation of chemokines by Cath-D possibly influences regulatory mechanisms in the tumoral extracellular microenvironment that in turn may affect the generation of the antitumoral immune response, the migration of cancer cells, or both processes. PMID:12651610

Structural and functional characterization of the CAP domain of pathogen-related yeast 1 (Pry1) protein

Science.gov (United States)

Darwiche, Rabih; Kelleher, Alan; Hudspeth, Elissa M.; Schneiter, Roger; Asojo, Oluwatoyin A.

2016-06-01

The production, crystal structure, and functional characterization of the C-terminal cysteine-rich secretory protein/antigen 5/pathogenesis related-1 (CAP) domain of pathogen-related yeast protein-1 (Pry1) from Saccharomyces cerevisiae is presented. The CAP domain of Pry1 (Pry1CAP) is functional in vivo as its expression restores cholesterol export to yeast mutants lacking endogenous Pry1 and Pry2. Recombinant Pry1CAP forms dimers in solution, is sufficient for in vitro cholesterol binding, and has comparable binding properties as full-length Pry1. Two crystal structures of Pry1CAP are reported, one with Mg2+ coordinated to the conserved CAP tetrad (His208, Glu215, Glu233 and His250) in spacegroup I41 and the other without divalent cations in spacegroup P6122. The latter structure contains four 1,4-dioxane molecules from the crystallization solution, one of which sits in the cholesterol binding site. Both structures reveal that the divalent cation and cholesterol binding sites are connected upon dimerization, providing a structural basis for the observed Mg2+-dependent sterol binding by Pry1.

Specific interaction with cardiolipin triggers functional activation of Dynamin-Related Protein 1.

Directory of Open Access Journals (Sweden)

Itsasne Bustillo-Zabalbeitia

Full Text Available Dynamin-Related Protein 1 (Drp1, a large GTPase of the dynamin superfamily, is required for mitochondrial fission in healthy and apoptotic cells. Drp1 activation is a complex process that involves translocation from the cytosol to the mitochondrial outer membrane (MOM and assembly into rings/spirals at the MOM, leading to membrane constriction/division. Similar to dynamins, Drp1 contains GTPase (G, bundle signaling element (BSE and stalk domains. However, instead of the lipid-interacting Pleckstrin Homology (PH domain present in the dynamins, Drp1 contains the so-called B insert or variable domain that has been suggested to play an important role in Drp1 regulation. Different proteins have been implicated in Drp1 recruitment to the MOM, although how MOM-localized Drp1 acquires its fully functional status remains poorly understood. We found that Drp1 can interact with pure lipid bilayers enriched in the mitochondrion-specific phospholipid cardiolipin (CL. Building on our previous study, we now explore the specificity and functional consequences of this interaction. We show that a four lysine module located within the B insert of Drp1 interacts preferentially with CL over other anionic lipids. This interaction dramatically enhances Drp1 oligomerization and assembly-stimulated GTP hydrolysis. Our results add significantly to a growing body of evidence indicating that CL is an important regulator of many essential mitochondrial functions.

Evidence for functional diversity between the voltage-gated proton channel Hv1 and its closest related protein HVRP1.

Directory of Open Access Journals (Sweden)

Iris H Kim

Full Text Available The Hv1 channel and voltage-sensitive phosphatases share with voltage-gated sodium, potassium, and calcium channels the ability to detect changes in membrane potential through voltage-sensing domains (VSDs. However, they lack the pore domain typical of these other channels. NaV, KV, and CaV proteins can be found in neurons and muscles, where they play important roles in electrical excitability. In contrast, VSD-containing proteins lacking a pore domain are found in non-excitable cells and are not involved in neuronal signaling. Here, we report the identification of HVRP1, a protein related to the Hv1 channel (from which the name Hv1 Related Protein 1 is derived, which we find to be expressed primarily in the central nervous system, and particularly in the cerebellum. Within the cerebellar tissue, HVRP1 is specifically expressed in granule neurons, as determined by in situ hybridization and immunohistochemistry. Analysis of subcellular distribution via electron microscopy and immunogold labeling reveals that the protein localizes on the post-synaptic side of contacts between glutamatergic mossy fibers and the granule cells. We also find that, despite the similarities in amino acid sequence and structural organization between Hv1 and HVRP1, the two proteins have distinct functional properties. The high conservation of HVRP1 in vertebrates and its cellular and subcellular localizations suggest an important function in the nervous system.

Application of encoded library technology (ELT) to a protein-protein interaction target: discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists.

Science.gov (United States)

Kollmann, Christopher S; Bai, Xiaopeng; Tsai, Ching-Hsuan; Yang, Hongfang; Lind, Kenneth E; Skinner, Steven R; Zhu, Zhengrong; Israel, David I; Cuozzo, John W; Morgan, Barry A; Yuki, Koichi; Xie, Can; Springer, Timothy A; Shimaoka, Motomu; Evindar, Ghotas

2014-04-01

The inhibition of protein-protein interactions remains a challenge for traditional small molecule drug discovery. Here we describe the use of DNA-encoded library technology for the discovery of small molecules that are potent inhibitors of the interaction between lymphocyte function-associated antigen 1 and its ligand intercellular adhesion molecule 1. A DNA-encoded library with a potential complexity of 4.1 billion compounds was exposed to the I-domain of the target protein and the bound ligands were affinity selected, yielding an enriched small-molecule hit family. Compounds representing this family were synthesized without their DNA encoding moiety and found to inhibit the lymphocyte function-associated antigen 1/intercellular adhesion molecule-1 interaction with submicromolar potency in both ELISA and cell adhesion assays. Re-synthesized compounds conjugated to DNA or a fluorophore were demonstrated to bind to cells expressing the target protein. Copyright © 2014 Elsevier Ltd. All rights reserved.

Structure and function of the TIR domain from the grape NLR protein RPV1

Directory of Open Access Journals (Sweden)

Simon John Williams

2016-12-01

Full Text Available The N-terminal Toll/interleukin-1 receptor/resistance protein (TIR domain has been shown to be both necessary and sufficient for defence signalling in the model plants flax and Arabidopsis. In examples from these organisms, TIR domain self-association is required for signalling function, albeit through distinct interfaces. Here, we investigate these properties in the TIR domain containing resistance protein RPV1 from the wild grapevine Muscadinia rotundifolia. The RPV1 TIR domain, without additional flanking sequence present, is autoactive when transiently expressed in tobacco, demonstrating that the TIR domain alone is capable of cell-death signalling. We determined the crystal structure of the RPV1 TIR domain at 2.3 Å resolution. In the crystals, the RPV1 TIR domain forms a dimer, mediated predominantly through residues in the αA and αE helices (AE interface. This interface is shared with the interface discovered in the dimeric complex of the TIR domains from the Arabidopsis RPS4/RRS1 resistance protein pair. We show that surface-exposed residues in the AE interface that mediate the dimer interaction in the crystals are highly conserved among plant TIR domain-containing proteins. While we were unable to demonstrate self-association of the RPV1 TIR domain in solution or using yeast 2-hybrid, mutations of surface-exposed residues in the AE interface prevent the cell-death autoactive phenotype. In addition, mutation of residues known to be important in the cell-death signalling function of the flax L6 TIR domain were also shown to be required for RPV1 TIR domain mediated cell-death. Our data demonstrate that multiple TIR domain surfaces control the cell-death function of the RPV1 TIR domain and we suggest that the conserved AE interface may have a general function in TIR-NLR signalling.

Downregulation of Protein 4.1R impairs centrosome function,bipolar spindle organization and anaphase

Energy Technology Data Exchange (ETDEWEB)

Spence, Jeffrey R.; Go, Minjoung M.; Bahmanyar, S.; Barth,A.I.M.; Krauss, Sharon Wald

2006-03-17

Centrosomes nucleate and organize interphase MTs and areinstrumental in the assembly of the mitotic bipolar spindle. Here wereport that two members of the multifunctional protein 4.1 family havedistinct distributions at centrosomes. Protein 4.1R localizes to maturecentrioles whereas 4.1G is a component of the pericentriolar matrixsurrounding centrioles. To selectively probe 4.1R function, we used RNAinterference-mediated depletion of 4.1R without decreasing 4.1Gexpression. 4.1R downregulation reduces MT anchoring and organization atinterphase and impairs centrosome separation during prometaphase.Metaphase chromosomes fail to properly condense/align and spindleorganization is aberrant. Notably 4.1R depletion causes mislocalizationof its binding partner NuMA (Nuclear Mitotic Apparatus Protein),essential for spindle pole focusing, and disrupts ninein. Duringanaphase/telophase, 4.1R-depleted cells have lagging chromosomes andaberrant MT bridges. Our data provide functional evidence that 4.1R makescrucial contributions to centrosome integrity and to mitotic spindlestructure enabling mitosis and anaphase to proceed with the coordinatedprecision required to avoid pathological events.

Expression, purification and functional characterization of human equilibrative nucleoside transporter subtype-1 (hENT1) protein from Sf9 insect cells.

Science.gov (United States)

Rehan, Shahid; Jaakola, Veli-Pekka

2015-10-01

Human equilibrative nucleoside transporter-1 (hENT1) is the major plasma membrane transporter involved in transportation of natural nucleosides as well as nucleoside analog drugs, used in anti-cancer and anti-viral therapies. Despite extensive biochemical and pharmacological studies, little is known about the structure-function relationship of this protein. The major obstacles to purification include a low endogenous expression level, the lack of an efficient expression and purification protocol, and the hydrophobic nature of the protein. Here, we report protein expression, purification and functional characterization of hENT1 from Sf9 insect cells. hENT1 expressed by Sf9 cells is functionally active as demonstrated by saturation binding with a Kd of 1.2±0.2nM and Bmax of 110±5pmol/mg for [(3)H]nitrobenzylmercaptopurine ribonucleoside ([(3)H]NBMPR). We also demonstrate purification of hENT1 using FLAG antibody affinity resin in lauryl maltose neopentyl glycol detergent with a Kd of 4.3±0.7nM. The yield of hENT1 from Sf9 cells was ∼0.5mg active transporter per liter of culture. The purified protein is functionally active, stable, homogenous and appropriate for further biophysical and structural studies. Copyright © 2015 Elsevier Inc. All rights reserved.

N-caffeoyltryptomine, a potent anti-inflammatory phenolic amide, suppressed MCP-1 expression in LPS-stimulated THP-1 cells and rats fed with a high fat diet

Science.gov (United States)

Monocyte chemoattractant protein-1 (MCP-1) is a well-known chemokine critically involved in the pathophysiological progression of cardiovascular diseases such as arthrosclerosis. N-caffeoyltryptamine is a phenolic amide with strong anti-inflammatory effects. Therefore, in this paper, the potential e...

Circulating monocyte chemoattractant protein‐1 (MCP‐1) is associated with cachexia in treatment‐naïve pancreatic cancer patients

Science.gov (United States)

Talbert, Erin E.; Lewis, Heather L.; Farren, Matthew R.; Ramsey, Mitchell L.; Chakedis, Jeffery M.; Rajasekera, Priyani; Haverick, Ericka; Sarna, Angela; Bloomston, Mark; Pawlik, Timothy M.; Zimmers, Teresa A.; Lesinski, Gregory B.; Hart, Phil A.; Dillhoff, Mary E.; Schmidt, Carl R.

2018-01-01

Abstract Background Cancer‐associated wasting, termed cancer cachexia, has a profound effect on the morbidity and mortality of cancer patients but remains difficult to recognize and diagnose. While increases in circulating levels of a number of inflammatory cytokines have been associated with cancer cachexia, these associations were generally made in patients with advanced disease and thus may be associated with disease progression rather than directly with the cachexia syndrome. Thus, we sought to assess potential biomarkers of cancer‐induced cachexia in patients with earlier stages of disease. Methods A custom multiplex array was used to measure circulating levels of 25 soluble factors from 70 pancreatic cancer patients undergoing attempted tumour resections. A high‐sensitivity multiplex was used for increased sensitivity for nine cytokines. Results Resectable pancreatic cancer patients with cachexia had low levels of canonical pro‐inflammatory cytokines including interleukin‐6 (IL‐6), interleukin‐1β (IL‐1β), interferon‐γ (IFN‐γ), and tumour necrosis factor (TNF). Even in our more sensitive analysis, these cytokines were not associated with cancer cachexia. Of the 25 circulating factors tested, only monocyte chemoattractant protein‐1 (MCP‐1) was increased in treatment‐naïve cachectic patients compared with weight stable patients and identified as a potential biomarker for cancer cachexia. Although circulating levels of leptin and granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) were found to be decreased in the same cohort of treatment‐naïve cachectic patients, these factors were closely associated with body mass index, limiting their utility as cancer cachexia biomarkers. Conclusions Unlike in advanced disease, it is possible that cachexia in patients with resectable pancreatic cancer is not associated with high levels of classical markers of systemic inflammation. However, cachectic, treatment‐naïve patients

Functional characterization of the ER stress induced X-box-binding protein-1 (Xbp-1 in the porcine system

Directory of Open Access Journals (Sweden)

Jin Dong-Il

2011-05-01

Full Text Available Abstract Background The unfolded protein response (UPR is an evolutionary conserved adaptive reaction for increasing cell survival under endoplasmic reticulum (ER stress conditions. X-box-binding protein-1 (Xbp1 is a key transcription factor of UPR that activates genes involved in protein folding, secretion, and degradation to restore ER function. The UPR induced by ER stress was extensively studied in diseases linked to protein misfolding and aggregations. However, in the porcine system, genes in the UPR pathway were not investigated. In this study, we isolated and characterized the porcine Xbp1 (pXbp1 gene in ER stress using porcine embryonic fibroblast (PEF cells and porcine organs. ER stress was induced by the treatment of tunicamycin and cell viability was investigated by the MTT assay. For cloning and analyzing the expression pattern of pXbp1, RT-PCR analysis and Western blot were used. Knock-down of pXbp1 was performed by the siRNA-mediated gene silencing. Results We found that the pXbp1 mRNA was the subject of the IRE1α-mediated unconventional splicing by ER stress. Knock-down of pXbp1 enhanced ER stress-mediated cell death in PEF cells. In adult organs, pXbp1 mRNA and protein were expressed and the spliced forms were detected. Conclusions It was first found that the UPR mechanisms and the function of pXbp1 in the porcine system. These results indicate that pXbp1 plays an important role during the ER stress response like other animal systems and open a new opportunity for examining the UPR pathway in the porcine model system.

Increasing cerebrospinal fluid chemokine concentrations despite undetectable cerebrospinal fluid HIV RNA in HIV-1-infected patients receiving antiretroviral therapy

NARCIS (Netherlands)

Gisolf, E. H.; van Praag, R. M.; Jurriaans, S.; Portegies, P.; Goudsmit, J.; Danner, S. A.; Lange, J. M.; Prins, J. M.

2000-01-01

Only limited data on cerebrospinal fluid (CSF) HIV-1 RNA responses and markers of local inflammation in CSF during antiretroviral therapy are available. HIV-RNA, soluble tumor necrosis factor (TNF)-receptor (sTNFr)-II, monocyte chemoattractant protein (MCP)-1, and interferon-gamma-inducible protein

Identification of a third protein 4.1 tumor suppressor, protein 4.1R, in meningioma pathogenesis

Energy Technology Data Exchange (ETDEWEB)

Robb, Victoria A.; Li, Wen; Gascard, Philippe; Perry, Arie; Mohandas, Narla; Gutmann, David H.

2003-06-11

Meningiomas are common tumors of the central nervous system, however, the mechanisms under lying their pathogenesis are largely undefined. Two members of the Protein 4.1 super family, the neuro fibromatosis 2 (NF2) gene product (merlin/schwannomin) and Protein 4.1B have been implicated as meningioma tumor suppressors. In this report, we demonstrate that another Protein 4.1 family member, Protein 4.1R, also functions as a meningioma tumor suppressor. Based on the assignment of the Protein 4.1R gene to chromosome 1p32-36, a common region of deletion observed in meningiomas, we analyzed Protein 4.1R expression in meningioma cell lines and surgical tumor specimens. We observed loss of Protein 4.1R protein expression in two meningioma cell lines (IOMM-Lee, CH157-MN) by Western blotting as well as in 6 of 15 sporadic meningioma as by immuno histo chemistry (IHC). Analysis of a subset of these sporadic meningiomas by fluorescent in situ hybridization (FISH) with a Protein 4.1R specific probe demonstrated 100 percent concordance with the IHC results. In support of a meningioma tumor suppressor function, over expression of Protein 4.1R resulted in suppression of IOMM-Lee and CH157MN cell proliferation. Similar to the Protein 4.1B and merlin meningioma tumor suppressors, Protein 4.1R localization in the membrane fraction increased significantly under conditions of growth arrest in vitro. Lastly, Protein 4.1R interacted with some known merlin/Protein 4.1B interactors such as CD44 and bII-spectrin, but did not associate with the Protein 4.1B interactors 14-3-3 and PRMT3 or the merlin binding proteins SCHIP-1 and HRS. Collectively, these results suggest that Protein 4.1R functions as an important tumor suppressor important in the molecular pathogenesis of meningioma.

An Amphipathic Helix Directs Cellular Membrane Curvature Sensing and Function of the BAR Domain Protein PICK1.

Science.gov (United States)

Herlo, Rasmus; Lund, Viktor K; Lycas, Matthew D; Jansen, Anna M; Khelashvili, George; Andersen, Rita C; Bhatia, Vikram; Pedersen, Thomas S; Albornoz, Pedro B C; Johner, Niklaus; Ammendrup-Johnsen, Ina; Christensen, Nikolaj R; Erlendsson, Simon; Stoklund, Mikkel; Larsen, Jannik B; Weinstein, Harel; Kjærulff, Ole; Stamou, Dimitrios; Gether, Ulrik; Madsen, Kenneth L

2018-05-15

BAR domains are dimeric protein modules that sense, induce, and stabilize lipid membrane curvature. Here, we show that membrane curvature sensing (MCS) directs cellular localization and function of the BAR domain protein PICK1. In PICK1, and the homologous proteins ICA69 and arfaptin2, we identify an amphipathic helix N-terminal to the BAR domain that mediates MCS. Mutational disruption of the helix in PICK1 impaired MCS without affecting membrane binding per se. In insulin-producing INS-1E cells, super-resolution microscopy revealed that disruption of the helix selectively compromised PICK1 density on insulin granules of high curvature during their maturation. This was accompanied by reduced hormone storage in the INS-1E cells. In Drosophila, disruption of the helix compromised growth regulation. By demonstrating size-dependent binding on insulin granules, our finding highlights the function of MCS for BAR domain proteins in a biological context distinct from their function, e.g., at the plasma membrane during endocytosis. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Mining the Human Complexome Database Identifies RBM14 as an XPO1-Associated Protein Involved in HIV-1 Rev Function

OpenAIRE

Budhiraja, Sona; Liu, Hongbing; Couturier, Jacob; Malovannaya, Anna; Qin, Jun; Lewis, Dorothy E.; Rice, Andrew P.

2015-01-01

By recruiting the host protein XPO1 (CRM1), the HIV-1 Rev protein mediates the nuclear export of incompletely spliced viral transcripts. We mined data from the recently described human nuclear complexome to identify a host protein, RBM14, which associates with XPO1 and Rev and is involved in Rev function. Using a Rev-dependent p24 reporter plasmid, we found that RBM14 depletion decreased Rev activity and Rev-mediated enhancement of the cytoplasmic levels of unspliced viral transcripts. RBM14 ...

Huntingtin-associated protein-1 (HAP1) regulates endocytosis and interacts with multiple trafficking-related proteins.

Science.gov (United States)

Mackenzie, Kimberly D; Lim, Yoon; Duffield, Michael D; Chataway, Timothy; Zhou, Xin-Fu; Keating, Damien J

2017-07-01

Huntingtin-associated protein 1 (HAP1) was initially identified as a binding partner of huntingtin, mutations in which underlie Huntington's disease. Subcellular localization and protein interaction data indicate that HAP1 may be important in vesicle trafficking, cell signalling and receptor internalization. In this study, a proteomics approach was used for the identification of novel HAP1-interacting partners to attempt to shed light on the physiological function of HAP1. Using affinity chromatography with HAP1-GST protein fragments bound to Sepharose columns, this study identified a number of trafficking-related proteins that bind to HAP1. Interestingly, many of the proteins that were identified by mass spectrometry have trafficking-related functions and include the clathrin light chain B and Sec23A, an ER to Golgi trafficking vesicle coat component. Using co-immunoprecipitation and GST-binding assays the association between HAP1 and clathrin light chain B has been validated in vitro. This study also finds that HAP1 co-localizes with clathrin light chain B. In line with a physiological function of the HAP1-clathrin interaction this study detected a dramatic reduction in vesicle retrieval and endocytosis in adrenal chromaffin cells. Furthermore, through examination of transferrin endocytosis in HAP1 -/- cortical neurons, this study has determined that HAP1 regulates neuronal endocytosis. In this study, the interaction between HAP1 and Sec23A was also validated through endogenous co-immunoprecipitation in rat brain homogenate. Through the identification of novel HAP1 binding partners, many of which have putative trafficking roles, this study provides us with new insights into the mechanisms underlying the important physiological function of HAP1 as an intracellular trafficking protein through its protein-protein interactions. Copyright © 2017 Elsevier Inc. All rights reserved.

Polycystin 1 loss of function is directly linked to an imbalance in G-protein signaling in the kidney.

Science.gov (United States)

Zhang, Bo; Tran, Uyen; Wessely, Oliver

2018-03-22

The development of the kidney relies on the establishment and maintenance of a precise tubular diameter of its functional units, the nephrons. This process is disrupted in polycystic kidney disease (PKD), resulting in dilations of the nephron and renal cyst formation. In the course of exploring G-protein-coupled signaling in the Xenopus pronephric kidney, we discovered that loss of the G-protein α subunit, Gnas, results in a PKD phenotype. Polycystin 1, one of the genes mutated in human PKD, encodes a protein resembling a G-protein-coupled receptor. Furthermore, deletion of the G-protein-binding domain present in the intracellular C terminus of polycystin 1 impacts functionality. A comprehensive analysis of all the G-protein α subunits expressed in the Xenopus pronephric kidney demonstrates that polycystin 1 recruits a select subset of G-protein α subunits and that their knockdown - as in the case of Gnas - results in a PKD phenotype. Mechanistically, the phenotype is caused by increased endogenous G-protein β/γ signaling and can be reversed by pharmacological inhibitors as well as knocking down Gnb1. Together, our data support the hypothesis that G proteins are recruited to the intracellular domain of PKD1 and that this interaction is crucial for its function in the kidney. © 2018. Published by The Company of Biologists Ltd.

Protein function prediction using neighbor relativity in protein-protein interaction network.

Science.gov (United States)

Moosavi, Sobhan; Rahgozar, Masoud; Rahimi, Amir

2013-04-01

There is a large gap between the number of discovered proteins and the number of functionally annotated ones. Due to the high cost of determining protein function by wet-lab research, function prediction has become a major task for computational biology and bioinformatics. Some researches utilize the proteins interaction information to predict function for un-annotated proteins. In this paper, we propose a novel approach called "Neighbor Relativity Coefficient" (NRC) based on interaction network topology which estimates the functional similarity between two proteins. NRC is calculated for each pair of proteins based on their graph-based features including distance, common neighbors and the number of paths between them. In order to ascribe function to an un-annotated protein, NRC estimates a weight for each neighbor to transfer its annotation to the unknown protein. Finally, the unknown protein will be annotated by the top score transferred functions. We also investigate the effect of using different coefficients for various types of functions. The proposed method has been evaluated on Saccharomyces cerevisiae and Homo sapiens interaction networks. The performance analysis demonstrates that NRC yields better results in comparison with previous protein function prediction approaches that utilize interaction network. Copyright © 2012 Elsevier Ltd. All rights reserved.

S1P receptor signalling and RGS proteins; expression and function in vascular smooth muscle cells and transfected CHO cells

NARCIS (Netherlands)

Hendriks-Balk, Mariëlle C.; van Loenen, Pieter B.; Hajji, Najat; Michel, Martin C.; Peters, Stephan L. M.; Alewijnse, Astrid E.

2009-01-01

Sphingosine-1-phosphate (S1P) signalling via G protein-coupled receptors is important for the regulation of cell function and differentiation. Specific Regulators of G protein Signalling (RGS) proteins modulate the function of these receptors in many cell types including vascular smooth muscle cells

Topology-function conservation in protein-protein interaction networks.

Science.gov (United States)

Davis, Darren; Yaveroğlu, Ömer Nebil; Malod-Dognin, Noël; Stojmirovic, Aleksandar; Pržulj, Nataša

2015-05-15

Proteins underlay the functioning of a cell and the wiring of proteins in protein-protein interaction network (PIN) relates to their biological functions. Proteins with similar wiring in the PIN (topology around them) have been shown to have similar functions. This property has been successfully exploited for predicting protein functions. Topological similarity is also used to guide network alignment algorithms that find similarly wired proteins between PINs of different species; these similarities are used to transfer annotation across PINs, e.g. from model organisms to human. To refine these functional predictions and annotation transfers, we need to gain insight into the variability of the topology-function relationships. For example, a function may be significantly associated with specific topologies, while another function may be weakly associated with several different topologies. Also, the topology-function relationships may differ between different species. To improve our understanding of topology-function relationships and of their conservation among species, we develop a statistical framework that is built upon canonical correlation analysis. Using the graphlet degrees to represent the wiring around proteins in PINs and gene ontology (GO) annotations to describe their functions, our framework: (i) characterizes statistically significant topology-function relationships in a given species, and (ii) uncovers the functions that have conserved topology in PINs of different species, which we term topologically orthologous functions. We apply our framework to PINs of yeast and human, identifying seven biological process and two cellular component GO terms to be topologically orthologous for the two organisms. © The Author 2015. Published by Oxford University Press.

Functionality of system components: Conservation of protein function in protein feature space

DEFF Research Database (Denmark)

Jensen, Lars Juhl; Ussery, David; Brunak, Søren

2003-01-01

well on organisms other than the one on which it was trained. We evaluate the performance of such a method, ProtFun, which relies on protein features as its sole input, and show that the method gives similar performance for most eukaryotes and performs much better than anticipated on archaea......Many protein features useful for prediction of protein function can be predicted from sequence, including posttranslational modifications, subcellular localization, and physical/chemical properties. We show here that such protein features are more conserved among orthologs than paralogs, indicating...... they are crucial for protein function and thus subject to selective pressure. This means that a function prediction method based on sequence-derived features may be able to discriminate between proteins with different function even when they have highly similar structure. Also, such a method is likely to perform...

Identification of the functional domains of the telomere protein Rap1 in Schizosaccharomyces pombe.

Directory of Open Access Journals (Sweden)

Ikumi Fujita

Full Text Available The telomere at the end of a linear chromosome plays crucial roles in genome stability. In the fission yeast Schizosaccharomyces pombe, the Rap1 protein, one of the central players at the telomeres, associates with multiple proteins to regulate various telomere functions, such as the maintenance of telomere DNA length, telomere end protection, maintenance of telomere heterochromatin, and telomere clustering in meiosis. The molecular bases of the interactions between Rap1 and its partners, however, remain largely unknown. Here, we describe the identification of the interaction domains of Rap1 with its partners. The Bqt1/Bqt2 complex, which is required for normal meiotic progression, Poz1, which is required for telomere length control, and Taz1, which is required for the recruitment of Rap1 to telomeres, bind to distinct domains in the C-terminal half of Rap1. Intriguingly, analyses of a series of deletion mutants for rap1(+ have revealed that the long N-terminal region (1-456 a.a. [amino acids] of Rap1 (full length: 693 a.a. is not required for telomere DNA length control, telomere end protection, and telomere gene silencing, whereas the C-terminal region (457-693 a.a. containing Poz1- and Taz1-binding domains plays important roles in those functions. Furthermore, the Bqt1/Bqt2- and Taz1-binding domains are essential for normal spore formation after meiosis. Our results suggest that the C-terminal half of Rap1 is critical for the primary telomere functions, whereas the N-terminal region containing the BRCT (BRCA1 C-terminus and Myb domains, which are evolutionally conserved among the Rap1 family proteins, does not play a major role at the telomeres.

The effect of metformin on monocyte secretory function in simvastatin-treated patients with impaired fasting glucose.

Science.gov (United States)

Krysiak, Robert; Okopien, Bogusław

2013-01-01

This study was designed to investigate whether metformin affects monocyte secretory function in patients with impaired fasting glucose receiving chronic statin therapy. The study included 48 patients with impaired fasting glucose treated for at least three months with simvastatin (40 mg daily). These patients were randomized to either metformin (3 g daily) or placebo, which was administered together with simvastatin for 90 days. Plasma lipids, glucose homeostasis markers, monocyte cytokine release and plasma C-reactive protein levels were determined before randomization and at the end of the treatment. Compared to placebo, metformin reduced monocyte release of tumor necrosis factor-α, interleukin-1β, interleukin-6, monocyte chemoattractant protein-1 and interleukin-8, as well as decreased plasma C-reactive protein levels, which were accompanied by an improvement in insulin sensitivity. The obtained results suggest that metformin may inhibit monocyte secretory function and reduce systemic inflammation in statin-treated patients with prediabetes. Impaired fasting glucose patients with high cardiovascular risk may receive the greatest benefits from concomitant treatment with a statin and metformin. Copyright © 2013 Elsevier Inc. All rights reserved.

Molecular interactions between MUC1 epithelial mucin, β-catenin, and CagA proteins

Directory of Open Access Journals (Sweden)

Wei eGuang

2012-05-01

Full Text Available Interleukin (IL-8-driven neutrophil infiltration of the gastric mucosa is pathognomonic of persistent Helicobacter pylori infection. Our prior study showed that ectopic over-expression of MUC1 in human AGS gastric epithelial cells reduced H. pylori-stimulated IL-8 production compared with cells expressing MUC1 endogenously. Conversely, Muc1 knockout (Muc1-/- mice displayed an increased level of transcripts encoding the keratinocyte chemoattractant (KC, the murine equivalent of human IL-8, in gastric mucosa compared with Muc1(+/+ mice during experimental H. pylori infection. The current study tested the hypothesis that a decreased IL-8 level observed following MUC1 over-expression is mediated through the ability of MUC1 to associate with β-catenin, thereby inhibiting H. pylori-induced β-catenin nuclear translocation. Increased neutrophil infiltration of the gastric mucosa of H. pylori-infected Muc1(-/- mice was observed compared with Muc1(+/+ wild type littermates, thus defining the functional consequences of increased KC expression in the Muc1-null animals. Protein co-immunoprecipitation (coIP studies using lysates of untreated or H. pylori-treated AGS cells demonstrated that (a MUC1 formed a coIP complex with β-catenin and CagA, (b MUC1 over-expression reduced CagA/β-catenin coIP, and (c in the absence of MUC1 over-expression, H. pylori infection increased the nuclear level of β-catenin, (d whereas MUC1 over-expression decreased bacteria-driven β-catenin nuclear localization. These results suggest that manipulation of MUC1 expression in gastric epithelia may be an effective therapeutic strategy to inhibit H. pylori-dependent IL-8 production, neutrophil infiltration, and stomach inflammation.

A highly conserved Poc1 protein characterized in embryos of the hydrozoan Clytia hemisphaerica: localization and functional studies.

Directory of Open Access Journals (Sweden)

Cécile Fourrage

Full Text Available Poc1 (Protein of Centriole 1 proteins are highly conserved WD40 domain-containing centriole components, well characterized in the alga Chlamydomonas, the ciliated protazoan Tetrahymena, the insect Drosophila and in vertebrate cells including Xenopus and zebrafish embryos. Functions and localizations related to the centriole and ciliary axoneme have been demonstrated for Poc1 in a range of species. The vertebrate Poc1 protein has also been reported to show an additional association with mitochondria, including enrichment in the specialized "germ plasm" region of Xenopus oocytes. We have identified and characterized a highly conserved Poc1 protein in the cnidarian Clytia hemisphaerica. Clytia Poc1 mRNA was found to be strongly expressed in eggs and early embryos, showing a punctate perinuclear localization in young oocytes. Fluorescence-tagged Poc1 proteins expressed in developing embryos showed strong localization to centrioles, including basal bodies. Anti-human Poc1 antibodies decorated mitochondria in Clytia, as reported in human cells, but failed to recognise endogenous or fluorescent-tagged Clytia Poc1. Injection of specific morpholino oligonucleotides into Clytia eggs prior to fertilization to repress Poc1 mRNA translation interfered with cell division from the blastula stage, likely corresponding to when neosynthesis normally takes over from maternally supplied protein. Cell cycle lengthening and arrest were observed, phenotypes consistent with an impaired centriolar biogenesis or function. The specificity of the defects could be demonstrated by injection of synthetic Poc1 mRNA, which restored normal development. We conclude that in Clytia embryos, Poc1 has an essentially centriolar localization and function.

Incorporating functional inter-relationships into protein function prediction algorithms

Directory of Open Access Journals (Sweden)

Kumar Vipin

2009-05-01

predictions made over a set of base classes. Finally, we provide qualitative and quantitative evidence that this incorporation of functional inter-relationships enables the discovery of interesting biology in the form of novel functional annotations for several yeast proteins, such as Sna4, Rtn1 and Lin1. Conclusion We implemented and evaluated a methodology for incorporating interrelationships between functional classes into a standard classification-based protein function prediction algorithm. Our results show that this incorporation can help improve the accuracy of such algorithms, and help uncover novel biology in the form of previously unknown functional annotations. The complete source code, a sample data set and the additional files for this paper are available free of charge for non-commercial use at http://www.cs.umn.edu/vk/gaurav/functionalsimilarity/.

Structure-function mapping of BbCRASP-1, the key complement factor H and FHL-1 binding protein of Borrelia burgdorferi.

Science.gov (United States)

Cordes, Frank S; Kraiczy, Peter; Roversi, Pietro; Simon, Markus M; Brade, Volker; Jahraus, Oliver; Wallis, Russell; Goodstadt, Leo; Ponting, Chris P; Skerka, Christine; Zipfel, Peter F; Wallich, Reinhard; Lea, Susan M

2006-05-01

Borrelia burgdorferi, a spirochaete transmitted to human hosts during feeding of infected Ixodes ticks, is the causative agent of Lyme disease, the most frequent vector-borne disease in Eurasia and North America. Sporadically Lyme disease develops into a chronic, multisystemic disorder. Serum-resistant B. burgdorferi strains bind complement factor H (FH) and FH-like protein 1 (FHL-1) on the spirochaete surface. This binding is dependent on the expression of proteins termed complement-regulator acquiring surface proteins (CRASPs). The atomic structure of BbCRASP-1, the key FHL-1/FH-binding protein of B. burgdorferi, has recently been determined. Our analysis indicates that its protein topology apparently evolved to provide a high affinity interaction site for FH/FHL-1 and leads to an atomic-level hypothesis for the functioning of BbCRASP-1. This work demonstrates that pathogens interact with complement regulators in ways that are distinct from the mechanisms used by the host and are thus obvious targets for drug design.

Recent insights into the biological functions of liver fatty acid binding protein 1

Science.gov (United States)

Wang, GuQi; Bonkovsky, Herbert L.; de Lemos, Andrew; Burczynski, Frank J.

2015-01-01

Over four decades have passed since liver fatty acid binding protein (FABP)1 was first isolated. There are few protein families for which most of the complete tertiary structures, binding properties, and tissue occurrences are described in such detail and yet new functions are being uncovered for this protein. FABP1 is known to be critical for fatty acid uptake and intracellular transport and also has an important role in regulating lipid metabolism and cellular signaling pathways. FABP1 is an important endogenous cytoprotectant, minimizing hepatocyte oxidative damage and interfering with ischemia-reperfusion and other hepatic injuries. The protein may be targeted for metabolic activation through the cross-talk among many transcriptional factors and their activating ligands. Deficiency or malfunction of FABP1 has been reported in several diseases. FABP1 also influences cell proliferation during liver regeneration and may be considered as a prognostic factor for hepatic surgery. FABP1 binds and modulates the action of many molecules such as fatty acids, heme, and other metalloporphyrins. The ability to bind heme is another cytoprotective property and one that deserves closer investigation. The role of FABP1 in substrate availability and in protection from oxidative stress suggests that FABP1 plays a pivotal role during intracellular bacterial/viral infections by reducing inflammation and the adverse effects of starvation (energy deficiency). PMID:26443794

The C1q family of proteins: insights into the emerging non-traditional functions

Directory of Open Access Journals (Sweden)

Berhane eGhebrehiwet

2012-04-01

Full Text Available Research conducted over the past 20 years have helped us unravel not only the hidden structural and functional subtleties of human C1q, but also has catapulted the molecule from a mere recognition unit of the classical pathway to a well-recognized molecular sensor of damage modified self or non-self antigens. Thus, C1q is involved in a rapidly expanding list of pathological disorders—including autoimmunity, trophoblast migration, preeclampsia and cancer. The results of two recent reports are provided to underscore the critical role C1q plays in health and disease. First is the observation by Singh and colleagues showing that pregnant C1q-/- mice recapitulate the key features of human preeclampsia that correlate with increased fetal death. Treatment of the C1q-/- mice with pravastatin restored trophoblast invasiveness, placental blood flow, and angiogenic balance and, thus, prevented the onset of preeclampsia. Second is the report by Hong et al., which showed that C1q can induce apoptosis of prostate cancer cells by activating the tumor suppressor molecule WW-domain containing oxydoreductase (WWOX or WOX1 and destabilizing cell adhesion. Downregulation of C1q on the other hand enhanced prostate hyperplasia and cancer formation due to failure of WOX1 activation. Recent evidence also shows that C1q belongs to a family of structurally and functionally related TNFα-like family of proteins that may have arisen from a common ancestral gene. Therefore C1q not only shares the diverse functions with the TNF family of proteins, but also explains why C1q has retained some of its ancestral cytokine-like activities. This review is intended to highlight some of the structural and functional aspects of C1q by underscoring the growing list of its non-traditional functions.

In vitro and in vivo dependency of chemokine generation on C5a and TNF-alpha

DEFF Research Database (Denmark)

Czermak, B J; Sarma, V; Bless, N M

1999-01-01

production in vitro and in vivo. Two rat CXC chemokines (macrophage inflammatory protein (MIP)-2 and cytokine-induced neutrophil chemoattractant (CINC)) as well as three rat CC chemokines (MIP-1alpha, MIP-1beta, and monocyte chemoattractant protein (MCP)-1) were investigated. Chemokine generation in vitro...

Dissociation of activated protein C functions by elimination of protein S cofactor enhancement.

LENUS (Irish Health Repository)

Harmon, Shona

2008-11-07

Activated protein C (APC) plays a critical anticoagulant role in vivo by inactivating procoagulant factor Va and factor VIIIa and thus down-regulating thrombin generation. In addition, APC bound to the endothelial cell protein C receptor can initiate protease-activated receptor-1 (PAR-1)-mediated cytoprotective signaling. Protein S constitutes a critical cofactor for the anticoagulant function of APC but is not known to be involved in regulating APC-mediated protective PAR-1 signaling. In this study we utilized a site-directed mutagenesis strategy to characterize a putative protein S binding region within the APC Gla domain. Three single amino acid substitutions within the APC Gla domain (D35T, D36A, and A39V) were found to mildly impair protein S-dependent anticoagulant activity (<2-fold) but retained entirely normal cytoprotective activity. However, a single amino acid substitution (L38D) ablated the ability of protein S to function as a cofactor for this APC variant. Consequently, in assays of protein S-dependent factor Va proteolysis using purified proteins or in the plasma milieu, APC-L38D variant exhibited minimal residual anticoagulant activity compared with wild type APC. Despite the location of Leu-38 in the Gla domain, APC-L38D interacted normally with endothelial cell protein C receptor and retained its ability to trigger PAR-1 mediated cytoprotective signaling in a manner indistinguishable from that of wild type APC. Consequently, elimination of protein S cofactor enhancement of APC anticoagulant function represents a novel and effective strategy by which to separate the anticoagulant and cytoprotective functions of APC for potential therapeutic gain.

A stable aspirin-triggered lipoxin A4 analog blocks phosphorylation of leukocyte-specific protein 1 in human neutrophils.

Science.gov (United States)

Ohira, Taisuke; Bannenberg, Gerard; Arita, Makoto; Takahashi, Minoru; Ge, Qingyuan; Van Dyke, Thomas E; Stahl, Gregory L; Serhan, Charles N; Badwey, John A

2004-08-01

Lipoxins and their aspirin-triggered 15-epimers are endogenous anti-inflammatory agents that block neutrophil chemotaxis in vitro and inhibit neutrophil influx in several models of acute inflammation. In this study, we examined the effects of 15-epi-16-(p-fluoro)-phenoxy-lipoxin A(4) methyl ester, an aspirin-triggered lipoxin A(4)-stable analog (ATLa), on the protein phosphorylation pattern of human neutrophils. Neutrophils stimulated with the chemoattractant fMLP were found to exhibit intense phosphorylation of a 55-kDa protein that was blocked by ATLa (10-50 nM). This 55-kDa protein was identified as leukocyte-specific protein 1, a downstream component of the p38-MAPK cascade in neutrophils, by mass spectrometry, Western blotting, and immunoprecipitation experiments. ATLa (50 nM) also reduced phosphorylation/activation of several components of the p38-MAPK pathway in these cells (MAPK kinase 3/MAPK kinase 6, p38-MAPK, MAPK-activated protein kinase-2). These results indicate that ATLa exerts its anti-inflammatory effects, at least in part, by blocking activation of the p38-MAPK cascade in neutrophils, which is known to promote chemotaxis and other proinflammatory responses by these cells.

The dengue vector Aedes aegypti contains a functional high mobility group box 1 (HMGB1 protein with a unique regulatory C-terminus.

Directory of Open Access Journals (Sweden)

Fabio Schneider Ribeiro

Full Text Available The mosquito Aedes aegypti can spread the dengue, chikungunya and yellow fever viruses. Thus, the search for key molecules involved in the mosquito survival represents today a promising vector control strategy. High Mobility Group Box (HMGB proteins are essential nuclear factors that maintain the high-order structure of chromatin, keeping eukaryotic cells viable. Outside the nucleus, secreted HMGB proteins could alert the innate immune system to foreign antigens and trigger the initiation of host defenses. In this work, we cloned and functionally characterized the HMGB1 protein from Aedes aegypti (AaHMGB1. The AaHMGB1 protein typically consists of two HMG-box DNA binding domains and an acidic C-terminus. Interestingly, AaHMGB1 contains a unique alanine/glutamine-rich (AQ-rich C-terminal region that seems to be exclusive of dipteran HMGB proteins. AaHMGB1 is localized to the cell nucleus, mainly associated with heterochromatin. Circular dichroism analyses of AaHMGB1 or the C-terminal truncated proteins revealed α-helical structures. We showed that AaHMGB1 can effectively bind and change the topology of DNA, and that the AQ-rich and the C-terminal acidic regions can modulate its ability to promote DNA supercoiling, as well as its preference to bind supercoiled DNA. AaHMGB1 is phosphorylated by PKA and PKC, but not by CK2. Importantly, phosphorylation of AaHMGB1 by PKA or PKC completely abolishes its DNA bending activity. Thus, our study shows that a functional HMGB1 protein occurs in Aedes aegypt and we provide the first description of a HMGB1 protein containing an AQ-rich regulatory C-terminus.

DMPD: Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. [Dynamic Macrophage Pathway CSML Database

Lifescience Database Archive (English)

Full Text Available 17667936 Structure, function and regulation of the Toll/IL-1 receptor adaptor prote... (.svg) (.html) (.csml) Show Structure, function and regulation of the Toll/IL-1 receptor adaptor proteins. ...PubmedID 17667936 Title Structure, function and regulation of the Toll/IL-1 recep

Protein disulfide isomerase-like protein 1-1 controls endosperm development through regulation of the amount and composition of seed proteins in rice.

Directory of Open Access Journals (Sweden)

Yeon Jeong Kim

Full Text Available Protein disulfide isomerase (PDI is a chaperone protein involved in oxidative protein folding by acting as a catalyst and assisting folding in the endoplasmic reticulum (ER. A genome database search showed that rice contains 19 PDI-like genes. However, their functions are not clearly identified. This paper shows possible functions of rice PDI-like protein 1-1 (PDIL1-1 during seed development. Seeds of the T-DNA insertion PDIL1-1 mutant, PDIL1-1Δ, identified by genomic DNA PCR and western blot analysis, display a chalky phenotype and a thick aleurone layer. Protein content per seed was significantly lower and free sugar content higher in PDIL1-1Δ mutant seeds than in the wild type. Proteomic analysis of PDIL1-1Δ mutant seeds showed that PDIL1-1 is post-translationally regulated, and its loss causes accumulation of many types of seed proteins including glucose/starch metabolism- and ROS (reactive oxygen species scavenging-related proteins. In addition, PDIL1-1 strongly interacts with the cysteine protease OsCP1. Our data indicate that the opaque phenotype of PDIL1-1Δ mutant seeds results from production of irregular starch granules and protein body through loss of regulatory activity for various proteins involved in the synthesis of seed components.

Phylogenetic and functional analysis of the bacteriophage P1 single-stranded DNA-binding protein

DEFF Research Database (Denmark)

Bendtsen, Jannick Dyrløv; Nilsson, A.S.; Lehnherr, H.

2002-01-01

and does not represent a recent acquirement of the phage. The P1 and E. coli SSB proteins are fully functionally interchangeable. SSB-P1 is nonessential for phage growth in an exponentially growing E. coli host, and it is sufficient to promote bacterial growth in the absence of the E. coli SSB protein....... Expression studies showed that the P1 ssb gene is transcribed only, in an rpoS-independent fashion, during stationary-phase growth in E. coli. Mixed infection experiments demonstrated that a wild-type phage has a selective advantage over an ssb-null mutant when exposed to a bacterial host in the stationary...

Functional aspects of protein flexibility

DEFF Research Database (Denmark)

Teilum, Kaare; Olsen, Johan G; Kragelund, Birthe B

2009-01-01

this into an intuitive perception of protein function is challenging. Flexibility is of overwhelming importance for protein function, and the changes in protein structure during interactions with binding partners can be dramatic. The present review addresses protein flexibility, focusing on protein-ligand interactions...

Loss of Niemann-Pick C1 or C2 protein results in similar biochemical changes suggesting that these proteins function in a common lysosomal pathway.

Directory of Open Access Journals (Sweden)

Sayali S Dixit

Full Text Available Niemann-Pick Type C (NPC disease is a lysosomal storage disorder characterized by accumulation of unesterified cholesterol and other lipids in the endolysosomal system. NPC disease results from a defect in either of two distinct cholesterol-binding proteins: a transmembrane protein, NPC1, and a small soluble protein, NPC2. NPC1 and NPC2 are thought to function closely in the export of lysosomal cholesterol with both proteins binding cholesterol in vitro but they may have unrelated lysosomal roles. To investigate this possibility, we compared biochemical consequences of the loss of either protein. Analyses of lysosome-enriched subcellular fractions from brain and liver revealed similar decreases in buoyant densities of lysosomes from NPC1 or NPC2 deficient mice compared to controls. The subcellular distribution of both proteins was similar and paralleled a lysosomal marker. In liver, absence of either NPC1 or NPC2 resulted in similar alterations in the carbohydrate processing of the lysosomal protease, tripeptidyl peptidase I. These results highlight biochemical alterations in the lysosomal system of the NPC-mutant mice that appear secondary to lipid storage. In addition, the similarity in biochemical phenotypes resulting from either NPC1 or NPC2 deficiency supports models in which the function of these two proteins within lysosomes are linked closely.

Protein regulation of induced pluripotent stem cells by transplanting in a Huntington's animal model.

Science.gov (United States)

Mu, S; Han, L; Zhou, G; Mo, C; Duan, J; He, Z; Wang, Z; Ren, L; Zhang, J

2016-10-01

The purpose of this study was to determine the functional recovery and protein regulation by transplanted induced pluripotent stem cells in a rat model of Huntington's disease (HD). In a quinolinic acid-induced rat model of striatal degeneration, induced pluripotent stem cells were transplanted into the ipsilateral lateral ventricle 10 days after the quinolinic acid injection. At 8 weeks after transplantation, fluorodeoxyglucose-PET/CT scan and balance-beam test were performed to evaluate the functional recovery of experimental rats. In addition, immunofluorescence and protein array analysis were used to investigate the regulation of stimulated protein expression in the striatum. At 8 weeks after induced pluripotent stem cell transplantation, motor function was improved in comparison with the quinolinic acid-treated rats. High fluorodeoxyglucose accumulation in the injured striatum was also observed by PET/CT scans. In addition, immunofluorescence analysis demonstrated that implanted cells migrated from the lateral ventricle into the lesioned striatum and differentiated into striatal projection neurons. Array analysis showed a significant upregulation of GFR (Glial cell line-derived neurotrophic factor receptor) alpha-1, Adiponectin/Acrp30, basic-fibroblast growth factors, MIP-1 (Macrophage-inflammatory protein) alpha and leptin, as well as downregulation of cytokine-induced neutrophil chemoattractant-3 in striatum after transplantatation of induced pluripotent stem cells in comparison with the quinolinic acid -treated rats. The findings in this work indicate that transplantation of induced pluripotent stem cells is a promising therapeutic candidate for HD. © 2016 British Neuropathological Society.

Function and structure of GFP-like proteins in the protein data bank.

Science.gov (United States)

Ong, Wayne J-H; Alvarez, Samuel; Leroux, Ivan E; Shahid, Ramza S; Samma, Alex A; Peshkepija, Paola; Morgan, Alicia L; Mulcahy, Shawn; Zimmer, Marc

2011-04-01

The RCSB protein databank contains 266 crystal structures of green fluorescent proteins (GFP) and GFP-like proteins. This is the first systematic analysis of all the GFP-like structures in the pdb. We have used the pdb to examine the function of fluorescent proteins (FP) in nature, aspects of excited state proton transfer (ESPT) in FPs, deformation from planarity of the chromophore and chromophore maturation. The conclusions reached in this review are that (1) The lid residues are highly conserved, particularly those on the "top" of the β-barrel. They are important to the function of GFP-like proteins, perhaps in protecting the chromophore or in β-barrel formation. (2) The primary/ancestral function of GFP-like proteins may well be to aid in light induced electron transfer. (3) The structural prerequisites for light activated proton pumps exist in many structures and it's possible that like bioluminescence, proton pumps are secondary functions of GFP-like proteins. (4) In most GFP-like proteins the protein matrix exerts a significant strain on planar chromophores forcing most GFP-like proteins to adopt non-planar chromophores. These chromophoric deviations from planarity play an important role in determining the fluorescence quantum yield. (5) The chemospatial characteristics of the chromophore cavity determine the isomerization state of the chromophore. The cavities of highlighter proteins that can undergo cis/trans isomerization have chemospatial properties that are common to both cis and trans GFP-like proteins.

Roles for text mining in protein function prediction.

Science.gov (United States)

Verspoor, Karin M

2014-01-01

The Human Genome Project has provided science with a hugely valuable resource: the blueprints for life; the specification of all of the genes that make up a human. While the genes have all been identified and deciphered, it is proteins that are the workhorses of the human body: they are essential to virtually all cell functions and are the primary mechanism through which biological function is carried out. Hence in order to fully understand what happens at a molecular level in biological organisms, and eventually to enable development of treatments for diseases where some aspect of a biological system goes awry, we must understand the functions of proteins. However, experimental characterization of protein function cannot scale to the vast amount of DNA sequence data now available. Computational protein function prediction has therefore emerged as a problem at the forefront of modern biology (Radivojac et al., Nat Methods 10(13):221-227, 2013).Within the varied approaches to computational protein function prediction that have been explored, there are several that make use of biomedical literature mining. These methods take advantage of information in the published literature to associate specific proteins with specific protein functions. In this chapter, we introduce two main strategies for doing this: association of function terms, represented as Gene Ontology terms (Ashburner et al., Nat Genet 25(1):25-29, 2000), to proteins based on information in published articles, and a paradigm called LEAP-FS (Literature-Enhanced Automated Prediction of Functional Sites) in which literature mining is used to validate the predictions of an orthogonal computational protein function prediction method.

Identification and functional analysis of novel phosphorylation sites in the RNA surveillance protein Upf1.

Science.gov (United States)

Lasalde, Clarivel; Rivera, Andrea V; León, Alfredo J; González-Feliciano, José A; Estrella, Luis A; Rodríguez-Cruz, Eva N; Correa, María E; Cajigas, Iván J; Bracho, Dina P; Vega, Irving E; Wilkinson, Miles F; González, Carlos I

2014-02-01

One third of inherited genetic diseases are caused by mRNAs harboring premature termination codons as a result of nonsense mutations. These aberrant mRNAs are degraded by the Nonsense-Mediated mRNA Decay (NMD) pathway. A central component of the NMD pathway is Upf1, an RNA-dependent ATPase and helicase. Upf1 is a known phosphorylated protein, but only portions of this large protein have been examined for phosphorylation sites and the functional relevance of its phosphorylation has not been elucidated in Saccharomyces cerevisiae. Using tandem mass spectrometry analyses, we report the identification of 11 putative phosphorylated sites in S. cerevisiae Upf1. Five of these phosphorylated residues are located within the ATPase and helicase domains and are conserved in higher eukaryotes, suggesting a biological significance for their phosphorylation. Indeed, functional analysis demonstrated that a small carboxy-terminal motif harboring at least three phosphorylated amino acids is important for three Upf1 functions: ATPase activity, NMD activity and the ability to promote translation termination efficiency. We provide evidence that two tyrosines within this phospho-motif (Y-738 and Y-742) act redundantly to promote ATP hydrolysis, NMD efficiency and translation termination fidelity.

Interaction of Berberine derivative with protein POT1 affect telomere function in cancer cells

Energy Technology Data Exchange (ETDEWEB)

Xiao, Nannan; Chen, Siqi; Ma, Yan; Qiu, Jun; Tan, Jia-Heng; Ou, Tian-Miao; Gu, Lian-Quan; Huang, Zhi-Shu [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou University City, Waihuan East Road 132, Guangzhou 510006 (China); Li, Ding, E-mail: liding@mail.sysu.edu.cn [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou University City, Waihuan East Road 132, Guangzhou 510006 (China)

2012-03-16

Highlights: Black-Right-Pointing-Pointer The protein POT1 plays an important role in telomere protection. Black-Right-Pointing-Pointer Functional POT1 was overexpressed in Escherichia coli for the first time, and purified. Black-Right-Pointing-Pointer Compound Sysu-00692 was found to be the first POT1-binding ligand. Black-Right-Pointing-Pointer Sysu-00692 could interfere with the binding activity of POT1 in vivo. Black-Right-Pointing-Pointer Sysu-00692 had inhibition on telomerase and cell proliferation. -- Abstract: The protein POT1 plays an important role in telomere protection, which is related with telomere elongation and cell immortality. The protein has been recognized as a promising drug target for cancer treatment. In the present study, we cloned, overexpressed in Escherichia coli for the first time, and purified recombinant human POT1. The protein was proved to be active through filter binding assay, FRET and CD experiments. In the initial screening for protein binding ligands using SPR, compound Sysu-00692 was found to bind well with the POT1, which was confirmed with EMSA. Its in vivo activity study showed that compound Sysu-00692 could interfere with the binding between human POT1 and the telomeric DNA through chromatin immunoprecipitation. Besides, the compound showed mild inhibition on telomerase and cell proliferation. As we know, compound Sysu-00692 is the first reported POT1-binding ligand, which could serve as a lead compound for further improvement. This work offered a potentially new approach for drug design for the treatment of cancers.

Role of AAA(+)-proteins in peroxisome biogenesis and function.

Science.gov (United States)

Grimm, Immanuel; Erdmann, Ralf; Girzalsky, Wolfgang

2016-05-01

Mutations in the PEX1 gene, which encodes a protein required for peroxisome biogenesis, are the most common cause of the Zellweger spectrum diseases. The recognition that Pex1p shares a conserved ATP-binding domain with p97 and NSF led to the discovery of the extended family of AAA+-type ATPases. So far, four AAA+-type ATPases are related to peroxisome function. Pex6p functions together with Pex1p in peroxisome biogenesis, ATAD1/Msp1p plays a role in membrane protein targeting and a member of the Lon-family of proteases is associated with peroxisomal quality control. This review summarizes the current knowledge on the AAA+-proteins involved in peroxisome biogenesis and function.

OFD1, as a Ciliary Protein, Exhibits Neuroprotective Function in Photoreceptor Degeneration Models.

Directory of Open Access Journals (Sweden)

Juan Wang

Full Text Available Ofd1 is a newly identified causative gene for Retinitis pigmentosa (RP, a photoreceptor degenerative disease. This study aimed to examine Ofd1 localization in retina and further to investigate its function in photoreceptor degeneration models. Ofd1 localization in rat retina was examined using immunofluorescence. N-methyl-N-nitrosourea (MNU-induced rats and Royal College of Surgeons (RCS rats were used as photoreceptor degeneration models. The expression pattern of Ofd1, other ciliary associated genes and Wnt signaling pathway genes were examined in rat models. Furthermore, pEGFP-Ofd1-CDS and pSUPER-Ofd1-shRNA were constructed to overexpress and knockdown the expression level in 661W and R28 cells. MNU was also used to induce cell death. Cilia formation was observed using immunocytochemistry (ICC. Reactive oxygen species (ROS were detected using the 2', 7'-Dichlorofluorescin diacetate (DCFH-DA assay. Apoptosis genes expression was examined using qRT-PCR, Western blotting and fluorescence-activated cell sorting (FACS. Ofd1 localized to outer segments of rat retina photoreceptors. Ofd1 and other ciliary proteins expression levels increased from the 1st and 4th postnatal weeks and decreased until the 6th week in the RCS rats, while their expression consistently decreased from the 1st and 7th day in the MNU rats. Moreover, Wnt signaling pathway proteins expression was significantly up-regulated in both rat models. Knockdown of Ofd1 expression resulted in a smaller population, shorter length of cell cilia, and lower cell viability. Ofd1 overexpression partially attenuated MNU toxic effects by reducing ROS levels and mitigating apoptosis. To the best of our knowledge, this is the first study demonstrating Ofd1 localization and its function in rat retina and in retinal degeneration rat models. Ofd1 plays a role in controlling photoreceptor cilium length and number. Importantly, it demonstrates a neuroprotective function by protecting the photoreceptor

Functional analysis of a novel KRAB/C2H2 zinc finger protein Mipu1

International Nuclear Information System (INIS)

Jiang, Lei; Tang, Daolin; Wang, Kangkai; Zhang, Huali; Yuan, Can; Duan, Dayue; Xiao, Xianzhong

2007-01-01

A novel rat gene, Mipu1, encodes a 608 amino acid protein with an amino-terminal KRAB domain and 14 carboxyl-terminal C 2 H 2 zinc finger motifs. Mipu1 is localized to the nucleus through its KRAB domain or the linker adjacent to its zinc finger region. Using the GST-Mipu1 bound to glutathione-Sepharose beads, a consensus putative DNA binding site (5'-TGTCTTATCGAA-3') was extracted from a random oligonucleotide library. EMSA and target detection assay showed that the probe containing the putative site can bind to purified GST-Mipu1 fusion protein. The oligonucleotide containing the putative site was inserted into the pGL3-promotor vector to produce a reporter construct. The expression of reporter gene was repressed by overexpression of Mipu1 in a dose-dependent manner. Mutation analysis of the consensus sequence indicated that the repression mediated by Mipu1 is sequence-dependent. These results suggest that Mipu1 is a nuclear protein, which functions as a transcriptional repressor

Architectures and Functional Coverage of Protein-Protein Interfaces

Science.gov (United States)

Tuncbag, Nurcan; Gursoy, Attila; Guney, Emre; Nussinov, Ruth; Keskin, Ozlem

2008-01-01

The diverse range of cellular functions is performed by a limited number of protein folds existing in nature. One may similarly expect that cellular functional diversity would be covered by a limited number of protein-protein interface architectures. Here, we present 8205 interface clusters, each representing unique interface architecture. This dataset of protein-protein interfaces is analyzed and compared with older datasets. We observe that the number of both biological and crystal interfaces increase significantly compared to the number of PDB entries. Further, we find that the number of distinct interface architectures grows at a much faster rate than the number of folds and is yet to level off. We further analyze the growth trend of the functional coverage by constructing functional interaction networks from interfaces. The functional coverage is also found to steadily increase. Interestingly, we also observe that despite the diversity of interface architectures, some are more favorable and frequently used, and of particular interest, those are the ones which are also preferred in single chains. PMID:18620705

Protein 4.1, a component of the erythrocyte membrane skeleton and its related homologue proteins forming the protein 4.1/FERM superfamily.

Directory of Open Access Journals (Sweden)

Aleksander F Sikorski

2007-01-01

Full Text Available The review is focused on the domain structure and function of protein 4.1, one of the proteins belonging to the membrane skeleton. The protein 4.1 of the red blood cells (4.1R is a multifunctional protein that localizes to the membrane skeleton and stabilizes erythrocyte shape and membrane mechanical properties, such as deformability and stability, via lateral interactions with spectrin, actin, glycophorin C and protein p55. Protein 4.1 binding is modulated through the action of kinases and/or calmodulin-Ca2+. Non-erythroid cells express the 4.1R homologues: 4.1G (general type, 4.1B (brain type, and 4.1N (neuron type, and the whole group belongs to the protein 4.1 superfamily, which is characterized by the presence of a highly conserved FERM domain at the N-terminus of the molecule. Proteins 4.1R, 4.1G, 4.1N and 4.1B are encoded by different genes. Most of the 4.1 superfamily proteins also contain an actin-binding domain. To date, more than 40 members have been identified. They can be divided into five groups: protein 4.1 molecules, ERM proteins, talin-related molecules, protein tyrosine phosphatase (PTPH proteins and NBL4 proteins. We have focused our attention on the main, well known representatives of 4.1 superfamily and tried to choose the proteins which are close to 4.1R or which have distinct functions. 4.1 family proteins are not just linkers between the plasma membrane and membrane skeleton; they also play an important role in various processes. Some, such as focal adhesion kinase (FAK, non-receptor tyrosine kinase that localizes to focal adhesions in adherent cells, play the role in cell adhesion. The other members control or take part in tumor suppression, regulation of cell cycle progression, inhibition of cell proliferation, downstream signaling of the glutamate receptors, and establishment of cell polarity; some are also involved in cell proliferation, cell motility, and/or cell-to-cell communication.

Targeting protein-protein interaction between MLL1 and reciprocal proteins for leukemia therapy.

Science.gov (United States)

Wang, Zhi-Hui; Li, Dong-Dong; Chen, Wei-Lin; You, Qi-Dong; Guo, Xiao-Ke

2018-01-15

The mixed lineage leukemia protein-1 (MLL1), as a lysine methyltransferase, predominantly regulates the methylation of histone H3 lysine 4 (H3K4) and functions in hematopoietic stem cell (HSC) self-renewal. MLL1 gene fuses with partner genes that results in the generation of MLL1 fusion proteins (MLL1-FPs), which are frequently detected in acute leukemia. In the progress of leukemogenesis, a great deal of proteins cooperate with MLL1 to form multiprotein complexes serving for the dysregulation of H3K4 methylation, the overexpression of homeobox (HOX) cluster genes, and the consequent generation of leukemia. Hence, disrupting the interactions between MLL1 and the reciprocal proteins has been considered to be a new treatment strategy for leukemia. Here, we reviewed potential protein-protein interactions (PPIs) between MLL1 and its reciprocal proteins, and summarized the inhibitors to target MLL1 PPIs. The druggability of MLL1 PPIs for leukemia were also discussed. Copyright © 2017. Published by Elsevier Ltd.

Functional Evolution of Influenza Virus NS1 Protein in Currently Circulating Human 2009 Pandemic H1N1 Viruses.

Science.gov (United States)

Clark, Amelia M; Nogales, Aitor; Martinez-Sobrido, Luis; Topham, David J; DeDiego, Marta L

2017-09-01

In 2009, a novel H1N1 influenza virus emerged in humans, causing a global pandemic. It was previously shown that the NS1 protein from this human 2009 pandemic H1N1 (pH1N1) virus was an effective interferon (IFN) antagonist but could not inhibit general host gene expression, unlike other NS1 proteins from seasonal human H1N1 and H3N2 viruses. Here we show that the NS1 protein from currently circulating pH1N1 viruses has evolved to encode 6 amino acid changes (E55K, L90I, I123V, E125D, K131E, and N205S) with respect to the original protein. Notably, these 6 residue changes restore the ability of pH1N1 NS1 to inhibit general host gene expression, mainly by their ability to restore binding to the cellular factor CPSF30. This is the first report describing the ability of the pH1N1 NS1 protein to naturally acquire mutations that restore this function. Importantly, a recombinant pH1N1 virus containing these 6 amino acid changes in the NS1 protein (pH1N1/NSs-6mut) inhibited host IFN and proinflammatory responses to a greater extent than that with the parental virus (pH1N1/NS1-wt), yet virus titers were not significantly increased in cell cultures or in mouse lungs, and the disease was partially attenuated. The pH1N1/NSs-6mut virus grew similarly to pH1N1/NSs-wt in mouse lungs, but infection with pH1N1/NSs-6mut induced lower levels of proinflammatory cytokines, likely due to a general inhibition of gene expression mediated by the mutated NS1 protein. This lower level of inflammation induced by the pH1N1/NSs-6mut virus likely accounts for the attenuated disease phenotype and may represent a host-virus adaptation affecting influenza virus pathogenesis. IMPORTANCE Seasonal influenza A viruses (IAVs) are among the most common causes of respiratory infections in humans. In addition, occasional pandemics are caused when IAVs circulating in other species emerge in the human population. In 2009, a swine-origin H1N1 IAV (pH1N1) was transmitted to humans, infecting people then and up

Functions of intrinsic disorder in transmembrane proteins

DEFF Research Database (Denmark)

Kjaergaard, Magnus; Kragelund, Birthe B.

2017-01-01

Intrinsic disorder is common in integral membrane proteins, particularly in the intracellular domains. Despite this observation, these domains are not always recognized as being disordered. In this review, we will discuss the biological functions of intrinsically disordered regions of membrane...... receptors. The functions of the disordered regions are many and varied. We will discuss selected examples including: (1) Organization of receptors, kinases, phosphatases and second messenger sources into signaling complexes. (2) Modulation of the membrane-embedded domain function by ball-and-chain like...... mechanisms. (3) Trafficking of membrane proteins. (4) Transient membrane associations. (5) Post-translational modifications most notably phosphorylation and (6) disorder-linked isoform dependent function. We finish the review by discussing the future challenges facing the membrane protein community regarding...

Correlation of serum MCP-1 and VE-cadherin levels with neural function and carotid atherosclerosis in patients with acute cerebral infarction

Directory of Open Access Journals (Sweden)

Yan-Bing Xi

2017-05-01

Full Text Available Objective: To study the correlation of serum monocyte chemoattractant protein-1 (MCP-1 and vascular endothelial cadherin (VE-cadherin levels with neural function and carotid atherosclerosis in patients with acute cerebral infarction. Methods: A total of 78 patients who were diagnosed with acute cerebral infarction in our hospital between May 2013 and August 2016 were selected as pathological group, and 80 healthy volunteers who received physical examination in our hospital during the same period were selected as control group. Serum was collected to determine the levels of MCP-1, VE-cadherin, nerve injury molecules, inflammatory mediators, proteases and their hydrolysate. Results: Serum MCP-1, VE-cadherin, NGB, NSE, S100β, HMGB-1, sCD40L, YKL-40, visfatin, CatK, MMP9 and ICTP levels of pathological group were significantly higher than those of control group; serum MCP-1 and VE-cadherin levels of pathological group were positively correlated with NGB, NSE, S100β, HMGB-1, sCD40L, YKL-40, visfatin, CatK, MMP9 and ICTP levels. Conclusion: Serum MCP-1 and VE-cadherin levels abnormally increase in patients with acute cerebral infarction, and are closely related to the nerve injury and atherosclerosis process.

Novel functions for the endocytic regulatory proteins MICAL-L1 and EHD1 in mitosis.

Science.gov (United States)

Reinecke, James B; Katafiasz, Dawn; Naslavsky, Naava; Caplan, Steve

2015-01-01

During interphase, recycling endosomes mediate the transport of internalized cargo back to the plasma membrane. However, in mitotic cells, recycling endosomes are essential for the completion of cytokinesis, the last phase of mitosis that promotes the physical separation the two daughter cells. Despite recent advances, our understanding of the molecular determinants that regulate recycling endosome dynamics during cytokinesis remains incomplete. We have previously demonstrated that Molecule Interacting with CasL Like-1 (MICAL-L1) and C-terminal Eps15 Homology Domain protein 1 (EHD1) coordinately regulate receptor transport from tubular recycling endosomes during interphase. However, their potential roles in controlling cytokinesis had not been addressed. In this study, we show that MICAL-L1 and EHD1 regulate mitosis. Depletion of either protein resulted in increased numbers of bi-nucleated cells. We provide evidence that bi-nucleation in MICAL-L1- and EHD1-depleted cells is a consequence of impaired recycling endosome transport during late cytokinesis. However, depletion of MICAL-L1, but not EHD1, resulted in aberrant chromosome alignment and lagging chromosomes, suggesting an EHD1-independent function for MICAL-L1 earlier in mitosis. Moreover, we provide evidence that MICAL-L1 and EHD1 differentially influence microtubule dynamics during early and late mitosis. Collectively, our new data suggest several unanticipated roles for MICAL-L1 and EHD1 during the cell cycle. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Molecular Evolution and Functional Diversification of Replication Protein A1 in Plants

Science.gov (United States)

Aklilu, Behailu B.; Culligan, Kevin M.

2016-01-01

Replication protein A (RPA) is a heterotrimeric, single-stranded DNA binding complex required for eukaryotic DNA replication, repair, and recombination. RPA is composed of three subunits, RPA1, RPA2, and RPA3. In contrast to single RPA subunit genes generally found in animals and yeast, plants encode multiple paralogs of RPA subunits, suggesting subfunctionalization. Genetic analysis demonstrates that five Arabidopsis thaliana RPA1 paralogs (RPA1A to RPA1E) have unique and overlapping functions in DNA replication, repair, and meiosis. We hypothesize here that RPA1 subfunctionalities will be reflected in major structural and sequence differences among the paralogs. To address this, we analyzed amino acid and nucleotide sequences of RPA1 paralogs from 25 complete genomes representing a wide spectrum of plants and unicellular green algae. We find here that the plant RPA1 gene family is divided into three general groups termed RPA1A, RPA1B, and RPA1C, which likely arose from two progenitor groups in unicellular green algae. In the family Brassicaceae the RPA1B and RPA1C groups have further expanded to include two unique sub-functional paralogs RPA1D and RPA1E, respectively. In addition, RPA1 groups have unique domains, motifs, cis-elements, gene expression profiles, and pattern of conservation that are consistent with proposed functions in monocot and dicot species, including a novel C-terminal zinc-finger domain found only in plant RPA1C-like sequences. These results allow for improved prediction of RPA1 subunit functions in newly sequenced plant genomes, and potentially provide a unique molecular tool to improve classification of Brassicaceae species. PMID:26858742

Arabidopsis Yak1 protein (AtYak1) is a dual specificity protein kinase

KAUST Repository

Kim, Dongjin; Ntui, Valentine Otang; Zhang, Nianshu; Xiong, Liming

2015-01-01

Yak1 is a member of dual-specificity Tyr phosphorylation-regulated kinases (DYRKs) that are evolutionarily conserved. The downstream targets of Yak1 and their functions are largely unknown. Here, a homologous protein AtYAK1 was identified in Arabidopsis thaliana and the phosphoprotein profiles of the wild type and an atyak1 mutant were compared on two-dimensional gel following Pro-Q Diamond phosphoprotein gel staining. Annexin1, Annexin2 and RBD were phosphorylated at serine/ threonine residues by the AtYak1 kinase. Annexin1, Annexin2 and Annexin4 were also phosphorylated at tyrosine residues. Our study demonstrated that AtYak1 is a dual specificity protein kinase in Arabidopsis that may regulate the phosphorylation status of the annexin family proteins.

Arabidopsis Yak1 protein (AtYak1) is a dual specificity protein kinase

KAUST Repository

Kim, Dongjin

2015-10-09

Yak1 is a member of dual-specificity Tyr phosphorylation-regulated kinases (DYRKs) that are evolutionarily conserved. The downstream targets of Yak1 and their functions are largely unknown. Here, a homologous protein AtYAK1 was identified in Arabidopsis thaliana and the phosphoprotein profiles of the wild type and an atyak1 mutant were compared on two-dimensional gel following Pro-Q Diamond phosphoprotein gel staining. Annexin1, Annexin2 and RBD were phosphorylated at serine/ threonine residues by the AtYak1 kinase. Annexin1, Annexin2 and Annexin4 were also phosphorylated at tyrosine residues. Our study demonstrated that AtYak1 is a dual specificity protein kinase in Arabidopsis that may regulate the phosphorylation status of the annexin family proteins.

Clueless, a protein required for mitochondrial function, interacts with the PINK1-Parkin complex in Drosophila

Directory of Open Access Journals (Sweden)

Aditya Sen

2015-06-01

Full Text Available Loss of mitochondrial function often leads to neurodegeneration and is thought to be one of the underlying causes of neurodegenerative diseases such as Parkinson's disease (PD. However, the precise events linking mitochondrial dysfunction to neuronal death remain elusive. PTEN-induced putative kinase 1 (PINK1 and Parkin (Park, either of which, when mutated, are responsible for early-onset PD, mark individual mitochondria for destruction at the mitochondrial outer membrane. The specific molecular pathways that regulate signaling between the nucleus and mitochondria to sense mitochondrial dysfunction under normal physiological conditions are not well understood. Here, we show that Drosophila Clueless (Clu, a highly conserved protein required for normal mitochondrial function, can associate with Translocase of the outer membrane (TOM 20, Porin and PINK1, and is thus located at the mitochondrial outer membrane. Previously, we found that clu genetically interacts with park in Drosophila female germ cells. Here, we show that clu also genetically interacts with PINK1, and our epistasis analysis places clu downstream of PINK1 and upstream of park. In addition, Clu forms a complex with PINK1 and Park, further supporting that Clu links mitochondrial function with the PINK1-Park pathway. Lack of Clu causes PINK1 and Park to interact with each other, and clu mutants have decreased mitochondrial protein levels, suggesting that Clu can act as a negative regulator of the PINK1-Park pathway. Taken together, these results suggest that Clu directly modulates mitochondrial function, and that Clu's function contributes to the PINK1-Park pathway of mitochondrial quality control.

Functional rescue of mutant ABCA1 proteins by sodium 4-phenylbutyrate.

Science.gov (United States)

Sorrenson, Brie; Suetani, Rachel J; Williams, Michael J A; Bickley, Vivienne M; George, Peter M; Jones, Gregory T; McCormick, Sally P A

2013-01-01

Mutations in the ATP-binding cassette transporter A1 (ABCA1) are a major cause of decreased HDL cholesterol (HDL-C), which infers an increased risk of cardiovascular disease (CVD). Many ABCA1 mutants show impaired localization to the plasma membrane. The aim of this study was to investigate whether the chemical chaperone, sodium 4-phenylbutyrate (4-PBA) could improve cellular localization and function of ABCA1 mutants. Nine different ABCA1 mutants (p.A594T, p.I659V, p.R1068H, p.T1512M, p.Y1767D, p.N1800H, p.R2004K, p.A2028V, p.Q2239N) expressed in HEK293 cells, displaying different degrees of mislocalization to the plasma membrane and discrete impacts on cholesterol efflux, were subject to treatment with 4-PBA. Treatment restored localization to the plasma membrane and increased cholesterol efflux function for the majority of mutants. Treatment with 4-PBA also increased ABCA1 protein expression in all transfected cell lines. In fibroblast cells obtained from low HDL-C subjects expressing two of the ABCA1 mutants (p.R1068H and p.N1800H), 4-PBA increased cholesterol efflux without any increase in ABCA1 expression. Our study is the first to investigate the effect of the chemical chaperone, 4-PBA on ABCA1 and shows that it is capable of restoring plasma membrane localization and enhancing the cholesterol efflux function of mutant ABCA1s both in vitro and ex vivo. These results suggest 4-PBA may warrant further investigation as a potential therapy for increasing cholesterol efflux and HDL-C levels.

Protein kinase substrate identification on functional protein arrays

Directory of Open Access Journals (Sweden)

Zhou Fang

2008-02-01

Full Text Available Abstract Background Over the last decade, kinases have emerged as attractive therapeutic targets for a number of different diseases, and numerous high throughput screening efforts in the pharmaceutical community are directed towards discovery of compounds that regulate kinase function. The emerging utility of systems biology approaches has necessitated the development of multiplex tools suitable for proteomic-scale experiments to replace lower throughput technologies such as mass spectroscopy for the study of protein phosphorylation. Recently, a new approach for identifying substrates of protein kinases has applied the miniaturized format of functional protein arrays to characterize phosphorylation for thousands of candidate protein substrates in a single experiment. This method involves the addition of protein kinases in solution to arrays of immobilized proteins to identify substrates using highly sensitive radioactive detection and hit identification algorithms. Results To date, the factors required for optimal performance of protein array-based kinase substrate identification have not been described. In the current study, we have carried out a detailed characterization of the protein array-based method for kinase substrate identification, including an examination of the effects of time, buffer compositions, and protein concentration on the results. The protein array approach was compared to standard solution-based assays for assessing substrate phosphorylation, and a correlation of greater than 80% was observed. The results presented here demonstrate how novel substrates for protein kinases can be quickly identified from arrays containing thousands of human proteins to provide new clues to protein kinase function. In addition, a pooling-deconvolution strategy was developed and applied that enhances characterization of specific kinase-substrate relationships and decreases reagent consumption. Conclusion Functional protein microarrays are an

The Ras suppressor Rsu-1 binds to the LIM 5 domain of the adaptor protein PINCH1 and participates in adhesion-related functions

International Nuclear Information System (INIS)

Dougherty, Gerard W.; Chopp, Treasa; Qi Shengmei; Cutler, Mary Lou

2005-01-01

Rsu-1 is a highly conserved leucine rich repeat (LRR) protein that is expressed ubiquitously in mammalian cells. Rsu-1 was identified based on its ability to inhibit transformation by Ras, and previous studies demonstrated that ectopic expression of Rsu-1 inhibited anchorage-independent growth of Ras-transformed cells and human tumor cell lines. Using GAL4-based yeast two-hybrid screening, the LIM domain protein, PINCH1, was identified as the binding partner of Rsu-1. PINCH1 is an adaptor protein that localizes to focal adhesions and it has been implicated in the regulation of adhesion functions. Subdomain mapping in yeast revealed that Rsu-1 binds to the LIM 5 domain of PINCH1, a region not previously identified as a specific binding domain for any other protein. Additional testing demonstrated that PINCH2, which is highly homologous to PINCH1, except in the LIM 5 domain, does not interact with Rsu-1. Glutathione transferase fusion protein binding studies determined that the LRR region of Rsu-1 interacts with PINCH1. Transient expression studies using epitope-tagged Rsu-1 and PINCH1 revealed that Rsu-1 co-immunoprecipitated with PINCH1 and colocalized with vinculin at sites of focal adhesions in mammalian cells. In addition, endogenous P33 Rsu-1 from 293T cells co-immunoprecipitated with transiently expressed myc-tagged PINCH1. Furthermore, RNAi-induced reduction in Rsu-1 RNA and protein inhibited cell attachment, and while previous studies demonstrated that ectopic expression of Rsu-1 inhibited Jun kinase activation, the depletion of Rsu-1 resulted in activation of Jun and p38 stress kinases. These studies demonstrate that Rsu-1 interacts with PINCH1 in mammalian cells and functions, in part, by altering cell adhesion

Induction of experimental autoimmune encephalomyelitis in C57BL/6 mice deficient in either the chemokine macrophage inflammatory protein-1alpha or its CCR5 receptor

DEFF Research Database (Denmark)

Tran, E H; Kuziel, W A; Owens, T

2000-01-01

-type mice in Th1 cytokine gene expression, the kinetics and severity of disease, and infiltration of the central nervous system by lymphocytes, macrophages and granulocytes. RNase protection assays showed comparable accumulation of mRNA for the chemokines interferon-inducible protein-10, RANTES, macrophage...... and its CCR5 receptor in the induction of EAE by immunizing C57BL / 6 mice deficient in either MIP-1alpha or CCR5 with myelin oligodendrocyte glycoprotein (MOG). We found that MIP-1alpha-deficient mice were fully susceptible to MOG-induced EAE. These knockout animals were indistinguishable from wild...... chemoattractant protein-1, MIP-1beta, MIP-2, lymphotactin and T cell activation gene-3 during the course of the disease. CCR5-deficient mice were also susceptible to disease induction by MOG. The dispensability of MIP-1alpha and CCR5 for MOG-induced EAE in C57BL / 6 mice supports the idea that differential...

Resuscitation Strategies for Burn Injuries Sustained in Austere Environments to Improve Renal Perfusion and Function

Science.gov (United States)

2017-10-01

monocyte chemoattractant protein 1; MAP, mean arterial pressure; HIF, hypoxic inducible factor; VEGF, vascular endothelial growth factor; ROS, reactive...TNF-α, tumor necrosis factor-α; AQP, aquaporin; GSH, glutathione; MDA, malondialdehyde; TBARS, thiobarbituric acid-reactive substances; MPO...shrunken glomeruli were present and tubular necrosis was demon- strated by detached basal lamina [42]. Similarly, while models of electrical burns

Imperfect DNA mirror repeats in the gag gene of HIV-1 (HXB2 identify key functional domains and coincide with protein structural elements in each of the mature proteins

Directory of Open Access Journals (Sweden)

Lang Dorothy M

2007-10-01

Full Text Available Abstract Background A DNA mirror repeat is a sequence segment delimited on the basis of its containing a center of symmetry on a single strand, e.g. 5'-GCATGGTACG-3'. It is most frequently described in association with a functionally significant site in a genomic sequence, and its occurrence is regarded as noteworthy, if not unusual. However, imperfect mirror repeats (IMRs having ≥ 50% symmetry are common in the protein coding DNA of monomeric proteins and their distribution has been found to coincide with protein structural elements – helices, β sheets and turns. In this study, the distribution of IMRs is evaluated in a polyprotein – to determine whether IMRs may be related to the position or order of protein cleavage or other hierarchal aspects of protein function. The gag gene of HIV-1 [GenBank:K03455] was selected for the study because its protein motifs and structural components are well documented. Results There is a highly specific relationship between IMRs and structural and functional aspects of the Gag polyprotein. The five longest IMRs in the polyprotein translate a key functional segment in each of the five cleavage products. Throughout the protein, IMRs coincide with functionally significant segments of the protein. A detailed annotation of the protein, which combines structural, functional and IMR data illustrates these associations. There is a significant statistical correlation between the ends of IMRs and the ends of PSEs in each of the mature proteins. Weakly symmetric IMRs (≥ 33% are related to cleavage positions and processes. Conclusion The frequency and distribution of IMRs in HIV-1 Gag indicates that DNA symmetry is a fundamental property of protein coding DNA and that different levels of symmetry are associated with different functional aspects of the gene and its protein. The interaction between IMRs and protein structure and function is precise and interwoven over the entire length of the polyprotein. The

Direct interaction of TFIIB and the IE protein of equine herpesvirus 1 is required for maximal trans-activation function

International Nuclear Information System (INIS)

Albrecht, Randy A.; Jang, Hyung K.; Kim, Seong K.; O'Callaghan, Dennis J.

2003-01-01

Recently, we reported that the immediate-early (IE) protein of equine herpesvirus 1 (EHV-1) associates with transcription factor TFIIB [J. Virol. 75 (2001), 10219]. In the current study, the IE protein purified as a glutathione-S-transferase (GST) fusion protein was shown to interact directly with purified TFIIB in GST-pulldown assays. A panel of TFIIB mutants employed in protein-binding assays revealed that residues 125 to 174 within the first direct repeat of TFIIB mediate its interaction with the IE protein. This interaction is physiologically relevant as transient transfection assays demonstrated that (1) exogenous native TFIIB did not perturb IE protein function, and (2) ectopic expression of a TFIIB mutant that lacked the IE protein interactive domain significantly diminished the ability of the IE protein to trans-activate EHV-1 promoters. These results suggest that an interaction of the IE protein with TFIIB is an important aspect of the regulatory role of the IE protein in the trans-activation of EHV-1 promoters

Canola/rapeseed protein-functionality and nutrition

Directory of Open Access Journals (Sweden)

Wanasundara Janitha P.D.

2016-07-01

Full Text Available Protein rich meal is a valuable co-product of canola/rapeseed oil extraction. Seed storage proteins that include cruciferin (11S and napin (2S dominate the protein complement of canola while oleosins, lipid transfer proteins and other minor proteins of non-storage nature are also found. Although oil-free canola meal contains 36–40% protein on a dry weight basis, non-protein components including fibre, polymeric phenolics, phytates and sinapine, etc. of the seed coat and cellular components make protein less suitable for food use. Separation of canola protein from non-protein components is a technical challenge but necessary to obtain full nutritional and functional potential of protein. Process conditions of raw material and protein preparation are critical of nutritional and functional value of the final protein product. The storage proteins of canola can satisfy many nutritional and functional requirements for food applications. Protein macromolecules of canola also provide functionalities required in applications beyond edible uses; there exists substantial potential as a source of plant protein and a renewable biopolymer. Available information at present is mostly based on the protein products that can be obtained as mixtures of storage protein types and other chemical constituents of the seed; therefore, full potential of canola storage proteins is yet to be revealed.

Proteomic and functional analyses reveal MAPK1 regulates milk protein synthesis.

Science.gov (United States)

Lu, Li-Min; Li, Qing-Zhang; Huang, Jian-Guo; Gao, Xue-Jun

2012-12-27

L-Lysine (L-Lys) is an essential amino acid that plays fundamental roles in protein synthesis. Many nuclear phosphorylated proteins such as Stat5 and mTOR regulate milk protein synthesis. However, the details of milk protein synthesis control at the transcript and translational levels are not well known. In this current study, a two-dimensional gel electrophoresis (2-DE)/MS-based proteomic technology was used to identify phosphoproteins responsible for milk protein synthesis in dairy cow mammary epithelial cells (DCMECs). The effect of L-Lys on DCMECs was analyzed by CASY technology and reversed phase high performance liquid chromatography (RP-HPLC). The results showed that cell proliferation ability and β-casein expression were enhanced in DCMECs treated with L-Lys. By phosphoproteomics analysis, six proteins, including MAPK1, were identified up-expressed in DCMECs treated with 1.2 mM L-Lys for 24 h, and were verified by quantitative real-time PCR (qRT-PCR) and western blot. Overexpression and siRNA inhibition of MAPK1 experiments showed that MAPK1 upregulated milk protein synthesis through Stat5 and mTOR pathway. These findings that MAPK1 involves in regulation of milk synthesis shed new insights for understanding the mechanisms of milk protein synthesis.

In silico studies on structure-function of DNA GCC- box binding domain of brassica napus DREB1 protein

International Nuclear Information System (INIS)

Qamarunnisa, S.; Hussain, M.

2012-01-01

DREB1 is a transcriptional factor, which selectively binds with the promoters of the genes involved in stress response in the plants. Homology of DREB protein and its binding element have been detected in the genome of many plants. However, only a few reports exist that discusses the binding properties of this protein with the gene (s) promoter. In the present study, we have undertaken studies exploring the structure-function relationship of Brassica napus DREB1. Multiple sequence alignment, protein homology modeling and intermolecular docking of GCC-box binding domain (GBD) of the said protein was carried out using atomic coordinates of GBD from Arabdiopsis thaliana and GCC-box containing DNA respectively. Similarities and/or identities in multiple, sequence alignment, particularly at the functionally important amino acids, strongly suggested the binding specificity of B. napus DREB1 to GCC-box. Similarly, despite 56% sequence homology, tertiary structures of both template and modeled protein were found to be extremely similar as indicated by root mean square deviation of 0.34 A. More similarities were established between GBD of both A. thaliana and B. napus DREB1 by conducting protein docking with the DNA containing GCC-box. It appears that both proteins interact through their beta-sheet with the major DNA groove including both nitrogen bases and phosphate and sugar moieties. Additionally, in most cases the interacting residues were also found to be identical. Briefly, this study attempts to elucidate the molecular basis of DREB1 interaction with its target sequence in the promoter. (author)

Origins of Protein Functions in Cells

Science.gov (United States)

Seelig, Burchard; Pohorille, Andrzej

2011-01-01

In modern organisms proteins perform a majority of cellular functions, such as chemical catalysis, energy transduction and transport of material across cell walls. Although great strides have been made towards understanding protein evolution, a meaningful extrapolation from contemporary proteins to their earliest ancestors is virtually impossible. In an alternative approach, the origin of water-soluble proteins was probed through the synthesis and in vitro evolution of very large libraries of random amino acid sequences. In combination with computer modeling and simulations, these experiments allow us to address a number of fundamental questions about the origins of proteins. Can functionality emerge from random sequences of proteins? How did the initial repertoire of functional proteins diversify to facilitate new functions? Did this diversification proceed primarily through drawing novel functionalities from random sequences or through evolution of already existing proto-enzymes? Did protein evolution start from a pool of proteins defined by a frozen accident and other collections of proteins could start a different evolutionary pathway? Although we do not have definitive answers to these questions yet, important clues have been uncovered. In one example (Keefe and Szostak, 2001), novel ATP binding proteins were identified that appear to be unrelated in both sequence and structure to any known ATP binding proteins. One of these proteins was subsequently redesigned computationally to bind GTP through introducing several mutations that introduce targeted structural changes to the protein, improve its binding to guanine and prevent water from accessing the active center. This study facilitates further investigations of individual evolutionary steps that lead to a change of function in primordial proteins. In a second study (Seelig and Szostak, 2007), novel enzymes were generated that can join two pieces of RNA in a reaction for which no natural enzymes are known

Designing sequence to control protein function in an EF-hand protein.

Science.gov (United States)

Bunick, Christopher G; Nelson, Melanie R; Mangahas, Sheryll; Hunter, Michael J; Sheehan, Jonathan H; Mizoue, Laura S; Bunick, Gerard J; Chazin, Walter J

2004-05-19

The extent of conformational change that calcium binding induces in EF-hand proteins is a key biochemical property specifying Ca(2+) sensor versus signal modulator function. To understand how differences in amino acid sequence lead to differences in the response to Ca(2+) binding, comparative analyses of sequence and structures, combined with model building, were used to develop hypotheses about which amino acid residues control Ca(2+)-induced conformational changes. These results were used to generate a first design of calbindomodulin (CBM-1), a calbindin D(9k) re-engineered with 15 mutations to respond to Ca(2+) binding with a conformational change similar to that of calmodulin. The gene for CBM-1 was synthesized, and the protein was expressed and purified. Remarkably, this protein did not exhibit any non-native-like molten globule properties despite the large number of mutations and the nonconservative nature of some of them. Ca(2+)-induced changes in CD intensity and in the binding of the hydrophobic probe, ANS, implied that CBM-1 does undergo Ca(2+) sensorlike conformational changes. The X-ray crystal structure of Ca(2+)-CBM-1 determined at 1.44 A resolution reveals the anticipated increase in hydrophobic surface area relative to the wild-type protein. A nascent calmodulin-like hydrophobic docking surface was also found, though it is occluded by the inter-EF-hand loop. The results from this first calbindomodulin design are discussed in terms of progress toward understanding the relationships between amino acid sequence, protein structure, and protein function for EF-hand CaBPs, as well as the additional mutations for the next CBM design.

Brittle Culm1, a COBRA-Like Protein, Functions in Cellulose Assembly through Binding Cellulose Microfibrils

Science.gov (United States)

Zhang, Baocai; Liu, Xiangling; Yan, Meixian; Zhang, Lanjun; Shi, Yanyun; Zhang, Mu; Qian, Qian; Li, Jiayang; Zhou, Yihua

2013-01-01

Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1), a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI) anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM) at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD) assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs) function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity. PMID:23990797

Brittle Culm1, a COBRA-like protein, functions in cellulose assembly through binding cellulose microfibrils.

Directory of Open Access Journals (Sweden)

Lifeng Liu

Full Text Available Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1, a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity.

Brittle Culm1, a COBRA-like protein, functions in cellulose assembly through binding cellulose microfibrils.

Science.gov (United States)

Liu, Lifeng; Shang-Guan, Keke; Zhang, Baocai; Liu, Xiangling; Yan, Meixian; Zhang, Lanjun; Shi, Yanyun; Zhang, Mu; Qian, Qian; Li, Jiayang; Zhou, Yihua

2013-01-01

Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1), a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI) anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM) at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD) assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs) function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity.

Synergistic enhancement of chemokine generation and lung injury by C5a or the membrane attack complex of complement

DEFF Research Database (Denmark)

Czermak, B J; Lentsch, A B; Bless, N M

1999-01-01

demonstrated synergistic production of C-X-C (macrophage inflammatory protein-2 and cytokine-induced neutrophil chemoattractant) and C-C (macrophage inflammatory protein-1alpha and monocyte chemoattractant-1) chemokines. In the absence of the costimulus, C5a or MAC did not induce chemokine generation....... In in vivo studies, C5a and MAC alone caused limited or no intrapulmonary generation of chemokines, but in the presence of a costimulus (IgG immune complexes) C5a and MAC caused synergistic intrapulmonary generation of C-X-C and C-C chemokines but not of tumor necrosis factor alpha. Under these conditions...... increased neutrophil accumulation occurred, as did lung injury. These observations suggest that C5a and MAC function synergistically with a costimulus to enhance chemokine generation and the intensity of the lung inflammatory response....

Confirmation of translatability and functionality certifies the dual endothelin1/VEGFsp receptor (DEspR) protein.

Science.gov (United States)

Herrera, Victoria L M; Steffen, Martin; Moran, Ann Marie; Tan, Glaiza A; Pasion, Khristine A; Rivera, Keith; Pappin, Darryl J; Ruiz-Opazo, Nelson

2016-06-14

In contrast to rat and mouse databases, the NCBI gene database lists the human dual-endothelin1/VEGFsp receptor (DEspR, formerly Dear) as a unitary transcribed pseudogene due to a stop [TGA]-codon at codon#14 in automated DNA and RNA sequences. However, re-analysis is needed given prior single gene studies detected a tryptophan [TGG]-codon#14 by manual Sanger sequencing, demonstrated DEspR translatability and functionality, and since the demonstration of actual non-translatability through expression studies, the standard-of-excellence for pseudogene designation, has not been performed. Re-analysis must meet UNIPROT criteria for demonstration of a protein's existence at the highest (protein) level, which a priori, would override DNA- or RNA-based deductions. To dissect the nucleotide sequence discrepancy, we performed Maxam-Gilbert sequencing and reviewed 727 RNA-seq entries. To comply with the highest level multiple UNIPROT criteria for determining DEspR's existence, we performed various experiments using multiple anti-DEspR monoclonal antibodies (mAbs) targeting distinct DEspR epitopes with one spanning the contested tryptophan [TGG]-codon#14, assessing: (a) DEspR protein expression, (b) predicted full-length protein size, (c) sequence-predicted protein-specific properties beyond codon#14: receptor glycosylation and internalization, (d) protein-partner interactions, and (e) DEspR functionality via DEspR-inhibition effects. Maxam-Gilbert sequencing and some RNA-seq entries demonstrate two guanines, hence a tryptophan [TGG]-codon#14 within a compression site spanning an error-prone compression sequence motif. Western blot analysis using anti-DEspR mAbs targeting distinct DEspR epitopes detect the identical glycosylated 17.5 kDa pull-down protein. Decrease in DEspR-protein size after PNGase-F digest demonstrates post-translational glycosylation, concordant with the consensus-glycosylation site beyond codon#14. Like other small single-transmembrane proteins, mass

Functional assignment to JEV proteins using SVM.

Science.gov (United States)

Sahoo, Ganesh Chandra; Dikhit, Manas Ranjan; Das, Pradeep

2008-01-01

Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP).

Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins.

Science.gov (United States)

Jong, A Y; Clark, M W; Gilbert, M; Oehm, A; Campbell, J L

1987-08-01

To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly homologous to a peptide in calf thymus UP1 and UP2 and a human heterogeneous nuclear ribonucleoprotein. Residues 125 to 162 constitute a fivefold tandem repeat of the sequence RGGFRG, the composition of which suggests a nucleic acid-binding site. Near the C terminus, residues 233 to 245 are homologous to several RNA-binding proteins. Of 18 C-terminal residues, 10 are acidic, a characteristic of the procaryotic single-stranded DNA-binding proteins and eucaryotic DNA- and RNA-binding proteins. In addition, examination of the subcellular distribution of SSB1 by immunofluorescence microscopy indicated that SSB1 is a nuclear protein, predominantly located in the nucleolus. Sequence homologies and the nucleolar localization make it likely that SSB1 functions in RNA metabolism in vivo, although an additional role in DNA metabolism cannot be excluded.

Protein domain recurrence and order can enhance prediction of protein functions

KAUST Repository

Abdel Messih, Mario A.

2012-09-07

Motivation: Burgeoning sequencing technologies have generated massive amounts of genomic and proteomic data. Annotating the functions of proteins identified in this data has become a big and crucial problem. Various computational methods have been developed to infer the protein functions based on either the sequences or domains of proteins. The existing methods, however, ignore the recurrence and the order of the protein domains in this function inference. Results: We developed two new methods to infer protein functions based on protein domain recurrence and domain order. Our first method, DRDO, calculates the posterior probability of the Gene Ontology terms based on domain recurrence and domain order information, whereas our second method, DRDO-NB, relies on the nave Bayes methodology using the same domain architecture information. Our large-scale benchmark comparisons show strong improvements in the accuracy of the protein function inference achieved by our new methods, demonstrating that domain recurrence and order can provide important information for inference of protein functions. The Author(s) 2012. Published by Oxford University Press.

Dual Role of Ancient Ubiquitous Protein 1 (AUP1) in Lipid Droplet Accumulation and Endoplasmic Reticulum (ER) Protein Quality Control

OpenAIRE

Klemm, Elizabeth J.; Spooner, Eric; Ploegh, Hidde L.

2011-01-01

Quality control of endoplasmic reticulum proteins involves the identification and engagement of misfolded proteins, dislocation of the misfolded protein across the endoplasmic reticulum (ER) membrane, and ubiquitin-mediated targeting to the proteasome for degradation. Ancient ubiquitous protein 1 (AUP1) physically associates with the mammalian HRD1-SEL1L complex, and AUP1 depletion impairs degradation of misfolded ER proteins. One of the functions of AUP1 in ER quality control is to recruit t...

Human Milk: Bioactive Proteins/Peptides and Functional Properties.

Science.gov (United States)

Lönnerdal, Bo

2016-06-23

Breastfeeding has been associated with many benefits, both in the short and in the long term. Infants being breastfed generally have less illness and have better cognitive development at 1 year of age than formula-fed infants. Later in life, they have a lower risk of obesity, diabetes and cardiovascular disease. Several components in breast milk may be responsible for these different outcomes, but bioactive proteins/peptides likely play a major role. Some proteins in breast milk are comparatively resistant towards digestion and may therefore exert their functions in the gastrointestinal tract in intact form or as larger fragments. Other milk proteins may be partially digested in the upper small intestine and the resulting peptides may exert functions in the lower small intestine. Lactoferrin, lysozyme and secretory IgA have been found intact in the stool of breastfed infants and are therefore examples of proteins that are resistant against proteolytic degradation in the gut. Together, these proteins serve protective roles against infection and support immune function in the immature infant. α-lactalbumin, β-casein, κ-casein and osteopontin are examples of proteins that are partially digested in the upper small intestine, and the resulting peptides influence functions in the gut. Such functions include stimulation of immune function, mineral and trace element absorption and defense against infection. © 2016 Nestec Ltd., Vevey/S. Karger AG, Basel.

Combining modularity, conservation, and interactions of proteins significantly increases precision and coverage of protein function prediction

Directory of Open Access Journals (Sweden)

Sers Christine T

2010-12-01

Full Text Available Abstract Background While the number of newly sequenced genomes and genes is constantly increasing, elucidation of their function still is a laborious and time-consuming task. This has led to the development of a wide range of methods for predicting protein functions in silico. We report on a new method that predicts function based on a combination of information about protein interactions, orthology, and the conservation of protein networks in different species. Results We show that aggregation of these independent sources of evidence leads to a drastic increase in number and quality of predictions when compared to baselines and other methods reported in the literature. For instance, our method generates more than 12,000 novel protein functions for human with an estimated precision of ~76%, among which are 7,500 new functional annotations for 1,973 human proteins that previously had zero or only one function annotated. We also verified our predictions on a set of genes that play an important role in colorectal cancer (MLH1, PMS2, EPHB4 and could confirm more than 73% of them based on evidence in the literature. Conclusions The combination of different methods into a single, comprehensive prediction method infers thousands of protein functions for every species included in the analysis at varying, yet always high levels of precision and very good coverage.

The BRCA1 variant p.Ser36Tyr abrogates BRCA1 protein function and potentially confers a moderate risk of breast cancer.

Science.gov (United States)

Christou, Charita M; Hadjisavvas, Andreas; Kyratzi, Maria; Flouri, Christina; Neophytou, Ioanna; Anastasiadou, Violetta; Loizidou, Maria A; Kyriacou, Kyriacos

2014-01-01

The identification of variants of unknown clinical significance (VUS) in the BRCA1 gene complicates genetic counselling and causes additional anxiety to carriers. In silico approaches currently used for VUS pathogenicity assessment are predictive and often produce conflicting data. Furthermore, functional assays are either domain or function specific, thus they do not examine the entire spectrum of BRCA1 functions and interpretation of individual assay results can be misleading. PolyPhen algorithm predicted that the BRCA1 p.Ser36Tyr VUS identified in the Cypriot population was damaging, whereas Align-GVGD predicted that it was possibly of no significance. In addition the BRCA1 p.Ser36Tyr variant was found to be associated with increased risk (OR = 3.47, 95% CI 1.13-10.67, P = 0.02) in a single case-control series of 1174 cases and 1109 controls. We describe a cellular system for examining the function of exogenous full-length BRCA1 and for classifying VUS. We achieved strong protein expression of full-length BRCA1 in transiently transfected HEK293T cells. The p.Ser36Tyr VUS exhibited low protein expression similar to the known pathogenic variant p.Cys61Gly. Co-precipitation analysis further demonstrated that it has a reduced ability to interact with BARD1. Further, co-precipitation analysis of nuclear and cytosolic extracts as well as immunofluorescence studies showed that a high proportion of the p.Ser36Tyr variant is withheld in the cytoplasm contrary to wild type protein. In addition the ability of p.Ser36Tyr to co-localize with conjugated ubiquitin foci in the nuclei of S-phase synchronized cells following genotoxic stress with hydroxyurea is impaired at more pronounced levels than that of the p.Cys61Gly pathogenic variant. The p.Ser36Tyr variant demonstrates abrogated function, and based on epidemiological, genetic, and clinical data we conclude that the p.Ser36Tyr variant is probably associated with a moderate breast cancer risk.

The BRCA1 variant p.Ser36Tyr abrogates BRCA1 protein function and potentially confers a moderate risk of breast cancer.

Directory of Open Access Journals (Sweden)

Charita M Christou

Full Text Available The identification of variants of unknown clinical significance (VUS in the BRCA1 gene complicates genetic counselling and causes additional anxiety to carriers. In silico approaches currently used for VUS pathogenicity assessment are predictive and often produce conflicting data. Furthermore, functional assays are either domain or function specific, thus they do not examine the entire spectrum of BRCA1 functions and interpretation of individual assay results can be misleading. PolyPhen algorithm predicted that the BRCA1 p.Ser36Tyr VUS identified in the Cypriot population was damaging, whereas Align-GVGD predicted that it was possibly of no significance. In addition the BRCA1 p.Ser36Tyr variant was found to be associated with increased risk (OR = 3.47, 95% CI 1.13-10.67, P = 0.02 in a single case-control series of 1174 cases and 1109 controls. We describe a cellular system for examining the function of exogenous full-length BRCA1 and for classifying VUS. We achieved strong protein expression of full-length BRCA1 in transiently transfected HEK293T cells. The p.Ser36Tyr VUS exhibited low protein expression similar to the known pathogenic variant p.Cys61Gly. Co-precipitation analysis further demonstrated that it has a reduced ability to interact with BARD1. Further, co-precipitation analysis of nuclear and cytosolic extracts as well as immunofluorescence studies showed that a high proportion of the p.Ser36Tyr variant is withheld in the cytoplasm contrary to wild type protein. In addition the ability of p.Ser36Tyr to co-localize with conjugated ubiquitin foci in the nuclei of S-phase synchronized cells following genotoxic stress with hydroxyurea is impaired at more pronounced levels than that of the p.Cys61Gly pathogenic variant. The p.Ser36Tyr variant demonstrates abrogated function, and based on epidemiological, genetic, and clinical data we conclude that the p.Ser36Tyr variant is probably associated with a moderate breast cancer risk.

Biases in the experimental annotations of protein function and their effect on our understanding of protein function space.

Directory of Open Access Journals (Sweden)

Alexandra M Schnoes

Full Text Available The ongoing functional annotation of proteins relies upon the work of curators to capture experimental findings from scientific literature and apply them to protein sequence and structure data. However, with the increasing use of high-throughput experimental assays, a small number of experimental studies dominate the functional protein annotations collected in databases. Here, we investigate just how prevalent is the "few articles - many proteins" phenomenon. We examine the experimentally validated annotation of proteins provided by several groups in the GO Consortium, and show that the distribution of proteins per published study is exponential, with 0.14% of articles providing the source of annotations for 25% of the proteins in the UniProt-GOA compilation. Since each of the dominant articles describes the use of an assay that can find only one function or a small group of functions, this leads to substantial biases in what we know about the function of many proteins. Mass-spectrometry, microscopy and RNAi experiments dominate high throughput experiments. Consequently, the functional information derived from these experiments is mostly of the subcellular location of proteins, and of the participation of proteins in embryonic developmental pathways. For some organisms, the information provided by different studies overlap by a large amount. We also show that the information provided by high throughput experiments is less specific than those provided by low throughput experiments. Given the experimental techniques available, certain biases in protein function annotation due to high-throughput experiments are unavoidable. Knowing that these biases exist and understanding their characteristics and extent is important for database curators, developers of function annotation programs, and anyone who uses protein function annotation data to plan experiments.

HIV-1 TAT protein enhances sensitization to methamphetamine by affecting dopaminergic function.

Science.gov (United States)

Kesby, James P; Najera, Julia A; Romoli, Benedetto; Fang, Yiding; Basova, Liana; Birmingham, Amanda; Marcondes, Maria Cecilia G; Dulcis, Davide; Semenova, Svetlana

2017-10-01

Methamphetamine abuse is common among humans with immunodeficiency virus (HIV). The HIV-1 regulatory protein TAT induces dysfunction of mesolimbic dopaminergic systems which may result in impaired reward processes and contribute to methamphetamine abuse. These studies investigated the impact of TAT expression on methamphetamine-induced locomotor sensitization, underlying changes in dopamine function and adenosine receptors in mesolimbic brain areas and neuroinflammation (microgliosis). Transgenic mice with doxycycline-induced TAT protein expression in the brain were tested for locomotor activity in response to repeated methamphetamine injections and methamphetamine challenge after a 7-day abstinence period. Dopamine function in the nucleus accumbens (Acb) was determined using high performance liquid chromatography. Expression of dopamine and/or adenosine A receptors (ADORA) in the Acb and caudate putamen (CPu) was assessed using RT-PCR and immunohistochemistry analyses. Microarrays with pathway analyses assessed dopamine and adenosine signaling in the CPu. Activity-dependent neurotransmitter switching of a reserve pool of non-dopaminergic neurons to a dopaminergic phenotype in the ventral tegmental area (VTA) was determined by immunohistochemistry and quantified with stereology. TAT expression enhanced methamphetamine-induced sensitization. TAT expression alone decreased striatal dopamine (D1, D2, D4, D5) and ADORA1A receptor expression, while increasing ADORA2A receptors expression. Moreover, TAT expression combined with methamphetamine exposure was associated with increased adenosine A receptors (ADORA1A) expression and increased recruitment of dopamine neurons in the VTA. TAT expression and methamphetamine exposure induced microglia activation with the largest effect after combined exposure. Our findings suggest that dopamine-adenosine receptor interactions and reserve pool neuronal recruitment may represent potential targets to develop new treatments for

Single proteins that serve linked functions in intracellular and extracellular microenvironments

Energy Technology Data Exchange (ETDEWEB)

Radisky, Derek C.; Stallings-Mann, Melody; Hirai, Yohei; Bissell, Mina J.

2009-06-03

protein secretion (as syntaxin-2), amphoterin/high mobility group box-1 (HMGB1), which may link inflammation (as amphoterin) with regulation of gene expression (as HMGB1), and tissue transglutaminase, which affects delivery of and response to apoptotic signals by serving a related function on both sides of the plasma membrane. As it is notable that all three of these proteins have been reported to transit the plasma membrane through non-classical secretory mechanisms, we will also discuss why coordinated inside/outside functions may be found in some examples of proteins which transit the plasma membrane through non-classical mechanisms and how this relationship can be used to identify additional proteins that share these characteristics.

Noroviruses Co-opt the Function of Host Proteins VAPA and VAPB for Replication via a Phenylalanine-Phenylalanine-Acidic-Tract-Motif Mimic in Nonstructural Viral Protein NS1/2.

Science.gov (United States)

McCune, Broc T; Tang, Wei; Lu, Jia; Eaglesham, James B; Thorne, Lucy; Mayer, Anne E; Condiff, Emily; Nice, Timothy J; Goodfellow, Ian; Krezel, Andrzej M; Virgin, Herbert W

2017-07-11

The Norovirus genus contains important human pathogens, but the role of host pathways in norovirus replication is largely unknown. Murine noroviruses provide the opportunity to study norovirus replication in cell culture and in small animals. The human norovirus nonstructural protein NS1/2 interacts with the host protein VAMP-associated protein A (VAPA), but the significance of the NS1/2-VAPA interaction is unexplored. Here we report decreased murine norovirus replication in VAPA- and VAPB-deficient cells. We characterized the role of VAPA in detail. VAPA was required for the efficiency of a step(s) in the viral replication cycle after entry of viral RNA into the cytoplasm but before the synthesis of viral minus-sense RNA. The interaction of VAPA with viral NS1/2 proteins is conserved between murine and human noroviruses. Murine norovirus NS1/2 directly bound the major sperm protein (MSP) domain of VAPA through its NS1 domain. Mutations within NS1 that disrupted interaction with VAPA inhibited viral replication. Structural analysis revealed that the viral NS1 domain contains a mimic of the phenylalanine-phenylalanine-acidic-tract (FFAT) motif that enables host proteins to bind to the VAPA MSP domain. The NS1/2-FFAT mimic region interacted with the VAPA-MSP domain in a manner similar to that seen with bona fide host FFAT motifs. Amino acids in the FFAT mimic region of the NS1 domain that are important for viral replication are highly conserved across murine norovirus strains. Thus, VAPA interaction with a norovirus protein that functionally mimics host FFAT motifs is important for murine norovirus replication. IMPORTANCE Human noroviruses are a leading cause of gastroenteritis worldwide, but host factors involved in norovirus replication are incompletely understood. Murine noroviruses have been studied to define mechanisms of norovirus replication. Here we defined the importance of the interaction between the hitherto poorly studied NS1/2 norovirus protein and the

Saccharomyces cerevisiae SSB1 protein and its relationship to nucleolar RNA-binding proteins.

OpenAIRE

Jong, A Y; Clark, M W; Gilbert, M; Oehm, A; Campbell, J L

1987-01-01

To better define the function of Saccharomyces cerevisiae SSB1, an abundant single-stranded nucleic acid-binding protein, we determined the nucleotide sequence of the SSB1 gene and compared it with those of other proteins of known function. The amino acid sequence contains 293 amino acid residues and has an Mr of 32,853. There are several stretches of sequence characteristic of other eucaryotic single-stranded nucleic acid-binding proteins. At the amino terminus, residues 39 to 54 are highly ...

Blimp-1 controls plasma cell function through regulation of immunoglobulin secretion and the unfolded protein response

Science.gov (United States)

Tellier, Julie; Shi, Wei; Minnich, Martina; Liao, Yang; Crawford, Simon; Smyth, Gordon K; Kallies, Axel; Busslinger, Meinrad; Nutt, Stephen L

2015-01-01

Plasma cell differentiation requires silencing of B cell transcription, while establishing antibody-secretory function and long-term survival. The transcription factors Blimp-1 and IRF4 are essential for plasma cell generation, however their function in mature plasma cells has remained elusive. We have found that while IRF4 was essential for plasma cell survival, Blimp-1 was dispensable. Blimp-1-deficient plasma cells retained their transcriptional identity, but lost the ability to secrete antibody. Blimp-1 regulated many components of the unfolded protein response (UPR), including XBP-1 and ATF6. The overlap of Blimp-1 and XBP-1 function was restricted to the UPR, with Blimp-1 uniquely regulating mTOR activity and plasma cell size. Thus, Blimp-1 is required for the unique physiological capacity of plasma cells that enables the secretion of protective antibody. PMID:26779600

Identifying the molecular functions of electron transport proteins using radial basis function networks and biochemical properties.

Science.gov (United States)

Le, Nguyen-Quoc-Khanh; Nguyen, Trinh-Trung-Duong; Ou, Yu-Yen

2017-05-01

The electron transport proteins have an important role in storing and transferring electrons in cellular respiration, which is the most proficient process through which cells gather energy from consumed food. According to the molecular functions, the electron transport chain components could be formed with five complexes with several different electron carriers and functions. Therefore, identifying the molecular functions in the electron transport chain is vital for helping biologists understand the electron transport chain process and energy production in cells. This work includes two phases for discriminating electron transport proteins from transport proteins and classifying categories of five complexes in electron transport proteins. In the first phase, the performances from PSSM with AAIndex feature set were successful in identifying electron transport proteins in transport proteins with achieved sensitivity of 73.2%, specificity of 94.1%, and accuracy of 91.3%, with MCC of 0.64 for independent data set. With the second phase, our method can approach a precise model for identifying of five complexes with different molecular functions in electron transport proteins. The PSSM with AAIndex properties in five complexes achieved MCC of 0.51, 0.47, 0.42, 0.74, and 1.00 for independent data set, respectively. We suggest that our study could be a power model for determining new proteins that belongs into which molecular function of electron transport proteins. Copyright © 2017 Elsevier Inc. All rights reserved.

Biases in the Experimental Annotations of Protein Function and Their Effect on Our Understanding of Protein Function Space

Science.gov (United States)

Schnoes, Alexandra M.; Ream, David C.; Thorman, Alexander W.; Babbitt, Patricia C.; Friedberg, Iddo

2013-01-01

The ongoing functional annotation of proteins relies upon the work of curators to capture experimental findings from scientific literature and apply them to protein sequence and structure data. However, with the increasing use of high-throughput experimental assays, a small number of experimental studies dominate the functional protein annotations collected in databases. Here, we investigate just how prevalent is the “few articles - many proteins” phenomenon. We examine the experimentally validated annotation of proteins provided by several groups in the GO Consortium, and show that the distribution of proteins per published study is exponential, with 0.14% of articles providing the source of annotations for 25% of the proteins in the UniProt-GOA compilation. Since each of the dominant articles describes the use of an assay that can find only one function or a small group of functions, this leads to substantial biases in what we know about the function of many proteins. Mass-spectrometry, microscopy and RNAi experiments dominate high throughput experiments. Consequently, the functional information derived from these experiments is mostly of the subcellular location of proteins, and of the participation of proteins in embryonic developmental pathways. For some organisms, the information provided by different studies overlap by a large amount. We also show that the information provided by high throughput experiments is less specific than those provided by low throughput experiments. Given the experimental techniques available, certain biases in protein function annotation due to high-throughput experiments are unavoidable. Knowing that these biases exist and understanding their characteristics and extent is important for database curators, developers of function annotation programs, and anyone who uses protein function annotation data to plan experiments. PMID:23737737

Characterization of SLCO5A1/OATP5A1, a solute carrier transport protein with non-classical function.

Directory of Open Access Journals (Sweden)

Katrin Sebastian

Full Text Available Organic anion transporting polypeptides (OATP/SLCO have been identified to mediate the uptake of a broad range of mainly amphipathic molecules. Human OATP5A1 was found to be expressed in the epithelium of many cancerous and non-cancerous tissues throughout the body but protein characterization and functional analysis have not yet been performed. This study focused on the biochemical characterization of OATP5A1 using Xenopus laevis oocytes and Flp-In T-REx-HeLa cells providing evidence regarding a possible OATP5A1 function. SLCO5A1 is highly expressed in mature dendritic cells compared to immature dendritic cells (∼6.5-fold and SLCO5A1 expression correlates with the differentiation status of primary blood cells. A core- and complex- N-glycosylated polypeptide monomer of ∼105 kDa and ∼130 kDa could be localized in intracellular membranes and on the plasma membrane, respectively. Inducible expression of SLCO5A1 in HeLa cells led to an inhibitory effect of ∼20% after 96 h on cell proliferation. Gene expression profiling with these cells identified immunologically relevant genes (e.g. CCL20 and genes implicated in developmental processes (e.g. TGM2. A single nucleotide polymorphism leading to the exchange of amino acid 33 (L→F revealed no differences regarding protein expression and function. In conclusion, we provide evidence that OATP5A1 might be a non-classical OATP family member which is involved in biological processes that require the reorganization of the cell shape, such as differentiation and migration.

Phosphorylation and interactions associated with the control of the Leishmania Poly-A Binding Protein 1 (PABP1) function during translation initiation.

Science.gov (United States)

de Melo Neto, Osvaldo P; da Costa Lima, Tamara D C; Merlo, Kleison C; Romão, Tatiany P; Rocha, Pollyanna O; Assis, Ludmila A; Nascimento, Larissa M; Xavier, Camila C; Rezende, Antonio M; Reis, Christian R S; Papadopoulou, Barbara

2018-03-23

The Poly-A Binding Protein (PABP) is a conserved eukaryotic polypeptide involved in many aspects of mRNA metabolism. During translation initiation, PABP interacts with the translation initiation complex eIF4F and enhances the translation of polyadenylated mRNAs. Schematically, most PABPs can be divided into an N-terminal RNA-binding region, a non-conserved linker segment and the C-terminal MLLE domain. In pathogenic Leishmania protozoans, three PABP homologues have been identified, with the first one (PABP1) targeted by phosphorylation and shown to co-immunoprecipitate with an eIF4F-like complex (EIF4E4/EIF4G3) implicated in translation initiation. Here, PABP1 phosphorylation was shown to be linked to logarithmic cell growth, reminiscent of EIF4E4 phosphorylation, and coincides with polysomal association. Phosphorylation targets multiple serine-proline (SP) or threonine-proline (TP) residues within the PABP1 linker region. This is an essential protein, but phosphorylation is not needed for its association with polysomes or cell viability. Mutations which do impair PABP1 polysomal association and are required for viability do not prevent phosphorylation, although further mutations lead to a presumed inactive protein largely lacking phosphorylated isoforms. Co-immunoprecipitation experiments were carried out to investigate PABP1 function further, identifying several novel protein partners and the EIF4E4/EIF4G3 complex, but no other eIF4F-like complex or subunit. A novel, direct interaction between PABP1 and EIF4E4 was also investigated and found to be mediated by the PABP1 MLLE binding to PABP Interacting Motifs (PAM2) within the EIF4E4 N-terminus. The results shown here are consistent with phosphorylation of PABP1 being part of a novel pathway controlling its function and possibly translation in Leishmania.

Conservation of Oxidative Protein Stabilization in an Insect Homologue of Parkinsonism-Associated Protein DJ-1

Energy Technology Data Exchange (ETDEWEB)

Lin, Jiusheng; Prahlad, Janani; Wilson, Mark A. (UNL)

2012-08-21

DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well-conserved in other DJ-1 homologues, including two (DJ-1{alpha} and DJ-1{beta}) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1{beta}. The structure of D. melanogaster DJ-1{beta} is similar to that of human DJ-1, although two important residues in the human protein, Met26 and His126, are not conserved in DJ-1{beta}. His126 in human DJ-1 is substituted with a tyrosine in DJ-1{beta}, and this residue is not able to compose a putative catalytic dyad with Cys106 that was proposed to be important in the human protein. The reactive cysteine in DJ-1 is oxidized readily to the cysteine-sulfinic acid in both flies and humans, and this may regulate the cytoprotective function of the protein. We show that the oxidation of this conserved cysteine residue to its sulfinate form (Cys-SO{sub 2{sup -}}) results in considerable thermal stabilization of both Drosophila DJ-1{beta} and human DJ-1. Therefore, protein stabilization is one potential mechanism by which cysteine oxidation may regulate DJ-1 function in vivo. More generally, most close DJ-1 homologues are likely stabilized by cysteine-sulfinic acid formation but destabilized by further oxidation, suggesting that they are biphasically regulated by oxidative modification.

Function-blocking antibodies to human vascular adhesion protein-1: a potential anti-inflammatory therapy.

Science.gov (United States)

Kirton, Christopher M; Laukkanen, Marja-Leena; Nieminen, Antti; Merinen, Marika; Stolen, Craig M; Armour, Kathryn; Smith, David J; Salmi, Marko; Jalkanen, Sirpa; Clark, Michael R

2005-11-01

Human vascular adhesion protein-1 (VAP-1) is a homodimeric 170-kDa sialoglycoprotein that is expressed on the surface of endothelial cells and functions as a semicarbazide-sensitive amine oxidase and as an adhesion molecule. Blockade of VAP-1 has been shown to reduce leukocyte adhesion and transmigration in in vivo and in vitro models, suggesting that VAP-1 is a potential target for anti-inflammatory therapy. In this study we have constructed mouse-human chimeric antibodies by genetic engineering in order to circumvent the potential problems involved in using murine antibodies in man. Our chimeric anti-VAP-1 antibodies, which were designed to lack Fc-dependent effector functions, bound specifically to cell surface-expressed recombinant human VAP-1 and recognized VAP-1 in different cell types in tonsil. Furthermore, the chimeric antibodies prevented leukocyte adhesion and transmigration in vitro and in vivo. Hence, these chimeric antibodies have the potential to be used as a new anti-inflammatory therapy.

Msp1 Is a Membrane Protein Dislocase for Tail-Anchored Proteins.

Science.gov (United States)

Wohlever, Matthew L; Mateja, Agnieszka; McGilvray, Philip T; Day, Kasey J; Keenan, Robert J

2017-07-20

Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical for TA protein extraction in vitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane. Copyright © 2017 Elsevier Inc. All rights reserved.

Identification of a 34 kDa protein altered in the LF-1 mutant as the herbicide-binding D1 protein of photosystem II

International Nuclear Information System (INIS)

Metz, J.; Pakrasi, H.; Seibert, M.; Arntzen, C.

1986-01-01

The LF-1 mutant of Scenedesmus has a complete block on the oxidizing side of its PSII reaction center. However, the reaction center as well as the reducing side of PSII is fully functional in this mutant. Compared to the wildtype (WT) the only detected protein difference in the PSII complex of LF-1 is the change in mobility of a 34 kDa protein to 36 kDa. This protein has been implicated to have a major role in Mn-binding and water-oxidation. The authors have recently shown that photoaffinity labeling of thylakoids with azido-[ 14 C]-atrazine tags the 34 kDa protein in WT and the 36 kDa protein in LF-1. It has been shown that the azido-atrazine labeled protein, called D1, functions in herbicide binding and Q/sub A/ to Q/sub B/ electron transfer on the reducing side of PSII. Polyclonal antibodies directed against the D1 protein of Amaranthus hybridus (Ohad, et al., EMBOJ 1985) were found to recognize the Scenedesmus 34 kDa (WT) and 36 kDa (LF-1) proteins. The implied dual function for the D1 protein on the reducing as well as the oxidizing side of PSII reaction center will be discussed

Usher protein functions in hair cells and photoreceptors.

Science.gov (United States)

Cosgrove, Dominic; Zallocchi, Marisa

2014-01-01

The 10 different genes associated with the deaf/blind disorder, Usher syndrome, encode a number of structurally and functionally distinct proteins, most expressed as multiple isoforms/protein variants. Functional characterization of these proteins suggests a role in stereocilia development in cochlear hair cells, likely owing to adhesive interactions in hair bundles. In mature hair cells, homodimers of the Usher cadherins, cadherin 23 and protocadherin 15, interact to form a structural fiber, the tip link, and the linkages that anchor the taller stereocilia's actin cytoskeleton core to the shorter adjacent stereocilia and the elusive mechanotransduction channels, explaining the deafness phenotype when these molecular interactions are perturbed. The conundrum is that photoreceptors lack a synonymous mechanotransduction apparatus, and so a common theory for Usher protein function in the two neurosensory cell types affected in Usher syndrome is lacking. Recent evidence linking photoreceptor cell dysfunction in the shaker 1 mouse model for Usher syndrome to light-induced protein translocation defects, combined with localization of an Usher protein interactome at the periciliary region of the photoreceptors suggests Usher proteins might regulate protein trafficking between the inner and outer segments of photoreceptors. A distinct Usher protein complex is trafficked to the ribbon synapses of hair cells, and synaptic defects have been reported in Usher mutants in both hair cells and photoreceptors. This review aims to clarify what is known about Usher protein function at the synaptic and apical poles of hair cells and photoreceptors and the prospects for identifying a unifying pathobiological mechanism to explain deaf/blindness in Usher syndrome. Copyright © 2013 Elsevier Ltd. All rights reserved.

Two novel WD40 domain–containing proteins, Ere1 and Ere2, function in the retromer-mediated endosomal recycling pathway

Science.gov (United States)

Shi, Yufeng; Stefan, Christopher J.; Rue, Sarah M.; Teis, David; Emr, Scott D.

2011-01-01

Regulated secretion, nutrient uptake, and responses to extracellular signals depend on cell-surface proteins that are internalized and recycled back to the plasma membrane. However, the underlying mechanisms that govern membrane protein recycling to the cell surface are not fully known. Using a chemical-genetic screen in yeast, we show that the arginine transporter Can1 is recycled back to the cell surface via two independent pathways mediated by the sorting nexins Snx4/41/42 and the retromer complex, respectively. In addition, we identify two novel WD40-domain endosomal recycling proteins, Ere1 and Ere2, that function in the retromer pathway. Ere1 is required for Can1 recycling via the retromer-mediated pathway, but it is not required for the transport of other retromer cargoes, such as Vps10 and Ftr1. Biochemical studies reveal that Ere1 physically interacts with internalized Can1. Ere2 is present in a complex containing Ere1 on endosomes and functions as a regulator of Ere1. Taken together, our results suggest that Snx4/41/42 and the retromer comprise two independent pathways for the recycling of internalized cell-surface proteins. Moreover, a complex containing the two novel proteins Ere1 and Ere2 mediates cargo-specific recognition by the retromer pathway. PMID:21880895

Interaction between a plasma membrane-localized ankyrin-repeat protein ITN1 and a nuclear protein RTV1

Energy Technology Data Exchange (ETDEWEB)

Sakamoto, Hikaru [Department of Bioproduction, Faculty of Bioindustry, Tokyo University of Agriculture, 196 Yasaka, Abashiri-shi, Hokkaido 093-2422 (Japan); Sakata, Keiko; Kusumi, Kensuke [Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Kojima, Mikiko; Sakakibara, Hitoshi [RIKEN Plant Science Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045 (Japan); Iba, Koh, E-mail: koibascb@kyushu-u.org [Department of Biology, Faculty of Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan)

2012-06-29

Highlights: Black-Right-Pointing-Pointer ITN1, a plasma membrane ankyrin protein, interacts with a nuclear DNA-binding protein RTV1. Black-Right-Pointing-Pointer The nuclear transport of RTV1 is partially inhibited by interaction with ITN1. Black-Right-Pointing-Pointer RTV1 can promote the nuclear localization of ITN1. Black-Right-Pointing-Pointer Both overexpression of RTV1 and the lack of ITN1 increase salicylic acids sensitivity in plants. -- Abstract: The increased tolerance to NaCl 1 (ITN1) protein is a plasma membrane (PM)-localized protein involved in responses to NaCl stress in Arabidopsis. The predicted structure of ITN1 is composed of multiple transmembrane regions and an ankyrin-repeat domain that is known to mediate protein-protein interactions. To elucidate the molecular functions of ITN1, we searched for interacting partners using a yeast two-hybrid assay, and a nuclear-localized DNA-binding protein, RTV1, was identified as a candidate. Bimolecular fluorescence complementation analysis revealed that RTV1 interacted with ITN1 at the PM and nuclei in vivo. RTV1 tagged with red fluorescent protein localized to nuclei and ITN1 tagged with green fluorescent protein localized to PM; however, both proteins localized to both nuclei and the PM when co-expressed. These findings suggest that RTV1 and ITN1 regulate the subcellular localization of each other.

Genome-wide identification, sequence characterization, and protein-protein interaction properties of DDB1 (damaged DNA binding protein-1)-binding WD40-repeat family members in Solanum lycopersicum.

Science.gov (United States)

Zhu, Yunye; Huang, Shengxiong; Miao, Min; Tang, Xiaofeng; Yue, Junyang; Wang, Wenjie; Liu, Yongsheng

2015-06-01

One hundred DDB1 (damaged DNA binding protein-1)-binding WD40-repeat domain (DWD) family genes were identified in the S. lycopersicum genome. The DWD genes encode proteins presumably functioning as the substrate recognition subunits of the cullin4-ring ubiquitin E3 ligase complex. These findings provide candidate genes and a research platform for further gene functionality and molecular breeding study. A subclass of DDB1 (damaged DNA binding protein-1)-binding WD40-repeat domain (DWD) family proteins has been demonstrated to function as the substrate recognition subunits of the cullin4-ring ubiquitin E3 ligase complex. However, little information is available about the cognate subfamily genes in tomato (S. lycopersicum). In this study, based on the recently released tomato genome sequences, 100 tomato genes encoding DWD proteins that potentially interact with DDB1 were identified and characterized, including analyses of the detailed annotations, chromosome locations and compositions of conserved amino acid domains. In addition, a phylogenetic tree, which comprises of three main groups, of the subfamily genes was constructed. The physical interaction between tomato DDB1 and 14 representative DWD proteins was determined by yeast two-hybrid and co-immunoprecipitation assays. The subcellular localization of these 14 representative DWD proteins was determined. Six of them were localized in both nucleus and cytoplasm, seven proteins exclusively in cytoplasm, and one protein either in nucleus and cytoplasm, or exclusively in cytoplasm. Comparative genomic analysis demonstrated that the expansion of these subfamily members in tomato predominantly resulted from two whole-genome triplication events in the evolution history.

Blow-up for a three dimensional Keller-Segel model with consumption of chemoattractant

Science.gov (United States)

Jiang, Jie; Wu, Hao; Zheng, Songmu

2018-04-01

We investigate blow-up properties for the initial-boundary value problem of a Keller-Segel model with consumption of chemoattractant when the spatial dimension is three. Through a kinetic reformulation of the Keller-Segel system, we first derive some higher-order estimates and obtain certain blow-up criteria for the local classical solutions. These blow-up criteria generalize the results in [4,5] from the whole space R3 to the case of bounded smooth domain Ω ⊂R3. Lower global blow-up estimate on ‖ n ‖ L∞ (Ω) is also obtained based on our higher-order estimates. Moreover, we prove local non-degeneracy for blow-up points.

ATP-binding motifs play key roles in Krp1p, kinesin-related protein 1, function for bi-polar growth control in fission yeast

International Nuclear Information System (INIS)

Rhee, Dong Keun; Cho, Bon A; Kim, Hyong Bai

2005-01-01

Kinesin is a microtubule-based motor protein with various functions related to the cell growth and division. It has been reported that Krp1p, kinesin-related protein 1, which belongs to the kinesin heavy chain superfamily, localizes on microtubules and may play an important role in cytokinesis. However, the function of Krp1p has not been fully elucidated. In this study, we overexpressed an intact form and three different mutant forms of Krp1p in fission yeast constructed by site-directed mutagenesis in two ATP-binding motifs or by truncation of the leucine zipper-like motif (LZiP). We observed hyper-extended microtubules and the aberrant nuclear shape in Krp1p-overexpressed fission yeast. As a functional consequence, a point mutation of ATP-binding domain 1 (G89E) in Krp1p reversed the effect of Krp1p overexpression in fission yeast, whereas the specific mutation in ATP-binding domain 2 (G238E) resulted in the altered cell polarity. Additionally, truncation of the leucine zipper-like domain (LZiP) at the C-terminal of Krp1p showed a normal nuclear division. Taken together, we suggest that krp1p is involved in regulation of cell-polarized growth through ATP-binding motifs in fission yeast

Structure-function of proteins interacting with the alpha1 pore-forming subunit of high voltage-activated calcium channel

Directory of Open Access Journals (Sweden)

Alan eNeely

2014-06-01

Full Text Available Openings of high-voltage-activated calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, high-voltage-activated calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1 associated with four additional polypeptide chains β, α2, δ and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of high-voltage-activated calcium channels.

Structure-function of proteins interacting with the α1 pore-forming subunit of high-voltage-activated calcium channels

Science.gov (United States)

Neely, Alan; Hidalgo, Patricia

2014-01-01

Openings of high-voltage-activated (HVA) calcium channels lead to a transient increase in calcium concentration that in turn activate a plethora of cellular functions, including muscle contraction, secretion and gene transcription. To coordinate all these responses calcium channels form supramolecular assemblies containing effectors and regulatory proteins that couple calcium influx to the downstream signal cascades and to feedback elements. According to the original biochemical characterization of skeletal muscle Dihydropyridine receptors, HVA calcium channels are multi-subunit protein complexes consisting of a pore-forming subunit (α1) associated with four additional polypeptide chains β, α2, δ, and γ, often referred to as accessory subunits. Twenty-five years after the first purification of a high-voltage calcium channel, the concept of a flexible stoichiometry to expand the repertoire of mechanisms that regulate calcium channel influx has emerged. Several other proteins have been identified that associate directly with the α1-subunit, including calmodulin and multiple members of the small and large GTPase family. Some of these proteins only interact with a subset of α1-subunits and during specific stages of biogenesis. More strikingly, most of the α1-subunit interacting proteins, such as the β-subunit and small GTPases, regulate both gating and trafficking through a variety of mechanisms. Modulation of channel activity covers almost all biophysical properties of the channel. Likewise, regulation of the number of channels in the plasma membrane is performed by altering the release of the α1-subunit from the endoplasmic reticulum, by reducing its degradation or enhancing its recycling back to the cell surface. In this review, we discuss the structural basis, interplay and functional role of selected proteins that interact with the central pore-forming subunit of HVA calcium channels. PMID:24917826

Protein phosphatase PPM1G regulates protein translation and cell growth by dephosphorylating 4E binding protein 1 (4E-BP1).

Science.gov (United States)

Liu, Jianyu; Stevens, Payton D; Eshleman, Nichole E; Gao, Tianyan

2013-08-09

Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Serving as one of the most important negative regulators of protein translation, 4E binding protein 1 (4E-BP1) binds to translation initiation factor 4E and inhibits cap-dependent translation in a phosphorylation-dependent manner. Although it has been demonstrated previously that the phosphorylation of 4E-BP1 is controlled by mammalian target of rapamycin in the mammalian target of rapamycin complex 1, the mechanism underlying the dephosphorylation of 4E-BP1 remains elusive. Here, we report the identification of PPM1G as the phosphatase of 4E-BP1. A coimmunoprecipitation experiment reveals that PPM1G binds to 4E-BP1 in cells and that purified PPM1G dephosphorylates 4E-BP1 in vitro. Knockdown of PPM1G in 293E and colon cancer HCT116 cells results in an increase in the phosphorylation of 4E-BP1 at both the Thr-37/46 and Ser-65 sites. Furthermore, the time course of 4E-BP1 dephosphorylation induced by amino acid starvation or mammalian target of rapamycin inhibition is slowed down significantly in PPM1G knockdown cells. Functionally, the amount of 4E-BP1 bound to the cap-dependent translation initiation complex is decreased when the expression of PPM1G is depleted. As a result, the rate of cap-dependent translation, cell size, and protein content are increased in PPM1G knockdown cells. Taken together, our study has identified protein phosphatase PPM1G as a novel regulator of cap-dependent protein translation by negatively controlling the phosphorylation of 4E-BP1.

Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses1[OPEN

Science.gov (United States)

Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; Munemasa, Shintaro; Booker, Matthew A.; Brandt, Benjamin; Waadt, Christian; Nusinow, Dmitri A.; Kay, Steve A.; Kunz, Hans-Henning; Schumacher, Karin; DeLong, Alison; Yates, John R.; Schroeder, Julian I.

2015-01-01

The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases. PMID:26175513

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis.

Science.gov (United States)

Du, Yushen; Wu, Nicholas C; Jiang, Lin; Zhang, Tianhao; Gong, Danyang; Shu, Sara; Wu, Ting-Ting; Sun, Ren

2016-11-01

Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp), we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available. To fully comprehend the diverse functions of a protein, it is essential to understand the functionality of individual residues. Current methods are highly dependent on evolutionary sequence conservation, which is

Hypothesis: NDL proteins function in stress responses by regulating microtubule organization.

Science.gov (United States)

Khatri, Nisha; Mudgil, Yashwanti

2015-01-01

N-MYC DOWNREGULATED-LIKE proteins (NDL), members of the alpha/beta hydrolase superfamily were recently rediscovered as interactors of G-protein signaling in Arabidopsis thaliana. Although the precise molecular function of NDL proteins is still elusive, in animals these proteins play protective role in hypoxia and expression is induced by hypoxia and nickel, indicating role in stress. Homology of NDL1 with animal counterpart N-MYC DOWNREGULATED GENE (NDRG) suggests similar functions in animals and plants. It is well established that stress responses leads to the microtubule depolymerization and reorganization which is crucial for stress tolerance. NDRG is a microtubule-associated protein which mediates the microtubule organization in animals by causing acetylation and increases the stability of α-tubulin. As NDL1 is highly homologous to NDRG, involvement of NDL1 in the microtubule organization during plant stress can also be expected. Discovery of interaction of NDL with protein kinesin light chain- related 1, enodomembrane family protein 70, syntaxin-23, tubulin alpha-2 chain, as a part of G protein interactome initiative encourages us to postulate microtubule stabilizing functions for NDL family in plants. Our search for NDL interactors in G protein interactome also predicts the role of NDL proteins in abiotic stress tolerance management. Based on published report in animals and predicted interacting partners for NDL in G protein interactome lead us to hypothesize involvement of NDL in the microtubule organization during abiotic stress management in plants.

Broadening the functionality of a J-protein/Hsp70 molecular chaperone system.

Science.gov (United States)

Schilke, Brenda A; Ciesielski, Szymon J; Ziegelhoffer, Thomas; Kamiya, Erina; Tonelli, Marco; Lee, Woonghee; Cornilescu, Gabriel; Hines, Justin K; Markley, John L; Craig, Elizabeth A

2017-10-01

By binding to a multitude of polypeptide substrates, Hsp70-based molecular chaperone systems perform a range of cellular functions. All J-protein co-chaperones play the essential role, via action of their J-domains, of stimulating the ATPase activity of Hsp70, thereby stabilizing its interaction with substrate. In addition, J-proteins drive the functional diversity of Hsp70 chaperone systems through action of regions outside their J-domains. Targeting to specific locations within a cellular compartment and binding of specific substrates for delivery to Hsp70 have been identified as modes of J-protein specialization. To better understand J-protein specialization, we concentrated on Saccharomyces cerevisiae SIS1, which encodes an essential J-protein of the cytosol/nucleus. We selected suppressors that allowed cells lacking SIS1 to form colonies. Substitutions changing single residues in Ydj1, a J-protein, which, like Sis1, partners with Hsp70 Ssa1, were isolated. These gain-of-function substitutions were located at the end of the J-domain, suggesting that suppression was connected to interaction with its partner Hsp70, rather than substrate binding or subcellular localization. Reasoning that, if YDJ1 suppressors affect Ssa1 function, substitutions in Hsp70 itself might also be able to overcome the cellular requirement for Sis1, we carried out a selection for SSA1 suppressor mutations. Suppressing substitutions were isolated that altered sites in Ssa1 affecting the cycle of substrate interaction. Together, our results point to a third, additional means by which J-proteins can drive Hsp70's ability to function in a wide range of cellular processes-modulating the Hsp70-substrate interaction cycle.

Purification of recombinant Chlamydia trachomatis histone H1-like protein Hc2, and comparative functional analysis of Hc2 and Hc1

DEFF Research Database (Denmark)

Pedersen, LB; Birkelund, Svend; Christiansen, Gunna

1996-01-01

The metabolically inactive developmental form of Chlamydia trachomatis, the elementary body, contains two very basic DNA-binding proteins with homology to eukaryotic histone H1. One of these, Hc1, is relatively well characterized and induces DNA condensation in vitro, whereas the other, Hc2......, is functionally virtually uncharacterized. In this study we describe the purification of Hc2, and a detailed comparative functional analysis of Hc2 and Hc1 is presented. By gel shift assays and electron microscopy, marked differences in the nucleic acid-binding properties of Hc2 and Hc1 were observed. Furthermore...

The F-box protein Fbp1 functions in the invasive growth and cell wall integrity mitogen-activated protein kinase (MAPK) pathways in Fusarium oxysporum.

Science.gov (United States)

Miguel-Rojas, Cristina; Hera, Concepcion

2016-01-01

F-box proteins determine substrate specificity of the ubiquitin-proteasome system. Previous work has demonstrated that the F-box protein Fbp1, a component of the SCF(Fbp1) E3 ligase complex, is essential for invasive growth and virulence of the fungal plant pathogen Fusarium oxysporum. Here, we show that, in addition to invasive growth, Fbp1 also contributes to vegetative hyphal fusion and fungal adhesion to tomato roots. All of these functions have been shown previously to require the mitogen-activated protein kinase (MAPK) Fmk1. We found that Fbp1 is required for full phosphorylation of Fmk1, indicating that Fbp1 regulates virulence and invasive growth via the Fmk1 pathway. Moreover, the Δfbp1 mutant is hypersensitive to sodium dodecylsulfate (SDS) and calcofluor white (CFW) and shows reduced phosphorylation levels of the cell wall integrity MAPK Mpk1 after SDS treatment. Collectively, these results suggest that Fbp1 contributes to both the invasive growth and cell wall integrity MAPK pathways of F. oxysporum. © 2015 BSPP AND JOHN WILEY & SONS LTD.

Only Acyl Carrier Protein 1 (AcpP1 Functions in Pseudomonas aeruginosa Fatty Acid Synthesis

Directory of Open Access Journals (Sweden)

Jin-Cheng Ma

2017-11-01

Full Text Available The genome of Pseudomonas aeruginosa contains three open reading frames, PA2966, PA1869, and PA3334, which encode putative acyl carrier proteins, AcpP1, AcpP2, and AcpP3, respectively. In this study, we found that, although these apo-ACPs were successfully phosphopantetheinylated by P. aeruginosa phosphopantetheinyl transferase (PcpS and all holo-forms of these proteins could be acylated by Vibrio harveyi acyl-ACP synthetase (AasS, only AcpP1 could be used as a substrate for the synthesis of fatty acids, catalyzed by P. aeruginosa cell free extracts in vitro, and only acpP1 gene could restore growth in the Escherichia coliacpP mutant strain CY1877. And P. aeruginosaacpP1 could not be deleted, while disruption of acpP2 or acpP3 in the P. aeruginosa genome allowed mutant strains to grow as well as the wild type strain. These findings confirmed that only P. aeruginosa AcpP1 functions in fatty acid biosynthesis, and that acpP2 and acpP3 do not play roles in the fatty acid synthetic pathway. Moreover, disruption of acpP2 and acpP3 did not affect the ability of P. aeruginosa to produce N-acylhomoserine lactones (AHL, but replacement of P. aeruginosaacpP1 with E. coliacpP caused P. aeruginosa to reduce the production of AHL molecules, which indicated that neither P. aeruginosa AcpP2 nor AcpP3 can act as a substrate for synthesis of AHL molecules in vivo. Furthermore, replacement of acpP1 with E. coliacpP reduced the ability of P. aeruginosa to produce some exo-products and abolished swarming motility in P. aeruginosa.

AVID: An integrative framework for discovering functional relationships among proteins

Directory of Open Access Journals (Sweden)

Keating Amy E

2005-06-01

Full Text Available Abstract Background Determining the functions of uncharacterized proteins is one of the most pressing problems in the post-genomic era. Large scale protein-protein interaction assays, global mRNA expression analyses and systematic protein localization studies provide experimental information that can be used for this purpose. The data from such experiments contain many false positives and false negatives, but can be processed using computational methods to provide reliable information about protein-protein relationships and protein function. An outstanding and important goal is to predict detailed functional annotation for all uncharacterized proteins that is reliable enough to effectively guide experiments. Results We present AVID, a computational method that uses a multi-stage learning framework to integrate experimental results with sequence information, generating networks reflecting functional similarities among proteins. We illustrate use of the networks by making predictions of detailed Gene Ontology (GO annotations in three categories: molecular function, biological process, and cellular component. Applied to the yeast Saccharomyces cerevisiae, AVID provides 37,451 pair-wise functional linkages between 4,191 proteins. These relationships are ~65–78% accurate, as assessed by cross-validation testing. Assignments of highly detailed functional descriptors to proteins, based on the networks, are estimated to be ~67% accurate for GO categories describing molecular function and cellular component and ~52% accurate for terms describing biological process. The predictions cover 1,490 proteins with no previous annotation in GO and also assign more detailed functions to many proteins annotated only with less descriptive terms. Predictions made by AVID are largely distinct from those made by other methods. Out of 37,451 predicted pair-wise relationships, the greatest number shared in common with another method is 3,413. Conclusion AVID provides

Inferring the Functions of Proteins from the Interrelationships between Functional Categories.

Science.gov (United States)

Taha, Kamal

2018-01-01

This study proposes a new method to determine the functions of an unannotated protein. The proteins and amino acid residues mentioned in biomedical texts associated with an unannotated protein can be considered as characteristics terms for , which are highly predictive of the potential functions of . Similarly, proteins and amino acid residues mentioned in biomedical texts associated with proteins annotated with a functional category can be considered as characteristics terms of . We introduce in this paper an information extraction system called IFP_IFC that predicts the functions of an unannotated protein by representing and each functional category by a vector of weights. Each weight reflects the degree of association between a characteristic term and (or a characteristic term and ). First, IFP_IFC constructs a network, whose nodes represent the different functional categories, and its edges the interrelationships between the nodes. Then, it determines the functions of by employing random walks with restarts on the mentioned network. The walker is the vector of . Finally, is assigned to the functional categories of the nodes in the network that are visited most by the walker. We evaluated the quality of IFP_IFC by comparing it experimentally with two other systems. Results showed marked improvement.

Exploring protein dynamics space: the dynasome as the missing link between protein structure and function.

Directory of Open Access Journals (Sweden)

Ulf Hensen

Full Text Available Proteins are usually described and classified according to amino acid sequence, structure or function. Here, we develop a minimally biased scheme to compare and classify proteins according to their internal mobility patterns. This approach is based on the notion that proteins not only fold into recurring structural motifs but might also be carrying out only a limited set of recurring mobility motifs. The complete set of these patterns, which we tentatively call the dynasome, spans a multi-dimensional space with axes, the dynasome descriptors, characterizing different aspects of protein dynamics. The unique dynamic fingerprint of each protein is represented as a vector in the dynasome space. The difference between any two vectors, consequently, gives a reliable measure of the difference between the corresponding protein dynamics. We characterize the properties of the dynasome by comparing the dynamics fingerprints obtained from molecular dynamics simulations of 112 proteins but our approach is, in principle, not restricted to any specific source of data of protein dynamics. We conclude that: 1. the dynasome consists of a continuum of proteins, rather than well separated classes. 2. For the majority of proteins we observe strong correlations between structure and dynamics. 3. Proteins with similar function carry out similar dynamics, which suggests a new method to improve protein function annotation based on protein dynamics.

Annotating Protein Functional Residues by Coupling High-Throughput Fitness Profile and Homologous-Structure Analysis

Directory of Open Access Journals (Sweden)

Yushen Du

2016-11-01

Full Text Available Identification and annotation of functional residues are fundamental questions in protein sequence analysis. Sequence and structure conservation provides valuable information to tackle these questions. It is, however, limited by the incomplete sampling of sequence space in natural evolution. Moreover, proteins often have multiple functions, with overlapping sequences that present challenges to accurate annotation of the exact functions of individual residues by conservation-based methods. Using the influenza A virus PB1 protein as an example, we developed a method to systematically identify and annotate functional residues. We used saturation mutagenesis and high-throughput sequencing to measure the replication capacity of single nucleotide mutations across the entire PB1 protein. After predicting protein stability upon mutations, we identified functional PB1 residues that are essential for viral replication. To further annotate the functional residues important to the canonical or noncanonical functions of viral RNA-dependent RNA polymerase (vRdRp, we performed a homologous-structure analysis with 16 different vRdRp structures. We achieved high sensitivity in annotating the known canonical polymerase functional residues. Moreover, we identified a cluster of noncanonical functional residues located in the loop region of the PB1 β-ribbon. We further demonstrated that these residues were important for PB1 protein nuclear import through the interaction with Ran-binding protein 5. In summary, we developed a systematic and sensitive method to identify and annotate functional residues that are not restrained by sequence conservation. Importantly, this method is generally applicable to other proteins about which homologous-structure information is available.

Structural and Functional Analysis of VQ Motif-Containing Proteins in Arabidopsis as Interacting Proteins of WRKY Transcription Factors1[W][OA

Science.gov (United States)

Cheng, Yuan; Zhou, Yuan; Yang, Yan; Chi, Ying-Jun; Zhou, Jie; Chen, Jian-Ye; Wang, Fei; Fan, Baofang; Shi, Kai; Zhou, Yan-Hong; Yu, Jing-Quan; Chen, Zhixiang

2012-01-01

WRKY transcription factors are encoded by a large gene superfamily with a broad range of roles in plants. Recently, several groups have reported that proteins containing a short VQ (FxxxVQxLTG) motif interact with WRKY proteins. We have recently discovered that two VQ proteins from Arabidopsis (Arabidopsis thaliana), SIGMA FACTOR-INTERACTING PROTEIN1 and SIGMA FACTOR-INTERACTING PROTEIN2, act as coactivators of WRKY33 in plant defense by specifically recognizing the C-terminal WRKY domain and stimulating the DNA-binding activity of WRKY33. In this study, we have analyzed the entire family of 34 structurally divergent VQ proteins from Arabidopsis. Yeast (Saccharomyces cerevisiae) two-hybrid assays showed that Arabidopsis VQ proteins interacted specifically with the C-terminal WRKY domains of group I and the sole WRKY domains of group IIc WRKY proteins. Using site-directed mutagenesis, we identified structural features of these two closely related groups of WRKY domains that are critical for interaction with VQ proteins. Quantitative reverse transcription polymerase chain reaction revealed that expression of a majority of Arabidopsis VQ genes was responsive to pathogen infection and salicylic acid treatment. Functional analysis using both knockout mutants and overexpression lines revealed strong phenotypes in growth, development, and susceptibility to pathogen infection. Altered phenotypes were substantially enhanced through cooverexpression of genes encoding interacting VQ and WRKY proteins. These findings indicate that VQ proteins play an important role in plant growth, development, and response to environmental conditions, most likely by acting as cofactors of group I and IIc WRKY transcription factors. PMID:22535423

Function of heterochromatin protein 1 during DNA repair

Czech Academy of Sciences Publication Activity Database

Bártová, Eva; Malyšková, Barbora; Komůrková, Denisa; Legartová, Soňa; Suchánková, Jana; Krejčí, Jana; Kozubek, Stanislav

2017-01-01

Roč. 254, č. 3 (2017), s. 1233-1240 ISSN 0033-183X R&D Projects: GA ČR GBP302/12/G157; GA MŠk 7F14369 Institutional support: RVO:68081707 Keywords : double-strand breaks * damage response * HP1 protein Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Cell biology Impact factor: 2.870, year: 2016

Molecular Characterization and Functional Analysis of PR-1-Like Proteins Identified from the Wheat Head Blight Fungus Fusarium graminearum.

Science.gov (United States)

Lu, Shunwen; Edwards, Michael C

2018-04-01

The group 1 pathogenesis-related (PR-1) proteins originally identified from plants and their homologs are also found in other eukaryotic kingdoms. Studies on nonplant PR-1-like (PR-1L) proteins have been pursued widely in humans and animals but rarely in filamentous ascomycetes. Here, we report the characterization of four PR-1L proteins identified from the ascomycete fungus Fusarium graminearum, the primary cause of Fusarium head blight of wheat and barley (designated FgPR-1L). Molecular cloning revealed that the four FgPR-1L proteins are all encoded by small open reading frames (612 to 909 bp) that are often interrupted by introns, in contrast to plant PR-1 genes that lack introns. Sequence analysis indicated that all FgPR-1L proteins contain the PR-1-specific three-dimensional structure, and one of them features a C-terminal transmembrane (TM) domain that has not been reported for any stand-alone PR-1 proteins. Transcriptional analysis revealed that the four FgPR-1L genes are expressed in axenic cultures and in planta with different spatial or temporal expression patterns. Phylogenetic analysis indicated that fungal PR-1L proteins fall into three major groups, one of which harbors FgPR-1L-2-related TM-containing proteins from both phytopathogenic and human-pathogenic ascomycetes. Low-temperature sodium dodecyl sulfate polyacrylamide gel electrophoresis and proteolytic assays indicated that the recombinant FgPR-1L-4 protein exists as a monomer and is resistant to subtilisin of the serine protease family. Functional analysis confirmed that deletion of the FgPR-1L-4 gene from the fungal genome results in significantly reduced virulence on susceptible wheat. This study provides the first example that the F. graminearum-wheat interaction involves a pathogen-derived PR-1L protein that affects fungal virulence on the host.

Multidrug resistance-associated protein-1 (MRP1 genetic variants, MRP1 protein levels and severity of COPD

Directory of Open Access Journals (Sweden)

Rutgers Bea

2010-05-01

Full Text Available Abstract Background Multidrug resistance-associated protein-1 (MRP1 protects against oxidative stress and toxic compounds generated by cigarette smoking, which is the main risk factor for chronic obstructive pulmonary disease (COPD. We have previously shown that single nucleotide polymorphisms (SNPs in MRP1 significantly associate with level of FEV1 in two independent population based cohorts. The aim of our study was to assess the associations of MRP1 SNPs with FEV1 level, MRP1 protein levels and inflammatory markers in bronchial biopsies and sputum of COPD patients. Methods Five SNPs (rs212093, rs4148382, rs504348, rs4781699, rs35621 in MRP1 were genotyped in 110 COPD patients. The effects of MRP1 SNPs were analyzed using linear regression models. Results One SNP, rs212093 was significantly associated with a higher FEV1 level and less airway wall inflammation. Another SNP, rs4148382 was significantly associated with a lower FEV1 level, higher number of inflammatory cells in induced sputum and with a higher MRP1 protein level in bronchial biopsies. Conclusions This is the first study linking MRP1 SNPs with lung function and inflammatory markers in COPD patients, suggesting a role of MRP1 SNPs in the severity of COPD in addition to their association with MRP1 protein level in bronchial biopsies.

Activation of the polyomavirus enhancer by a murine activator protein 1 (AP1) homolog and two contiguous proteins.

OpenAIRE

Martin, M E; Piette, J; Yaniv, M; Tang, W J; Folk, W R

1988-01-01

The polyomavirus enhancer is composed of multiple DNA sequence elements serving as binding sites for proteins present in mouse nuclear extracts that activate transcription and DNA replication. We have identified three such proteins and their binding sites and correlate them with enhancer function. Mutation of nucleotide (nt) 5140 in the enhancer alters the binding site (TGACTAA, nt 5139-5145) for polyomavirus enhancer A binding protein 1 (PEA1), a murine homolog of the human transcription fac...

Hypothesis: NDL proteins function in stress responses by regulating microtubule organization

OpenAIRE

Khatri, Nisha; Mudgil, Yashwanti

2015-01-01

N-MYC DOWNREGULATED-LIKE proteins (NDL), members of the alpha/beta hydrolase superfamily were recently rediscovered as interactors of G-protein signaling in Arabidopsis thaliana. Although the precise molecular function of NDL proteins is still elusive, in animals these proteins play protective role in hypoxia and expression is induced by hypoxia and nickel, indicating role in stress. Homology of NDL1 with animal counterpart N-MYC DOWNREGULATED GENE (NDRG) suggests similar functions in animals...

Structural and Function Prediction of Musa acuminata subsp. Malaccensis Protein

Directory of Open Access Journals (Sweden)

Anum Munir

2016-03-01

Full Text Available Hypothetical proteins (HPs are the proteins whose presence has been anticipated, yet in vivo function has not been built up. Illustrating the structural and functional privileged insights of these HPs might likewise prompt a superior comprehension of the protein-protein associations or networks in diverse types of life. Bananas (Musa acuminata spp., including sweet and cooking types, are giant perennial monocotyledonous herbs of the order Zingiberales, a sister grouped to the all-around considered Poales, which incorporate oats. Bananas are crucial for nourishment security in numerous tropical and subtropical nations and the most prominent organic product in industrialized nations. In the present study, the hypothetical protein of M. acuminata (Banana was chosen for analysis and modeling by distinctive bioinformatics apparatuses and databases. As indicated by primary and secondary structure analysis, XP_009393594.1 is a stable hydrophobic protein containing a noteworthy extent of α-helices; Homology modeling was done utilizing SWISS-MODEL server where the templates identity with XP_009393594.1 protein was less which demonstrated novelty of our protein. Ab initio strategy was conducted to produce its 3D structure. A few evaluations of quality assessment and validation parameters determined the generated protein model as stable with genuinely great quality. Functional analysis was completed by ProtFun 2.2, and KEGG (KAAS, recommended that the hypothetical protein is a transcription factor with cytoplasmic domain as zinc finger. The protein was observed to be vital for translation process, involved in metabolism, signaling and cellular processes, genetic information processing and Zinc ion binding. It is suggested that further test approval would help to anticipate the structures and functions of other uncharacterized proteins of different plants and living being.

UNC-89 (obscurin) binds to MEL-26, a BTB-domain protein, and affects the function of MEI-1 (katanin) in striated muscle of Caenorhabditis elegans.

Science.gov (United States)

Wilson, Kristy J; Qadota, Hiroshi; Mains, Paul E; Benian, Guy M

2012-07-01

The ubiquitin proteasome system is involved in degradation of old or damaged sarcomeric proteins. Most E3 ubiquitin ligases are associated with cullins, which function as scaffolds for assembly of the protein degradation machinery. Cullin 3 uses an adaptor to link to substrates; in Caenorhabditis elegans, one of these adaptors is the BTB-domain protein MEL-26 (maternal effect lethal). Here we show that MEL-26 interacts with the giant sarcomeric protein UNC-89 (obscurin). MEL-26 and UNC-89 partially colocalize at sarcomeric M-lines. Loss of function or gain of function of mel-26 results in disorganization of myosin thick filaments similar to that found in unc-89 mutants. It had been reported that in early C. elegans embryos, a target of the CUL-3/MEL-26 ubiquitylation complex is the microtubule-severing enzyme katanin (MEI-1). Loss of function or gain of function of mei-1 also results in disorganization of thick filaments similar to unc-89 mutants. Genetic data indicate that at least some of the mel-26 loss-of-function phenotype in muscle can be attributed to increased microtubule-severing activity of MEI-1. The level of MEI-1 protein is reduced in an unc-89 mutant, suggesting that the normal role of UNC-89 is to inhibit the CUL-3/MEL-26 complex toward MEI-1.

Structuring detergents for extracting and stabilizing functional membrane proteins.

Directory of Open Access Journals (Sweden)

Rima Matar-Merheb

Full Text Available BACKGROUND: Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. METHODOLOGY/PRINCIPAL FINDINGS: Anionic calix[4]arene based detergents (C4Cn, n=1-12 were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein, a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM. They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux much more efficiently than SDS (sodium dodecyl sulphate, FC12 (Foscholine 12 or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. CONCLUSION/SIGNIFICANCE: These compounds seem promising to extract in a functional state

Identification of functional candidates amongst hypothetical proteins of Treponema pallidum ssp. pallidum.

Science.gov (United States)

Naqvi, Ahmad Abu Turab; Shahbaaz, Mohd; Ahmad, Faizan; Hassan, Md Imtaiyaz

2015-01-01

Syphilis is a globally occurring venereal disease, and its infection is propagated through sexual contact. The causative agent of syphilis, Treponema pallidum ssp. pallidum, a Gram-negative sphirochaete, is an obligate human parasite. Genome of T. pallidum ssp. pallidum SS14 strain (RefSeq NC_010741.1) encodes 1,027 proteins, of which 444 proteins are known as hypothetical proteins (HPs), i.e., proteins of unknown functions. Here, we performed functional annotation of HPs of T. pallidum ssp. pallidum using various database, domain architecture predictors, protein function annotators and clustering tools. We have analyzed the sequences of 444 HPs of T. pallidum ssp. pallidum and subsequently predicted the function of 207 HPs with a high level of confidence. However, functions of 237 HPs are predicted with less accuracy. We found various enzymes, transporters, binding proteins in the annotated group of HPs that may be possible molecular targets, facilitating for the survival of pathogen. Our comprehensive analysis helps to understand the mechanism of pathogenesis to provide many novel potential therapeutic interventions.

Protein Functionalized Nanodiamond Arrays

Directory of Open Access Journals (Sweden)

Liu YL

2010-01-01

Full Text Available Abstract Various nanoscale elements are currently being explored for bio-applications, such as in bio-images, bio-detection, and bio-sensors. Among them, nanodiamonds possess remarkable features such as low bio-cytotoxicity, good optical property in fluorescent and Raman spectra, and good photostability for bio-applications. In this work, we devise techniques to position functionalized nanodiamonds on self-assembled monolayer (SAMs arrays adsorbed on silicon and ITO substrates surface using electron beam lithography techniques. The nanodiamond arrays were functionalized with lysozyme to target a certain biomolecule or protein specifically. The optical properties of the nanodiamond-protein complex arrays were characterized by a high throughput confocal microscope. The synthesized nanodiamond-lysozyme complex arrays were found to still retain their functionality in interacting with E. coli.

Intracellular Transport and Kinesin Superfamily Proteins: Structure, Function and Dynamics

Science.gov (United States)

Hirokawa, N.; Takemura, R.

Using various molecular cell biological and molecular genetic approaches, we identified kinesin superfamily proteins (KIFs) and characterized their significant functions in intracellular transport, which is fundamental for cellular morphogenesis, functioning, and survival. We showed that KIFs not only transport various membranous organelles, proteins complexes and mRNAs fundamental for cellular functions but also play significant roles in higher brain functions such as memory and learning, determination of important developmental processes such as left-right asymmetry formation and brain wiring. We also elucidated that KIFs recognize and bind to their specific cargoes using scaffolding or adaptor protein complexes. Concerning the mechanism of motility, we discovered the simplest unique monomeric motor KIF1A and determined by molecular biophysics, cryoelectron microscopy and X-ray crystallography that KIF1A can move on a microtubule processively as a monomer by biased Brownian motion and by hydolyzing ATP.

Identification of novel putative-binding proteins for cellular prion protein and a specific interaction with the STIP1 homology and U-Box-containing protein 1

Science.gov (United States)

Gimenez, Ana Paula Lappas; Richter, Larissa Morato Luciani; Atherino, Mariana Campos; Beirão, Breno Castello Branco; Fávaro, Celso; Costa, Michele Dietrich Moura; Zanata, Silvio Marques; Malnic, Bettina; Mercadante, Adriana Frohlich

2015-01-01

ABSTRACT Prion diseases involve the conversion of the endogenous cellular prion protein, PrPC, into a misfolded infectious isoform, PrPSc. Several functions have been attributed to PrPC, and its role has also been investigated in the olfactory system. PrPC is expressed in both the olfactory bulb (OB) and olfactory epithelium (OE) and the nasal cavity is an important route of transmission of diseases caused by prions. Moreover, Prnp−/− mice showed impaired behavior in olfactory tests. Given the high PrPC expression in OE and its putative role in olfaction, we screened a mouse OE cDNA library to identify novel PrPC-binding partners. Ten different putative PrPC ligands were identified, which were involved in functions such as cellular proliferation and apoptosis, cytoskeleton and vesicle transport, ubiquitination of proteins, stress response, and other physiological processes. In vitro binding assays confirmed the interaction of PrPC with STIP1 homology and U-Box containing protein 1 (Stub1) and are reported here for the first time. Stub1 is a co-chaperone with ubiquitin E3-ligase activity, which is associated with neurodegenerative diseases characterized by protein misfolding and aggregation. Physiological and pathological implications of PrPC-Stub1 interaction are under investigation. The PrPC-binding proteins identified here are not exclusive to the OE, suggesting that these interactions may occur in other tissues and play general biological roles. These data corroborate the proposal that PrPC is part of a multiprotein complex that modulates several cellular functions and provide a platform for further studies on the physiological and pathological roles of prion protein. PMID:26237451

Herpes Simplex Virus Type 1 Infects Enteric Neurons and Triggers Gut Dysfunction via Macrophage Recruitment.

Science.gov (United States)

Brun, Paola; Qesari, Marsela; Marconi, Peggy C; Kotsafti, Andromachi; Porzionato, Andrea; Macchi, Veronica; Schwendener, Reto A; Scarpa, Marco; Giron, Maria C; Palù, Giorgio; Calistri, Arianna; Castagliuolo, Ignazio

2018-01-01

Herpes Simplex Virus type 1 (HSV-1), a neurotropic pathogen widespread in human population, infects the enteric nervous system (ENS) in humans and rodents and causes intestinal neuromuscular dysfunction in rats. Although infiltration of inflammatory cells in the myenteric plexus and neurodegeneration of enteric nerves are common features of patients suffering from functional intestinal disorders, the proof of a pathogenic link with HSV-1 is still unsettled mainly because the underlying mechanisms are largely unknown. In this study we demonstrated that following intragastrical administration HSV-1 infects neurons within the myenteric plexus resulting in functional and structural alterations of the ENS. By infecting mice with HSV-1 replication-defective strain we revealed that gastrointestinal neuromuscular anomalies were however independent of viral replication. Indeed, enteric neurons exposed to UV-inactivated HSV-1 produced monocyte chemoattractant protein-1 (MCP-1/CCL2) to recruit activated macrophages in the longitudinal muscle myenteric plexus. Infiltrating macrophages produced reactive oxygen and nitrogen species and directly harmed enteric neurons resulting in gastrointestinal dysmotility. In HSV-1 infected mice intestinal neuromuscular dysfunctions were ameliorated by in vivo administration of (i) liposomes containing dichloromethylene bisphosphonic acid (clodronate) to deplete tissue macrophages, (ii) CCR2 chemokine receptor antagonist RS504393 to block the CCL2/CCR2 pathway, (iii) Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and AR-C 102222 to quench production of nitrogen reactive species produced via iNOS. Overall these data demonstrate that HSV-1 infection makes enteric neurons recruit macrophages via production of a specific chemoattractant factor. The resulting inflammatory reaction is mandatory for intestinal dysmotility. These findings provide insights into the neuro-immune communication that occurs in the ENS following HSV-1 infection

Herpes Simplex Virus Type 1 Infects Enteric Neurons and Triggers Gut Dysfunction via Macrophage Recruitment

Directory of Open Access Journals (Sweden)

Paola Brun

2018-03-01

Full Text Available Herpes Simplex Virus type 1 (HSV-1, a neurotropic pathogen widespread in human population, infects the enteric nervous system (ENS in humans and rodents and causes intestinal neuromuscular dysfunction in rats. Although infiltration of inflammatory cells in the myenteric plexus and neurodegeneration of enteric nerves are common features of patients suffering from functional intestinal disorders, the proof of a pathogenic link with HSV-1 is still unsettled mainly because the underlying mechanisms are largely unknown. In this study we demonstrated that following intragastrical administration HSV-1 infects neurons within the myenteric plexus resulting in functional and structural alterations of the ENS. By infecting mice with HSV-1 replication-defective strain we revealed that gastrointestinal neuromuscular anomalies were however independent of viral replication. Indeed, enteric neurons exposed to UV-inactivated HSV-1 produced monocyte chemoattractant protein-1 (MCP-1/CCL2 to recruit activated macrophages in the longitudinal muscle myenteric plexus. Infiltrating macrophages produced reactive oxygen and nitrogen species and directly harmed enteric neurons resulting in gastrointestinal dysmotility. In HSV-1 infected mice intestinal neuromuscular dysfunctions were ameliorated by in vivo administration of (i liposomes containing dichloromethylene bisphosphonic acid (clodronate to deplete tissue macrophages, (ii CCR2 chemokine receptor antagonist RS504393 to block the CCL2/CCR2 pathway, (iii Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME and AR-C 102222 to quench production of nitrogen reactive species produced via iNOS. Overall these data demonstrate that HSV-1 infection makes enteric neurons recruit macrophages via production of a specific chemoattractant factor. The resulting inflammatory reaction is mandatory for intestinal dysmotility. These findings provide insights into the neuro-immune communication that occurs in the ENS following HSV-1

Proteomic analysis of the herpes simplex virus 1 virion protein 16 transactivator protein in infected cells.

Science.gov (United States)

Suk, Hyung; Knipe, David M

2015-06-01

The herpes simplex virus 1 virion protein 16 (VP16) tegument protein forms a transactivation complex with the cellular proteins host cell factor 1 (HCF-1) and octamer-binding transcription factor 1 (Oct-1) upon entry into the host cell. VP16 has also been shown to interact with a number of virion tegument proteins and viral glycoprotein H to promote viral assembly, but no comprehensive study of the VP16 proteome has been performed at early times postinfection. We therefore performed a proteomic analysis of VP16-interacting proteins at 3 h postinfection. We confirmed the interaction of VP16 with HCF-1 and a large number of cellular Mediator complex proteins, but most surprisingly, we found that the major viral protein associating with VP16 is the infected cell protein 4 (ICP4) immediate-early (IE) transactivator protein. These results raise the potential for a new function for VP16 in associating with the IE ICP4 and playing a role in transactivation of early and late gene expression, in addition to its well-documented function in transactivation of IE gene expression. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

UET: a database of evolutionarily-predicted functional determinants of protein sequences that cluster as functional sites in protein structures.

Science.gov (United States)

Lua, Rhonald C; Wilson, Stephen J; Konecki, Daniel M; Wilkins, Angela D; Venner, Eric; Morgan, Daniel H; Lichtarge, Olivier

2016-01-04

The structure and function of proteins underlie most aspects of biology and their mutational perturbations often cause disease. To identify the molecular determinants of function as well as targets for drugs, it is central to characterize the important residues and how they cluster to form functional sites. The Evolutionary Trace (ET) achieves this by ranking the functional and structural importance of the protein sequence positions. ET uses evolutionary distances to estimate functional distances and correlates genotype variations with those in the fitness phenotype. Thus, ET ranks are worse for sequence positions that vary among evolutionarily closer homologs but better for positions that vary mostly among distant homologs. This approach identifies functional determinants, predicts function, guides the mutational redesign of functional and allosteric specificity, and interprets the action of coding sequence variations in proteins, people and populations. Now, the UET database offers pre-computed ET analyses for the protein structure databank, and on-the-fly analysis of any protein sequence. A web interface retrieves ET rankings of sequence positions and maps results to a structure to identify functionally important regions. This UET database integrates several ways of viewing the results on the protein sequence or structure and can be found at http://mammoth.bcm.tmc.edu/uet/. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

An early function of the adenoviral E1B 55 kDa protein is required for the nuclear relocalization of the cellular p53 protein in adenovirus-infected normal human cells

International Nuclear Information System (INIS)

Cardoso, F.M.; Kato, Sayuri E.M.; Huang Wenying; Flint, S. Jane; Gonzalez, Ramon A.

2008-01-01

It is well established that the human subgroup C adenovirus type 5 (Ad5) E1B 55 kDa protein can regulate the activity and concentration of the cellular tumor suppressor, p53. However, the contribution(s) of these functions of the E1B protein to viral reproduction remains unclear. To investigate this issue, we examined properties of p53 in normal human cells infected by E1B mutant viruses that display defective entry into the late phase or viral late mRNA export. The steady-state concentrations of p53 were significantly higher in cells infected by the E1B 55 kDa null mutant Hr6 or three mutants carrying small insertions in the E1B 55 kDa protein coding sequence than in Ad5-infected cells. Nevertheless, none of the mutants induced apoptosis in infected cells. Rather, the localization of p53 to E1B containing nuclear sites observed during infection by Ad5 was prevented by mutations that impair interaction of the E1B protein with p53 and/or with the E4 Orf6 protein. These results indicate that the E1B protein fulfills an early function that correlates efficient entry into the late phase with the localization of E1B and p53 in the nucleus of Ad5-infected normal human cells

MM-ISMSA: An Ultrafast and Accurate Scoring Function for Protein-Protein Docking.

Science.gov (United States)

Klett, Javier; Núñez-Salgado, Alfonso; Dos Santos, Helena G; Cortés-Cabrera, Álvaro; Perona, Almudena; Gil-Redondo, Rubén; Abia, David; Gago, Federico; Morreale, Antonio

2012-09-11

An ultrafast and accurate scoring function for protein-protein docking is presented. It includes (1) a molecular mechanics (MM) part based on a 12-6 Lennard-Jones potential; (2) an electrostatic component based on an implicit solvent model (ISM) with individual desolvation penalties for each partner in the protein-protein complex plus a hydrogen bonding term; and (3) a surface area (SA) contribution to account for the loss of water contacts upon protein-protein complex formation. The accuracy and performance of the scoring function, termed MM-ISMSA, have been assessed by (1) comparing the total binding energies, the electrostatic term, and its components (charge-charge and individual desolvation energies), as well as the per residue contributions, to results obtained with well-established methods such as APBSA or MM-PB(GB)SA for a set of 1242 decoy protein-protein complexes and (2) testing its ability to recognize the docking solution closest to the experimental structure as that providing the most favorable total binding energy. For this purpose, a test set consisting of 15 protein-protein complexes with known 3D structure mixed with 10 decoys for each complex was used. The correlation between the values afforded by MM-ISMSA and those from the other methods is quite remarkable (r(2) ∼ 0.9), and only 0.2-5.0 s (depending on the number of residues) are spent on a single calculation including an all vs all pairwise energy decomposition. On the other hand, MM-ISMSA correctly identifies the best docking solution as that closest to the experimental structure in 80% of the cases. Finally, MM-ISMSA can process molecular dynamics trajectories and reports the results as averaged values with their standard deviations. MM-ISMSA has been implemented as a plugin to the widely used molecular graphics program PyMOL, although it can also be executed in command-line mode. MM-ISMSA is distributed free of charge to nonprofit organizations.

Alcohol modulates circulating levels of interleukin-6 and monocyte chemoattractant protein-1 in chronic pancreatitis

DEFF Research Database (Denmark)

Pedersen, N; Larsen, S; Seidelin, J B

2004-01-01

was to evaluate the circulating levels of IL-6, MCP-1, TGF-beta1, IGF-1 and IGFBP-3 in patients with alcoholic chronic pancreatitis (CP). METHODS: Twelve male patients with severe CP and 11 matched controls ingested 40 g alcohol. Plasma cytokine concentrations were measured for 24 h and assessed by sandwich ELISA......BACKGROUND: Cytokines are markers of acute pancreatic inflammation and essential for distant organ injury, but they also stimulate pancreatic fibrogenesis and are thus involved in the progression from acute pancreatitis to chronic pancreatic injury and fibrosis. The aim of this study...... techniques. RESULTS: IL-6 was higher in CP at fasting and 1, 4 and 24 h after alcohol intake (P

Structural symmetry and protein function.

Science.gov (United States)

Goodsell, D S; Olson, A J

2000-01-01

The majority of soluble and membrane-bound proteins in modern cells are symmetrical oligomeric complexes with two or more subunits. The evolutionary selection of symmetrical oligomeric complexes is driven by functional, genetic, and physicochemical needs. Large proteins are selected for specific morphological functions, such as formation of rings, containers, and filaments, and for cooperative functions, such as allosteric regulation and multivalent binding. Large proteins are also more stable against denaturation and have a reduced surface area exposed to solvent when compared with many individual, smaller proteins. Large proteins are constructed as oligomers for reasons of error control in synthesis, coding efficiency, and regulation of assembly. Symmetrical oligomers are favored because of stability and finite control of assembly. Several functions limit symmetry, such as interaction with DNA or membranes, and directional motion. Symmetry is broken or modified in many forms: quasisymmetry, in which identical subunits adopt similar but different conformations; pleomorphism, in which identical subunits form different complexes; pseudosymmetry, in which different molecules form approximately symmetrical complexes; and symmetry mismatch, in which oligomers of different symmetries interact along their respective symmetry axes. Asymmetry is also observed at several levels. Nearly all complexes show local asymmetry at the level of side chain conformation. Several complexes have reciprocating mechanisms in which the complex is asymmetric, but, over time, all subunits cycle through the same set of conformations. Global asymmetry is only rarely observed. Evolution of oligomeric complexes may favor the formation of dimers over complexes with higher cyclic symmetry, through a mechanism of prepositioned pairs of interacting residues. However, examples have been found for all of the crystallographic point groups, demonstrating that functional need can drive the evolution of

Diversity and functions of protein glycosylation in insects.

Science.gov (United States)

Walski, Tomasz; De Schutter, Kristof; Van Damme, Els J M; Smagghe, Guy

2017-04-01

The majority of proteins is modified with carbohydrate structures. This modification, called glycosylation, was shown to be crucial for protein folding, stability and subcellular location, as well as protein-protein interactions, recognition and signaling. Protein glycosylation is involved in multiple physiological processes, including embryonic development, growth, circadian rhythms, cell attachment as well as maintenance of organ structure, immunity and fertility. Although the general principles of glycosylation are similar among eukaryotic organisms, insects synthesize a distinct repertoire of glycan structures compared to plants and vertebrates. Consequently, a number of unique insect glycans mediate functions specific to this class of invertebrates. For instance, the core α1,3-fucosylation of N-glycans is absent in vertebrates, while in insects this modification is crucial for the development of wings and the nervous system. At present, most of the data on insect glycobiology comes from research in Drosophila. Yet, progressively more information on the glycan structures and the importance of glycosylation in other insects like beetles, caterpillars, aphids and bees is becoming available. This review gives a summary of the current knowledge and recent progress related to glycan diversity and function(s) of protein glycosylation in insects. We focus on N- and O-glycosylation, their synthesis, physiological role(s), as well as the molecular and biochemical basis of these processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Novel mutation predicted to disrupt SGOL1 protein function

African Journals Online (AJOL)

Rohit Gupta

2012-11-02

Nov 2, 2012 ... structural consequences of mutation over folding conformation of the 3rd exon. Further we carried .... Coiled Coil domain [PDB IDs: 3FGA] was retrieved from. Protein Data ... 1.0 nm of 216 SPC water molecules. We used 2CLА ...

Genome wide identification of wheat and Brachypodium type one protein phosphatases and functional characterization of durum wheat TdPP1a

OpenAIRE

Bradai, Mariem; Mahjoubi, Habib; Chini, Andrea; Chabouté, Marie-Edith; Hanin, Moez; Ebel, Chantal

2018-01-01

Reversible phosphorylation is an essential mechanism regulating signal transduction during development and environmental stress responses. An important number of dephosphorylation events in the cell are catalyzed by type one protein phosphatases (PP1), which catalytic activity is driven by the binding of regulatory proteins that control their substrate specificity or subcellular localization. Plants harbor several PP1 isoforms accounting for large functional redundancies. While animal PP1s we...

In silico functional elucidation of uncharacterized proteins of Chlamydia abortus strain LLG.

Science.gov (United States)

Singh, Gagandeep; Sharma, Dixit; Singh, Vikram; Rani, Jyoti; Marotta, Francessco; Kumar, Manoj; Mal, Gorakh; Singh, Birbal

2017-03-01

This study reports structural modeling, molecular dynamics profiling of hypothetical proteins in Chlamydia abortus genome database. The hypothetical protein sequences were extracted from C. abortus LLG Genome Database for functional elucidation using in silico methods. Fifty-one proteins with their roles in defense, binding and transporting other biomolecules were unraveled. Forty-five proteins were found to be nonhomologous to proteins present in hosts infected by C. abortus . Of these, 31 proteins were related to virulence. The structural modeling of two proteins, first, WP_006344020.1 (phosphorylase) and second, WP_006344325.1 (chlamydial protease/proteasome-like activity factor) were accomplished. The conserved active sites necessary for the catalytic function were analyzed. The finally concluded proteins are envisioned as possible targets for developing drugs to curtail chlamydial infections, however, and should be validated by molecular biological methods.

Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein.

Directory of Open Access Journals (Sweden)

Andrea Cerutti

Full Text Available Hepatitis C virus (HCV infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS, but no nuclear export signal (NES has yet been identified.We show here that the aa(109-133 region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126 in the identified NES or in the sequence encoding the mature core aa(1-173 significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication.Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection.

Identification of a functional, CRM-1-dependent nuclear export signal in hepatitis C virus core protein.

Science.gov (United States)

Cerutti, Andrea; Maillard, Patrick; Minisini, Rosalba; Vidalain, Pierre-Olivier; Roohvand, Farzin; Pecheur, Eve-Isabelle; Pirisi, Mario; Budkowska, Agata

2011-01-01

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified.We show here that the aa(109-133) region directs the translocation of core from the nucleus to the cytoplasm by the CRM-1-mediated nuclear export pathway. Mutagenesis of the three hydrophobic residues (L119, I123 and L126) in the identified NES or in the sequence encoding the mature core aa(1-173) significantly enhanced the nuclear localisation of the corresponding proteins in transfected Huh7 cells. Both the NES and the adjacent hydrophobic sequence in domain II of core were required to maintain the core protein or its fragments in the cytoplasmic compartment. Electron microscopy studies of the JFH1 replication model demonstrated that core was translocated into the nucleus a few minutes after the virus entered the cell. The blockade of nucleocytoplasmic export by leptomycin B treatment early in infection led to the detection of core protein in the nucleus by confocal microscopy and coincided with a decrease in virus replication.Our data suggest that the functional NLS and NES direct HCV core protein shuttling between the cytoplasmic and nuclear compartments, with at least some core protein transported to the nucleus. These new properties of HCV core may be essential for virus multiplication and interaction with nuclear molecules, influence cell signaling and the pathogenesis of HCV infection.

Insights into Hox protein function from a large scale combinatorial analysis of protein domains.

Directory of Open Access Journals (Sweden)

Samir Merabet

2011-10-01

Full Text Available Protein function is encoded within protein sequence and protein domains. However, how protein domains cooperate within a protein to modulate overall activity and how this impacts functional diversification at the molecular and organism levels remains largely unaddressed. Focusing on three domains of the central class Drosophila Hox transcription factor AbdominalA (AbdA, we used combinatorial domain mutations and most known AbdA developmental functions as biological readouts to investigate how protein domains collectively shape protein activity. The results uncover redundancy, interactivity, and multifunctionality of protein domains as salient features underlying overall AbdA protein activity, providing means to apprehend functional diversity and accounting for the robustness of Hox-controlled developmental programs. Importantly, the results highlight context-dependency in protein domain usage and interaction, allowing major modifications in domains to be tolerated without general functional loss. The non-pleoitropic effect of domain mutation suggests that protein modification may contribute more broadly to molecular changes underlying morphological diversification during evolution, so far thought to rely largely on modification in gene cis-regulatory sequences.

Role of Shwachman-Bodian-Diamond syndrome protein in translation machinery and cell chemotaxis: a comparative genomics approach

Directory of Open Access Journals (Sweden)

Vasieva O

2011-09-01

Full Text Available Olga VasievaInstitute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom; Fellowship for the Interpretation of Genomes, Burr Ridge, IL, USAAbstract: Shwachman-Bodian-Diamond syndrome (SBDS is linked to a mutation in a single gene. The SBDS proinvolved in RNA metabolism and ribosome-associated functions, but SBDS mutation is primarily linked to a defect in polymorphonuclear leukocytes unable to orient correctly in a spatial gradient of chemoattractants. Results of data mining and comparative genomic approaches undertaken in this study suggest that SBDS protein is also linked to tRNA metabolism and translation initiation. Analysis of crosstalk between translation machinery and cytoskeletal dynamics provides new insights into the cellular chemotactic defects caused by SBDS protein malfunction. The proposed functional interactions provide a new approach to exploit potential targets in the treatment and monitoring of this disease.Keywords: Shwachman-Bodian-Diamond syndrome, wybutosine, tRNA, chemotaxis, translation, genomics, gene proximity

Genome wide identification of wheat and Brachypodium type one protein phosphatases and functional characterization of durum wheat TdPP1a.

Directory of Open Access Journals (Sweden)

Mariem Bradai

Full Text Available Reversible phosphorylation is an essential mechanism regulating signal transduction during development and environmental stress responses. An important number of dephosphorylation events in the cell are catalyzed by type one protein phosphatases (PP1, which catalytic activity is driven by the binding of regulatory proteins that control their substrate specificity or subcellular localization. Plants harbor several PP1 isoforms accounting for large functional redundancies. While animal PP1s were reported to play relevant roles in controlling multiple cellular processes, plant orthologs remain poorly studied. To decipher the role of plant PP1s, we compared PP1 genes from three monocot species, Brachypodium, common wheat and rice at the genomic and transcriptomic levels. To gain more insight into the wheat PP1 proteins, we identified and characterized TdPP1a, the first wheat type one protein phosphatase from a Tunisian durum wheat variety Oum Rabiaa3. TdPP1a is highly conserved in sequence and structure when compared to mammalian, yeast and other plant PP1s. We demonstrate that TdPP1a is an active, metallo-dependent phosphatase in vitro and is able to interact with AtI2, a typical regulator of PP1 functions. Also, TdPP1a is capable to complement the heat stress sensitivity of the yeast mutant indicating that TdPP1a is functional also in vivo. Moreover, transient expression of TdPP1a::GFP in tobacco leaves revealed that it is ubiquitously distributed within the cell, with a strong accumulation in the nucleus. Finally, transcriptional analyses showed similar expression levels in roots and leaves of durum wheat seedlings. Interestingly, the expression in leaves is significantly induced following salinity stress, suggesting a potential role of TdPP1a in wheat salt stress response.

Quantitative protein localization signatures reveal an association between spatial and functional divergences of proteins.

Science.gov (United States)

Loo, Lit-Hsin; Laksameethanasan, Danai; Tung, Yi-Ling

2014-03-01

Protein subcellular localization is a major determinant of protein function. However, this important protein feature is often described in terms of discrete and qualitative categories of subcellular compartments, and therefore it has limited applications in quantitative protein function analyses. Here, we present Protein Localization Analysis and Search Tools (PLAST), an automated analysis framework for constructing and comparing quantitative signatures of protein subcellular localization patterns based on microscopy images. PLAST produces human-interpretable protein localization maps that quantitatively describe the similarities in the localization patterns of proteins and major subcellular compartments, without requiring manual assignment or supervised learning of these compartments. Using the budding yeast Saccharomyces cerevisiae as a model system, we show that PLAST is more accurate than existing, qualitative protein localization annotations in identifying known co-localized proteins. Furthermore, we demonstrate that PLAST can reveal protein localization-function relationships that are not obvious from these annotations. First, we identified proteins that have similar localization patterns and participate in closely-related biological processes, but do not necessarily form stable complexes with each other or localize at the same organelles. Second, we found an association between spatial and functional divergences of proteins during evolution. Surprisingly, as proteins with common ancestors evolve, they tend to develop more diverged subcellular localization patterns, but still occupy similar numbers of compartments. This suggests that divergence of protein localization might be more frequently due to the development of more specific localization patterns over ancestral compartments than the occupation of new compartments. PLAST enables systematic and quantitative analyses of protein localization-function relationships, and will be useful to elucidate protein

Scoring functions for protein-protein interactions.

Science.gov (United States)

Moal, Iain H; Moretti, Rocco; Baker, David; Fernández-Recio, Juan

2013-12-01

The computational evaluation of protein-protein interactions will play an important role in organising the wealth of data being generated by high-throughput initiatives. Here we discuss future applications, report recent developments and identify areas requiring further investigation. Many functions have been developed to quantify the structural and energetic properties of interacting proteins, finding use in interrelated challenges revolving around the relationship between sequence, structure and binding free energy. These include loop modelling, side-chain refinement, docking, multimer assembly, affinity prediction, affinity change upon mutation, hotspots location and interface design. Information derived from models optimised for one of these challenges can be used to benefit the others, and can be unified within the theoretical frameworks of multi-task learning and Pareto-optimal multi-objective learning. Copyright © 2013 Elsevier Ltd. All rights reserved.

JNK Signaling: Regulation and Functions Based on Complex Protein-Protein Partnerships

Science.gov (United States)

Zeke, András; Misheva, Mariya

2016-01-01

SUMMARY The c-Jun N-terminal kinases (JNKs), as members of the mitogen-activated protein kinase (MAPK) family, mediate eukaryotic cell responses to a wide range of abiotic and biotic stress insults. JNKs also regulate important physiological processes, including neuronal functions, immunological actions, and embryonic development, via their impact on gene expression, cytoskeletal protein dynamics, and cell death/survival pathways. Although the JNK pathway has been under study for >20 years, its complexity is still perplexing, with multiple protein partners of JNKs underlying the diversity of actions. Here we review the current knowledge of JNK structure and isoforms as well as the partnerships of JNKs with a range of intracellular proteins. Many of these proteins are direct substrates of the JNKs. We analyzed almost 100 of these target proteins in detail within a framework of their classification based on their regulation by JNKs. Examples of these JNK substrates include a diverse assortment of nuclear transcription factors (Jun, ATF2, Myc, Elk1), cytoplasmic proteins involved in cytoskeleton regulation (DCX, Tau, WDR62) or vesicular transport (JIP1, JIP3), cell membrane receptors (BMPR2), and mitochondrial proteins (Mcl1, Bim). In addition, because upstream signaling components impact JNK activity, we critically assessed the involvement of signaling scaffolds and the roles of feedback mechanisms in the JNK pathway. Despite a clarification of many regulatory events in JNK-dependent signaling during the past decade, many other structural and mechanistic insights are just beginning to be revealed. These advances open new opportunities to understand the role of JNK signaling in diverse physiological and pathophysiological states. PMID:27466283

Alcohol modulates circulating levels of interleukin-6 and monocyte chemoattractant protein-1 in chronic pancreatitis

DEFF Research Database (Denmark)

Pedersen, N; Larsen, S; Seidelin, J B

2004-01-01

Cytokines are markers of acute pancreatic inflammation and essential for distant organ injury, but they also stimulate pancreatic fibrogenesis and are thus involved in the progression from acute pancreatitis to chronic pancreatic injury and fibrosis. The aim of this study was to evaluate the circ...... the circulating levels of IL-6, MCP-1, TGF-beta1, IGF-1 and IGFBP-3 in patients with alcoholic chronic pancreatitis (CP).......Cytokines are markers of acute pancreatic inflammation and essential for distant organ injury, but they also stimulate pancreatic fibrogenesis and are thus involved in the progression from acute pancreatitis to chronic pancreatic injury and fibrosis. The aim of this study was to evaluate...

Recognition of functional sites in protein structures.

Science.gov (United States)

Shulman-Peleg, Alexandra; Nussinov, Ruth; Wolfson, Haim J

2004-06-04

Recognition of regions on the surface of one protein, that are similar to a binding site of another is crucial for the prediction of molecular interactions and for functional classifications. We first describe a novel method, SiteEngine, that assumes no sequence or fold similarities and is able to recognize proteins that have similar binding sites and may perform similar functions. We achieve high efficiency and speed by introducing a low-resolution surface representation via chemically important surface points, by hashing triangles of physico-chemical properties and by application of hierarchical scoring schemes for a thorough exploration of global and local similarities. We proceed to rigorously apply this method to functional site recognition in three possible ways: first, we search a given functional site on a large set of complete protein structures. Second, a potential functional site on a protein of interest is compared with known binding sites, to recognize similar features. Third, a complete protein structure is searched for the presence of an a priori unknown functional site, similar to known sites. Our method is robust and efficient enough to allow computationally demanding applications such as the first and the third. From the biological standpoint, the first application may identify secondary binding sites of drugs that may lead to side-effects. The third application finds new potential sites on the protein that may provide targets for drug design. Each of the three applications may aid in assigning a function and in classification of binding patterns. We highlight the advantages and disadvantages of each type of search, provide examples of large-scale searches of the entire Protein Data Base and make functional predictions.

Dual Role of Ancient Ubiquitous Protein 1 (AUP1) in Lipid Droplet Accumulation and Endoplasmic Reticulum (ER) Protein Quality Control

Science.gov (United States)

Klemm, Elizabeth J.; Spooner, Eric; Ploegh, Hidde L.

2011-01-01

Quality control of endoplasmic reticulum proteins involves the identification and engagement of misfolded proteins, dislocation of the misfolded protein across the endoplasmic reticulum (ER) membrane, and ubiquitin-mediated targeting to the proteasome for degradation. Ancient ubiquitous protein 1 (AUP1) physically associates with the mammalian HRD1-SEL1L complex, and AUP1 depletion impairs degradation of misfolded ER proteins. One of the functions of AUP1 in ER quality control is to recruit the soluble E2 ubiquitin-conjugating enzyme UBE2G2. We further show that the CUE domain of AUP1 regulates polyubiquitylation and facilitates the interaction of AUP1 with the HRD1 complex and with dislocation substrates. AUP1 localizes both to the ER and to lipid droplets. The AUP1 expression level affects the abundance of cellular lipid droplets and as such represents the first protein with lipid droplet regulatory activity to be linked to ER quality control. These findings indicate a possible connection between ER protein quality control and lipid droplets. PMID:21857022

The function of the inner nuclear envelope protein SUN1 in mRNA export is regulated by phosphorylation.

Science.gov (United States)

Li, Ping; Stumpf, Maria; Müller, Rolf; Eichinger, Ludwig; Glöckner, Gernot; Noegel, Angelika A

2017-08-22

SUN1, a component of the LINC (Linker of Nucleoskeleton and Cytoskeleton) complex, functions in mammalian mRNA export through the NXF1-dependent pathway. It associates with mRNP complexes by direct interaction with NXF1. It also binds to the NPC through association with the nuclear pore component Nup153, which is involved in mRNA export. The SUN1-NXF1 association is at least partly regulated by a protein kinase C (PKC) which phosphorylates serine 113 (S113) in the N-terminal domain leading to reduced interaction. The phosphorylation appears to be important for the SUN1 function in nuclear mRNA export since GFP-SUN1 carrying a S113A mutation was less efficient in restoring mRNA export after SUN1 knockdown as compared to the wild type protein. By contrast, GFP-SUN1-S113D resembling the phosphorylated state allowed very efficient export of poly(A)+RNA. Furthermore, probing a possible role of the LINC complex component Nesprin-2 in this process we observed impaired mRNA export in Nesprin-2 knockdown cells. This effect might be independent of SUN1 as expression of a GFP tagged SUN-domain deficient SUN1, which no longer can interact with Nesprin-2, did not affect mRNA export.

Overlapping functions of argonaute proteins in patterning and morphogenesis of Drosophila embryos.

Directory of Open Access Journals (Sweden)

Wibke J Meyer

2006-08-01

Full Text Available Argonaute proteins are essential components of the molecular machinery that drives RNA silencing. In Drosophila, different members of the Argonaute family of proteins have been assigned to distinct RNA silencing pathways. While Ago1 is required for microRNA function, Ago2 is a crucial component of the RNA-induced silencing complex in siRNA-triggered RNA interference. Drosophila Ago2 contains an unusual amino-terminus with two types of imperfect glutamine-rich repeats (GRRs of unknown function. Here we show that the GRRs of Ago2 are essential for the normal function of the protein. Alleles with reduced numbers of GRRs cause specific disruptions in two morphogenetic processes associated with the midblastula transition: membrane growth and microtubule-based organelle transport. These defects do not appear to result from disruption of siRNA-dependent processes but rather suggest an interference of the mutant Ago2 proteins in an Ago1-dependent pathway. Using loss-of-function alleles, we further demonstrate that Ago1 and Ago2 act in a partially redundant manner to control the expression of the segment-polarity gene wingless in the early embryo. Our findings argue against a strict separation of Ago1 and Ago2 functions and suggest that these proteins act in concert to control key steps of the midblastula transition and of segmental patterning.

Binding proteins of somatomedins and their functions

International Nuclear Information System (INIS)

Kostelecka, Z.; Blahovec, J.

1998-01-01

In this paper the functions of binding proteins are discussed. One variable that provides insulin-like growth factors (IGFs) control at the extracellular level is the presence of high-affinity, soluble insulin-like growth factor proteins (IGFBPs). IGFBP-1 inhibits IGF effect on human osteosarcoma cells. Increased concentration of IGFBP-3 inhibits the proliferation of breast cancer cell line MCF 7 either directly or by competition for IGF receptors. Maybe IGFBPs work as anti-mitogens and IGFs are potential promotors of cancer growth

Tet protein function during Drosophila development.

Directory of Open Access Journals (Sweden)

Fei Wang

Full Text Available The TET (Ten-eleven translocation 1, 2 and 3 proteins have been shown to function as DNA hydroxymethylases in vertebrates and their requirements have been documented extensively. Recently, the Tet proteins have been shown to also hydroxylate 5-methylcytosine in RNA. 5-hydroxymethylcytosine (5hmrC is enriched in messenger RNA but the function of this modification has yet to be elucidated. Because Cytosine methylation in DNA is barely detectable in Drosophila, it serves as an ideal model to study the biological function of 5hmrC. Here, we characterized the temporal and spatial expression and requirement of Tet throughout Drosophila development. We show that Tet is essential for viability as Tet complete loss-of-function animals die at the late pupal stage. Tet is highly expressed in neuronal tissues and at more moderate levels in somatic muscle precursors in embryos and larvae. Depletion of Tet in muscle precursors at early embryonic stages leads to defects in larval locomotion and late pupal lethality. Although Tet knock-down in neuronal tissue does not cause lethality, it is essential for neuronal function during development through its affects upon locomotion in larvae and the circadian rhythm of adult flies. Further, we report the function of Tet in ovarian morphogenesis. Together, our findings provide basic insights into the biological function of Tet in Drosophila, and may illuminate observed neuronal and muscle phenotypes observed in vertebrates.

Prediction of functional sites in proteins using conserved functional group analysis.

Science.gov (United States)

Innis, C Axel; Anand, A Prem; Sowdhamini, R

2004-04-02

A detailed knowledge of a protein's functional site is an absolute prerequisite for understanding its mode of action at the molecular level. However, the rapid pace at which sequence and structural information is being accumulated for proteins greatly exceeds our ability to determine their biochemical roles experimentally. As a result, computational methods are required which allow for the efficient processing of the evolutionary information contained in this wealth of data, in particular that related to the nature and location of functionally important sites and residues. The method presented here, referred to as conserved functional group (CFG) analysis, relies on a simplified representation of the chemical groups found in amino acid side-chains to identify functional sites from a single protein structure and a number of its sequence homologues. We show that CFG analysis can fully or partially predict the location of functional sites in approximately 96% of the 470 cases tested and that, unlike other methods available, it is able to tolerate wide variations in sequence identity. In addition, we discuss its potential in a structural genomics context, where automation, scalability and efficiency are critical, and an increasing number of protein structures are determined with no prior knowledge of function. This is exemplified by our analysis of the hypothetical protein Ydde_Ecoli, whose structure was recently solved by members of the North East Structural Genomics consortium. Although the proposed active site for this protein needs to be validated experimentally, this example illustrates the scope of CFG analysis as a general tool for the identification of residues likely to play an important role in a protein's biochemical function. Thus, our method offers a convenient solution to rapidly and automatically process the vast amounts of data that are beginning to emerge from structural genomics projects.

Integrative approaches to the prediction of protein functions based on the feature selection

Directory of Open Access Journals (Sweden)

Lee Hyunju

2009-12-01

Full Text Available Abstract Background Protein function prediction has been one of the most important issues in functional genomics. With the current availability of various genomic data sets, many researchers have attempted to develop integration models that combine all available genomic data for protein function prediction. These efforts have resulted in the improvement of prediction quality and the extension of prediction coverage. However, it has also been observed that integrating more data sources does not always increase the prediction quality. Therefore, selecting data sources that highly contribute to the protein function prediction has become an important issue. Results We present systematic feature selection methods that assess the contribution of genome-wide data sets to predict protein functions and then investigate the relationship between genomic data sources and protein functions. In this study, we use ten different genomic data sources in Mus musculus, including: protein-domains, protein-protein interactions, gene expressions, phenotype ontology, phylogenetic profiles and disease data sources to predict protein functions that are labelled with Gene Ontology (GO terms. We then apply two approaches to feature selection: exhaustive search feature selection using a kernel based logistic regression (KLR, and a kernel based L1-norm regularized logistic regression (KL1LR. In the first approach, we exhaustively measure the contribution of each data set for each function based on its prediction quality. In the second approach, we use the estimated coefficients of features as measures of contribution of data sources. Our results show that the proposed methods improve the prediction quality compared to the full integration of all data sources and other filter-based feature selection methods. We also show that contributing data sources can differ depending on the protein function. Furthermore, we observe that highly contributing data sets can be similar among

Identification of herpesvirus proteins that contribute to G1/S arrest.

Science.gov (United States)

Paladino, Patrick; Marcon, Edyta; Greenblatt, Jack; Frappier, Lori

2014-04-01

Lytic infection by herpesviruses induces cell cycle arrest at the G1/S transition. This appears to be a function of multiple herpesvirus proteins, but only a minority of herpesvirus proteins have been examined for cell cycle effects. To gain a more comprehensive understanding of the viral proteins that contribute to G1/S arrest, we screened a library of over 200 proteins from herpes simplex virus type 1, human cytomegalovirus, and Epstein-Barr virus (EBV) for effects on the G1/S interface, using HeLa fluorescent, ubiquitination-based cell cycle indicator (Fucci) cells in which G1/S can be detected colorimetrically. Proteins from each virus were identified that induce accumulation of G1/S cells, predominantly tegument, early, and capsid proteins. The identification of several capsid proteins in this screen suggests that incoming viral capsids may function to modulate cellular processes. The cell cycle effects of selected EBV proteins were further verified and examined for effects on p53 and p21 as regulators of the G1/S transition. Two EBV replication proteins (BORF2 and BMRF1) were found to induce p53 but not p21, while a previously uncharacterized tegument protein (BGLF2) was found to induce p21 protein levels in a p53-independent manner. Proteomic analyses of BGLF2-interacting proteins identified interactions with the NIMA-related protein kinase (NEK9) and GEM-interacting protein (GMIP). Silencing of either NEK9 or GMIP induced p21 without affecting p53 and abrogated the ability of BGLF2 to further induce p21. Collectively, these results suggest multiple viral proteins contribute to G1/S arrest, including BGLF2, which induces p21 levels likely by interfering with the functions of NEK9 and GMIP. Most people are infected with multiple herpesviruses, whose proteins alter the infected cells in several ways. During lytic infection, the viral proteins block cell proliferation just before the cellular DNA replicates. We used a novel screening method to identify proteins

Functions and structures of eukaryotic recombination proteins

International Nuclear Information System (INIS)

Ogawa, Tomoko

1994-01-01

We have found that Rad51 and RecA Proteins form strikingly similar structures together with dsDNA and ATP. Their right handed helical nucleoprotein filaments extend the B-form DNA double helixes to 1.5 times in length and wind the helix. The similarity and uniqueness of their structures must reflect functional homologies between these proteins. Therefore, it is highly probable that similar recombination proteins are present in various organisms of different evolutional states. We have succeeded to clone RAD51 genes from human, mouse, chicken and fission yeast genes, and found that the homologues are widely distributed in eukaryotes. The HsRad51 and MmRad51 or ChRad51 proteins consist of 339 amino acids differing only by 4 or 12 amino acids, respectively, and highly homologous to both yeast proteins, but less so to Dmcl. All of these proteins are homologous to the region from residues 33 to 240 of RecA which was named ''homologous core. The homologous core is likely to be responsible for functions common for all of them, such as the formation of helical nucleoprotein filament that is considered to be involved in homologous pairing in the recombination reaction. The mouse gene is transcribed at a high level in thymus, spleen, testis, and ovary, at lower level in brain and at a further lower level in some other tissues. It is transcribed efficiently in recombination active tissues. A clear functional difference of Rad51 homologues from RecA was suggested by the failure of heterologous genes to complement the deficiency of Scrad51 mutants. This failure seems to reflect the absence of a compatible partner, such as ScRad52 protein in the case of ScRad51 protein, between different species. Thus, these discoveries play a role of the starting point to understand the fundamental gene targeting in mammalian cells and in gene therapy. (J.P.N.)

The functional properties, modification and utilization of whey proteins

Directory of Open Access Journals (Sweden)

B. G. Venter

1986-03-01

Full Text Available Whey protein has an excellent nutritional value and exhibits a functional potential. In comparison with certain other food proteins, the whey protein content of essential amino acids is extremely favourable for human consumption. Depending on the heat-treatment history thereof, soluble whey proteins with utilizable functional properties, apart from high biological value, true digestibility, protein efficiency ratio and nett protein utilization, can be recovered. Various technological and chemical recovery processes have been designed. Chemically and enzymatically modified whey protein is manufactured to obtain technological and functional advantages. The important functional properties of whey proteins, namely hydration, gelation, emulsifying and foaming properties, are reviewed.

Myocardial ischemic preconditioning upregulated protein 1(Mipu1):zinc finger protein 667 - a multifunctional KRAB/C{sub 2}H{sub 2} zinc finger protein

Energy Technology Data Exchange (ETDEWEB)

Han, D.; Zhang, C. [Institute of Cardiovascular Disease, Key Lab for Arteriosclerology of Hunan Province, Post-doctoral Mobile Stations for Basic Medicine, University of South China, Hengyang City, Hunan Province (China); Fan, W.J. [Institute of Cardiovascular Disease, Key Lab for Arteriosclerology of Hunan Province, Post-doctoral Mobile Stations for Basic Medicine, University of South China, Hengyang City, Hunan Province (China); The Second Affiliated Hospital, University of South China, Hengyang City, Hunan Province (China); Pan, W.J.; Feng, D.M.; Qu, S.L.; Jiang, Z.S. [Institute of Cardiovascular Disease, Key Lab for Arteriosclerology of Hunan Province, Post-doctoral Mobile Stations for Basic Medicine, University of South China, Hengyang City, Hunan Province (China)

2014-10-31

Myocardial ischemic preconditioning upregulated protein 1 (Mipu1) is a newly discovered upregulated gene produced in rats during the myocardial ischemic preconditioning process. Mipu1 cDNA contains a 1824-base pair open reading frame and encodes a 608 amino acid protein with an N-terminal Krüppel-associated box (KRAB) domain and classical zinc finger C{sub 2}H{sub 2} motifs in the C-terminus. Mipu1 protein is located in the cell nucleus. Recent studies found that Mipu1 has a protective effect on the ischemia-reperfusion injury of heart, brain, and other organs. As a nuclear factor, Mipu1 may perform its protective function through directly transcribing and repressing the expression of proapoptotic genes to repress cell apoptosis. In addition, Mipu1 also plays an important role in regulating the gene expression of downstream inflammatory mediators by inhibiting the activation of activator protein-1 and serum response element.

From Lipid Homeostasis to Differentiation: Old and New Functions of the Zinc Cluster Proteins Ecm22, Upc2, Sut1 and Sut2

Directory of Open Access Journals (Sweden)

Ifeoluwapo Matthew Joshua

2017-04-01

Full Text Available Zinc cluster proteins are a large family of transcriptional regulators with a wide range of biological functions. The zinc cluster proteins Ecm22, Upc2, Sut1 and Sut2 have initially been identified as regulators of sterol import in the budding yeast Saccharomyces cerevisiae. These proteins also control adaptations to anaerobic growth, sterol biosynthesis as well as filamentation and mating. Orthologs of these zinc cluster proteins have been identified in several species of Candida. Upc2 plays a critical role in antifungal resistance in these important human fungal pathogens. Upc2 is therefore an interesting potential target for novel antifungals. In this review we discuss the functions, mode of actions and regulation of Ecm22, Upc2, Sut1 and Sut2 in budding yeast and Candida.

Functional modulation of the glutamate transporter variant GLT1b by the PDZ domain protein PICK1

DEFF Research Database (Denmark)

Søgaard, Rikke; Borre, Lars; Braunstein, Thomas H

2013-01-01

The dominant glutamate transporter isoform in the mammalian brain, GLT1, exists as at least three splice variants, GLT1a, GLT1b, and GLT1c. GLT1b interacts with the scaffold protein PICK1 (protein interacting with kinase C1), which is implicated in glutamatergic neurotransmission via its regulato...

A computational model of the LGI1 protein suggests a common binding site for ADAM proteins.

Directory of Open Access Journals (Sweden)

Emanuela Leonardi

Full Text Available Mutations of human leucine-rich glioma inactivated (LGI1 gene encoding the epitempin protein cause autosomal dominant temporal lateral epilepsy (ADTLE, a rare familial partial epileptic syndrome. The LGI1 gene seems to have a role on the transmission of neuronal messages but the exact molecular mechanism remains unclear. In contrast to other genes involved in epileptic disorders, epitempin shows no homology with known ion channel genes but contains two domains, composed of repeated structural units, known to mediate protein-protein interactions.A three dimensional in silico model of the two epitempin domains was built to predict the structure-function relationship and propose a functional model integrating previous experimental findings. Conserved and electrostatic charged regions of the model surface suggest a possible arrangement between the two domains and identifies a possible ADAM protein binding site in the β-propeller domain and another protein binding site in the leucine-rich repeat domain. The functional model indicates that epitempin could mediate the interaction between proteins localized to different synaptic sides in a static way, by forming a dimer, or in a dynamic way, by binding proteins at different times.The model was also used to predict effects of known disease-causing missense mutations. Most of the variants are predicted to alter protein folding while several other map to functional surface regions. In agreement with experimental evidence, this suggests that non-secreted LGI1 mutants could be retained within the cell by quality control mechanisms or by altering interactions required for the secretion process.

Wiki-pi: a web-server of annotated human protein-protein interactions to aid in discovery of protein function.

Directory of Open Access Journals (Sweden)

Naoki Orii

Full Text Available Protein-protein interactions (PPIs are the basis of biological functions. Knowledge of the interactions of a protein can help understand its molecular function and its association with different biological processes and pathways. Several publicly available databases provide comprehensive information about individual proteins, such as their sequence, structure, and function. There also exist databases that are built exclusively to provide PPIs by curating them from published literature. The information provided in these web resources is protein-centric, and not PPI-centric. The PPIs are typically provided as lists of interactions of a given gene with links to interacting partners; they do not present a comprehensive view of the nature of both the proteins involved in the interactions. A web database that allows search and retrieval based on biomedical characteristics of PPIs is lacking, and is needed. We present Wiki-Pi (read Wiki-π, a web-based interface to a database of human PPIs, which allows users to retrieve interactions by their biomedical attributes such as their association to diseases, pathways, drugs and biological functions. Each retrieved PPI is shown with annotations of both of the participant proteins side-by-side, creating a basis to hypothesize the biological function facilitated by the interaction. Conceptually, it is a search engine for PPIs analogous to PubMed for scientific literature. Its usefulness in generating novel scientific hypotheses is demonstrated through the study of IGSF21, a little-known gene that was recently identified to be associated with diabetic retinopathy. Using Wiki-Pi, we infer that its association to diabetic retinopathy may be mediated through its interactions with the genes HSPB1, KRAS, TMSB4X and DGKD, and that it may be involved in cellular response to external stimuli, cytoskeletal organization and regulation of molecular activity. The website also provides a wiki-like capability allowing users

The E4 protein; structure, function and patterns of expression

Energy Technology Data Exchange (ETDEWEB)

Doorbar, John, E-mail: jdoorba@nimr.mrc.ac.uk

2013-10-15

The papillomavirus E4 open reading frame (ORF) is contained within the E2 ORF, with the primary E4 gene-product (E1{sup ∧}E4) being translated from a spliced mRNA that includes the E1 initiation codon and adjacent sequences. E4 is located centrally within the E2 gene, in a region that encodes the E2 protein′s flexible hinge domain. Although a number of minor E4 transcripts have been reported, it is the product of the abundant E1{sup ∧}E4 mRNA that has been most extensively analysed. During the papillomavirus life cycle, the E1{sup ∧}E4 gene products generally become detectable at the onset of vegetative viral genome amplification as the late stages of infection begin. E4 contributes to genome amplification success and virus synthesis, with its high level of expression suggesting additional roles in virus release and/or transmission. In general, E4 is easily visualised in biopsy material by immunostaining, and can be detected in lesions caused by diverse papillomavirus types, including those of dogs, rabbits and cattle as well as humans. The E4 protein can serve as a biomarker of active virus infection, and in the case of high-risk human types also disease severity. In some cutaneous lesions, E4 can be expressed at higher levels than the virion coat proteins, and can account for as much as 30% of total lesional protein content. The E4 proteins of the Beta, Gamma and Mu HPV types assemble into distinctive cytoplasmic, and sometimes nuclear, inclusion granules. In general, the E4 proteins are expressed before L2 and L1, with their structure and function being modified, first by kinases as the infected cell progresses through the S and G2 cell cycle phases, but also by proteases as the cell exits the cell cycle and undergoes true terminal differentiation. The kinases that regulate E4 also affect other viral proteins simultaneously, and include protein kinase A, Cyclin-dependent kinase, members of the MAP Kinase family and protein kinase C. For HPV16 E1{sup �

Ion Binding Energies Determining Functional Transport of ClC Proteins

Science.gov (United States)

Yu, Tao; Guo, Xu; Zou, Xian-Wu; Sang, Jian-Ping

2014-06-01

The ClC-type proteins, a large family of chloride transport proteins ubiquitously expressed in biological organisms, have been extensively studied for decades. Biological function of ClC proteins can be reflected by analyzing the binding situation of Cl- ions. We investigate ion binding properties of ClC-ec1 protein with the atomic molecular dynamics simulation approach. The calculated electrostatic binding energy results indicate that Cl- at the central binding site Scen has more binding stability than the internal binding site Sint. Quantitative comparison between the latest experimental heat release data isothermal titration calorimetry (ITC) and our calculated results demonstrates that chloride ions prefer to bind at Scen than Sint in the wild-type ClC-ec1 structure and prefer to bind at Sext and Scen than Sint in mutant E148A/E148Q structures. Even though the chloride ions make less contribution to heat release when binding to Sint and are relatively unstable in the Cl- pathway, they are still part contributors for the Cl- functional transport. This work provides a guide rule to estimate the importance of Cl- at the binding sites and how chloride ions have influences on the function of ClC proteins.

Uncoupling proteins (UCP) in unicellular eukaryotes: true UCPs or UCP1-like acting proteins?

Science.gov (United States)

Luévano-Martínez, Luis Alberto

2012-04-05

Uncoupling proteins belong to the superfamily of mitochondrial anion carriers. They are apparently present throughout the Eukarya domain in which only some members have an established physiological function, i.e. UCP1 from brown adipose tissue is involved in non-shivering thermogenesis. However, the proteins responsible for the phenotype observed in unicellular organisms have not been characterized. In this report we analyzed functional evidence concerning unicellular UCPs and found that true UCPs are restricted to some taxonomical groups while proteins conferring a UCP1-like phenotype to fungi and most protists are the result of a promiscuous activity exerted by other mitochondrial anion carriers. We describe a possible evolutionary route followed by these proteins by which they acquire this promiscuous mechanism. Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

International Nuclear Information System (INIS)

Nielsen, Anders Lade

2009-01-01

Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer's disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of γ-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as β-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.

The coat protein complex II, COPII, protein Sec13 directly interacts with presenilin-1

Energy Technology Data Exchange (ETDEWEB)

Nielsen, Anders Lade, E-mail: aln@humgen.au.dk [Department of Human Genetics, The Bartholin Building, University of Aarhus, DK-8000 Aarhus C (Denmark)

2009-10-23

Mutations in the human gene encoding presenilin-1, PS1, account for most cases of early-onset familial Alzheimer's disease. PS1 has nine transmembrane domains and a large loop orientated towards the cytoplasm. PS1 locates to cellular compartments as endoplasmic reticulum (ER), Golgi apparatus, vesicular structures, and plasma membrane, and is an integral member of {gamma}-secretase, a protein protease complex with specificity for intra-membranous cleavage of substrates such as {beta}-amyloid precursor protein. Here, an interaction between PS1 and the Sec13 protein is described. Sec13 takes part in coat protein complex II, COPII, vesicular trafficking, nuclear pore function, and ER directed protein sequestering and degradation control. The interaction maps to the N-terminal part of the large hydrophilic PS1 loop and the first of the six WD40-repeats present in Sec13. The identified Sec13 interaction to PS1 is a new candidate interaction for linking PS1 to secretory and protein degrading vesicular circuits.

Synthesis and functioning of the colicin E1 lysis protein: Comparison with the colicin A lysis protein

International Nuclear Information System (INIS)

Cavard, D.

1991-01-01

The colicin E1 lysis protein, CelA, was identified as a 3-kDa protein in induced cells of Escherichia coli K-12 carrying pColE1 by pulse-chase labeling with either [ 35 S]cysteine or [ 3 H]lysine. This 3-kDa protein was acylated, as shown by [2- 3 H]glycerol labeling, and seemed to correspond to the mature CelA protein. The rate of modification and processing of CelA was different from that observed for Cal, the colicin A lysis protein. In contrast to Cal, no intermediate form was detected for CelA, no signal peptide accumulated, and no modified precursor form was observed after globomycin treatment. Thus, the rate of synthesis would not be specific to lysis proteins. Solubilization in sodium dodecyl sulfate of the mature forms of both CelA and Cal varied similarly at the time of colicin release, indicating a change in lysis protein structure. This particular property would play a role in the mechanism of colicin export. The accumulation of the signal peptide seems to be a factor determining the toxicity of the lysis proteins since CelA provoked less cell damage than Cal. Quasi-lysis and killing due to CelA were higher in degP mutants than in wild-type cells. They were minimal in pldA mutants

Rift Valley fever virus NSs protein functions and the similarity to other bunyavirus NSs proteins.

Science.gov (United States)

Ly, Hoai J; Ikegami, Tetsuro

2016-07-02

Rift Valley fever is a mosquito-borne zoonotic disease that affects both ruminants and humans. The nonstructural (NS) protein, which is a major virulence factor for Rift Valley fever virus (RVFV), is encoded on the S-segment. Through the cullin 1-Skp1-Fbox E3 ligase complex, the NSs protein promotes the degradation of at least two host proteins, the TFIIH p62 and the PKR proteins. NSs protein bridges the Fbox protein with subsequent substrates, and facilitates the transfer of ubiquitin. The SAP30-YY1 complex also bridges the NSs protein with chromatin DNA, affecting cohesion and segregation of chromatin DNA as well as the activation of interferon-β promoter. The presence of NSs filaments in the nucleus induces DNA damage responses and causes cell-cycle arrest, p53 activation, and apoptosis. Despite the fact that NSs proteins have poor amino acid similarity among bunyaviruses, the strategy utilized to hijack host cells are similar. This review will provide and summarize an update of recent findings pertaining to the biological functions of the NSs protein of RVFV as well as the differences from those of other bunyaviruses.

Linking structural features of protein complexes and biological function.

Science.gov (United States)

Sowmya, Gopichandran; Breen, Edmond J; Ranganathan, Shoba

2015-09-01

Protein-protein interaction (PPI) establishes the central basis for complex cellular networks in a biological cell. Association of proteins with other proteins occurs at varying affinities, yet with a high degree of specificity. PPIs lead to diverse functionality such as catalysis, regulation, signaling, immunity, and inhibition, playing a crucial role in functional genomics. The molecular principle of such interactions is often elusive in nature. Therefore, a comprehensive analysis of known protein complexes from the Protein Data Bank (PDB) is essential for the characterization of structural interface features to determine structure-function relationship. Thus, we analyzed a nonredundant dataset of 278 heterodimer protein complexes, categorized into major functional classes, for distinguishing features. Interestingly, our analysis has identified five key features (interface area, interface polar residue abundance, hydrogen bonds, solvation free energy gain from interface formation, and binding energy) that are discriminatory among the functional classes using Kruskal-Wallis rank sum test. Significant correlations between these PPI interface features amongst functional categories are also documented. Salt bridges correlate with interface area in regulator-inhibitors (r = 0.75). These representative features have implications for the prediction of potential function of novel protein complexes. The results provide molecular insights for better understanding of PPIs and their relation to biological functions. © 2015 The Protein Society.

Functional analyses of the three simian hemorrhagic fever virus nonstructural protein 1 papain-like proteases.

Science.gov (United States)

Vatter, Heather A; Di, Han; Donaldson, Eric F; Radu, Gertrud U; Maines, Taronna R; Brinton, Margo A

2014-08-01

The N-terminal region of simian hemorrhagic fever virus (SHFV) nonstructural polyprotein 1a is predicted to encode three papain-like proteases (PLP1α, PLP1β, and PLP1γ). Catalytic residues and cleavage sites for each of the SHFV PLP1s were predicted by alignment of the SHFV PLP1 region sequences with each other as well as with those of other arteriviruses, and the predicted catalytic residues were shown to be proximal by homology modeling of the SHFV nsp1s on porcine respiratory and reproductive syndrome virus (PRRSV) nsp1 crystal structures. The functionality of the predicted catalytic Cys residues and cleavage sites was tested by analysis of the autoproteolytic products generated in in vitro transcription/translation reactions done with wild-type or mutant SHFV nsp1 constructs. Cleavage sites were also analyzed by mass spectroscopy analysis of selected immunoprecipitated cleavage products. The data showed that each of the three SHFV PLP1s is an active protease. Cys63 was identified as the catalytic Cys of SHFV PLP1α and is adjacent to an Ala instead of the canonical Tyr observed in other arterivirus PLP1s. SHFV PLP1γ is able to cleave at both downstream and upstream nsp1 junction sites. Although intermediate precursor polyproteins as well as alternative products generated by each of the SHFV PLP1s cleaving at sites within the N-terminal region of nsp1β were produced in the in vitro reactions, Western blotting of SHFV-infected, MA104 cell lysates with SHFV nsp1 protein-specific antibodies detected only the three mature nsp1 proteins. SHFV is unique among arteriviruses in having three N-terminal papain-like protease 1 (PLP1) domains. Other arteriviruses encode one or two active PLP1s. This is the first functional study of the SHFV PLP1s. Analysis of the products of in vitro autoprocessing of an N-terminal SHFV nonstructural 1a polypeptide fragment showed that each of the three SHFV PLP1s is active, and the predicted catalytic Cys residues and cleavage sites

Exploring Protein Function Using the Saccharomyces Genome Database.

Science.gov (United States)

Wong, Edith D

2017-01-01

Elucidating the function of individual proteins will help to create a comprehensive picture of cell biology, as well as shed light on human disease mechanisms, possible treatments, and cures. Due to its compact genome, and extensive history of experimentation and annotation, the budding yeast Saccharomyces cerevisiae is an ideal model organism in which to determine protein function. This information can then be leveraged to infer functions of human homologs. Despite the large amount of research and biological data about S. cerevisiae, many proteins' functions remain unknown. Here, we explore ways to use the Saccharomyces Genome Database (SGD; http://www.yeastgenome.org ) to predict the function of proteins and gain insight into their roles in various cellular processes.

C-Terminal Fluorescent Labeling Impairs Functionality of DNA Mismatch Repair Proteins

Science.gov (United States)

Brieger, Angela; Plotz, Guido; Hinrichsen, Inga; Passmann, Sandra; Adam, Ronja; Zeuzem, Stefan

2012-01-01

The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency. PMID:22348133

C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins.

Directory of Open Access Journals (Sweden)

Angela Brieger

Full Text Available The human DNA mismatch repair (MMR process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2. Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.

The cAMP-induced G protein subunits dissociation monitored in live Dictyostelium cells by BRET reveals two activation rates, a positive effect of caffeine and potential role of microtubules.

Science.gov (United States)

Tariqul Islam, A F M; Yue, Haicen; Scavello, Margarethakay; Haldeman, Pearce; Rappel, Wouter-Jan; Charest, Pascale G

2018-08-01

To study the dynamics and mechanisms controlling activation of the heterotrimeric G protein Gα2βγ in Dictyostelium in response to stimulation by the chemoattractant cyclic AMP (cAMP), we monitored the G protein subunit interaction in live cells using bioluminescence resonance energy transfer (BRET). We found that cAMP induces the cAR1-mediated dissociation of the G protein subunits to a similar extent in both undifferentiated and differentiated cells, suggesting that only a small number of cAR1 (as expressed in undifferentiated cells) is necessary to induce the full activation of Gα2βγ. In addition, we found that treating cells with caffeine increases the potency of cAMP-induced Gα2βγ activation; and that disrupting the microtubule network but not F-actin inhibits the cAMP-induced dissociation of Gα2βγ. Thus, microtubules are necessary for efficient cAR1-mediated activation of the heterotrimeric G protein. Finally, kinetics analyses of Gα2βγ subunit dissociation induced by different cAMP concentrations indicate that there are two distinct rates at which the heterotrimeric G protein subunits dissociate when cells are stimulated with cAMP concentrations above 500 nM versus only one rate at lower cAMP concentrations. Quantitative modeling suggests that the kinetics profile of Gα2βγ subunit dissociation results from the presence of both uncoupled and G protein pre-coupled cAR1 that have differential affinities for cAMP and, consequently, induce G protein subunit dissociation through different rates. We suggest that these different signaling kinetic profiles may play an important role in initial chemoattractant gradient sensing. Copyright © 2018 Elsevier Inc. All rights reserved.

Structural domains required for channel function of the mouse transient receptor potential protein homologue TRP1beta.

Science.gov (United States)

Engelke, Michael; Friedrich, Olaf; Budde, Petra; Schäfer, Christina; Niemann, Ursula; Zitt, Christof; Jüngling, Eberhard; Rocks, Oliver; Lückhoff, Andreas; Frey, Jürgen

2002-07-17

Transient receptor potential proteins (TRP) are supposed to participate in the formation of store-operated Ca(2+) influx channels by co-assembly. However, little is known which domains facilitate the interaction of subunits. Contribution of the N-terminal coiled-coil domain and ankyrin-like repeats and the putative pore region of the mouse TRP1beta (mTRP1beta) variant to the formation of functional cation channels were analyzed following overexpression in HEK293 (human embryonic kidney) cells. MTRP1beta expressing cells exhibited enhanced Ca(2+) influx and enhanced whole-cell membrane currents compared to mTRP1beta deletion mutants. Using a yeast two-hybrid assay only the coiled-coil domain facilitated homodimerization of the N-terminus. These results suggest that the N-terminus of mTRP1beta is required for structural organization thus forming functional channels.

Nuclear HMGA1 nonhistone chromatin proteins directly influence mitochondrial transcription, maintenance, and function

International Nuclear Information System (INIS)

Dement, Gregory A.; Maloney, Scott C.; Reeves, Raymond

2007-01-01

We have previously demonstrated that HMGA1 proteins translocate from the nucleus to mitochondria and bind to mitochondrial DNA (mtDNA) at the D-loop control region [G.A. Dement, N.R. Treff, N.S. Magnuson, V. Franceschi, R. Reeves, Dynamic mitochondrial localization of nuclear transcription factor HMGA1, Exp. Cell Res. 307 (2005) 388-401.] [11]. To elucidate possible physiological roles for such binding, we employed methods to analyze mtDNA transcription, mitochondrial maintenance, and other organelle functions in transgenic human MCF-7 cells (HA7C) induced to over-express an HA-tagged HMGA1 protein and control (parental) MCF-7 cells. Quantitative real-time (RT) PCR analyses demonstrated that mtDNA levels were reduced approximately 2-fold in HMGA1 over-expressing HA7C cells and flow cytometric analyses further revealed that mitochondrial mass was significantly reduced in these cells. Cellular ATP levels were also reduced in HA7C cells and survival studies showed an increased sensitivity to killing by 2-deoxy-D-glucose, a glycolysis-specific inhibitor. Flow cytometric analyses revealed additional mitochondrial abnormalities in HA7C cells that are consistent with a cancerous phenotype: namely, increased reactive oxygen species (ROS) and increased mitochondrial membrane potential (ΔΨ m ). Additional RT-PCR analyses demonstrated that gene transcripts from both the heavy (ND2, COXI, ATP6) and light (ND6) strands of mtDNA were up-regulated approximately 3-fold in HA7C cells. Together, these mitochondrial changes are consistent with many previous reports and reveal several possible mechanisms by which HMGA1 over-expression, a common feature of naturally occurring cancers, may affect tumor progression

Protein Function Prediction Based on Sequence and Structure Information

KAUST Repository

Smaili, Fatima Z.

2016-05-25

The number of available protein sequences in public databases is increasing exponentially. However, a significant fraction of these sequences lack functional annotation which is essential to our understanding of how biological systems and processes operate. In this master thesis project, we worked on inferring protein functions based on the primary protein sequence. In the approach we follow, 3D models are first constructed using I-TASSER. Functions are then deduced by structurally matching these predicted models, using global and local similarities, through three independent enzyme commission (EC) and gene ontology (GO) function libraries. The method was tested on 250 “hard” proteins, which lack homologous templates in both structure and function libraries. The results show that this method outperforms the conventional prediction methods based on sequence similarity or threading. Additionally, our method could be improved even further by incorporating protein-protein interaction information. Overall, the method we use provides an efficient approach for automated functional annotation of non-homologous proteins, starting from their sequence.

Dengue Virus Non-structural Protein 1 Modulates Infectious Particle Production via Interaction with the Structural Proteins.

Directory of Open Access Journals (Sweden)

Pietro Scaturro

Full Text Available Non-structural protein 1 (NS1 is one of the most enigmatic proteins of the Dengue virus (DENV, playing distinct functions in immune evasion, pathogenesis and viral replication. The recently reported crystal structure of DENV NS1 revealed its peculiar three-dimensional fold; however, detailed information on NS1 function at different steps of the viral replication cycle is still missing. By using the recently reported crystal structure, as well as amino acid sequence conservation, as a guide for a comprehensive site-directed mutagenesis study, we discovered that in addition to being essential for RNA replication, DENV NS1 is also critically required for the production of infectious virus particles. Taking advantage of a trans-complementation approach based on fully functional epitope-tagged NS1 variants, we identified previously unreported interactions between NS1 and the structural proteins Envelope (E and precursor Membrane (prM. Interestingly, coimmunoprecipitation revealed an additional association with capsid, arguing that NS1 interacts via the structural glycoproteins with DENV particles. Results obtained with mutations residing either in the NS1 Wing domain or in the β-ladder domain suggest that NS1 might have two distinct functions in the assembly of DENV particles. By using a trans-complementation approach with a C-terminally KDEL-tagged ER-resident NS1, we demonstrate that the secretion of NS1 is dispensable for both RNA replication and infectious particle production. In conclusion, our results provide an extensive genetic map of NS1 determinants essential for viral RNA replication and identify a novel role of NS1 in virion production that is mediated via interaction with the structural proteins. These studies extend the list of NS1 functions and argue for a central role in coordinating replication and assembly/release of infectious DENV particles.

The function of the RNA-binding protein TEL1 in moss reveals ancient regulatory mechanisms of shoot development.

Science.gov (United States)

Vivancos, Julien; Spinner, Lara; Mazubert, Christelle; Charlot, Florence; Paquet, Nicolas; Thareau, Vincent; Dron, Michel; Nogué, Fabien; Charon, Céline

2012-03-01

The shoot represents the basic body plan in land plants. It consists of a repeated structure composed of stems and leaves. Whereas vascular plants generate a shoot in their diploid phase, non-vascular plants such as mosses form a shoot (called the gametophore) in their haploid generation. The evolution of regulatory mechanisms or genetic networks used in the development of these two kinds of shoots is unclear. TERMINAL EAR1-like genes have been involved in diploid shoot development in vascular plants. Here, we show that disruption of PpTEL1 from the moss Physcomitrella patens, causes reduced protonema growth and gametophore initiation, as well as defects in gametophore development. Leafy shoots formed on ΔTEL1 mutants exhibit shorter stems with more leaves per shoot, suggesting an accelerated leaf initiation (shortened plastochron), a phenotype shared with the Poaceae vascular plants TE1 and PLA2/LHD2 mutants. Moreover, the positive correlation between plastochron length and leaf size observed in ΔTEL1 mutants suggests a conserved compensatory mechanism correlating leaf growth and leaf initiation rate that would minimize overall changes in plant biomass. The RNA-binding protein encoded by PpTEL1 contains two N-terminus RNA-recognition motifs, and a third C-terminus non-canonical RRM, specific to TEL proteins. Removal of the PpTEL1 C-terminus (including this third RRM) or only 16-18 amino acids within it seriously impairs PpTEL1 function, suggesting a critical role for this third RRM. These results show a conserved function of the RNA-binding PpTEL1 protein in the regulation of shoot development, from early ancestors to vascular plants, that depends on the third TEL-specific RRM.

Unveiling protein functions through the dynamics of the interaction network.

Directory of Open Access Journals (Sweden)

Irene Sendiña-Nadal

Full Text Available Protein interaction networks have become a tool to study biological processes, either for predicting molecular functions or for designing proper new drugs to regulate the main biological interactions. Furthermore, such networks are known to be organized in sub-networks of proteins contributing to the same cellular function. However, the protein function prediction is not accurate and each protein has traditionally been assigned to only one function by the network formalism. By considering the network of the physical interactions between proteins of the yeast together with a manual and single functional classification scheme, we introduce a method able to reveal important information on protein function, at both micro- and macro-scale. In particular, the inspection of the properties of oscillatory dynamics on top of the protein interaction network leads to the identification of misclassification problems in protein function assignments, as well as to unveil correct identification of protein functions. We also demonstrate that our approach can give a network representation of the meta-organization of biological processes by unraveling the interactions between different functional classes.

Partial dispensability of Djp1's J domain in peroxisomal protein import in Saccharomyces cerevisiae results from genetic redundancy with another class II J protein, Caj1.

Science.gov (United States)

Dobriyal, Neha; Tripathi, Prerna; Sarkar, Susrita; Tak, Yogesh; Verma, Amit K; Sahi, Chandan

2017-05-01

J proteins are obligate co-chaperones of Hsp70s. Via their signature J domain, all J proteins interact with their partner Hsp70s and stimulate their weak ATPase activity, which is vital for Hsp70 functions. The dependency of J proteins on their J domain is such that mutations in critical amino acids in the J domain often results into a null phenotype for a particular J protein. Here, we show that the J domain of Djp1, a cytosolic J protein important for peroxisomal protein import in Saccharomyces cerevisiae, is partially dispensable. A complete deletion of Djp1 J domain resulted into only partial loss in peroxisomal protein import function. Instead, the C-terminal domain of Djp1 was found to be essential for proper localization of the peroxisomal targeted GFP-PTS1. Furthermore, we show that Caj1, another cytosolic J protein, also has some role in peroxisomal protein import. Caj1 was found to be partially redundant with Djp1 as cells lacking both Djp1 and Caj1 resulted into a much more severe defect in GFP-PTS1 localization. Based on these results, we propose that dispensability of J domains could be attributed to genetic redundancy between different J proteins sharing common structural topology and cellular localization.

Arabidopsis protein phosphatase DBP1 nucleates a protein network with a role in regulating plant defense.

Directory of Open Access Journals (Sweden)

José Luis Carrasco

Full Text Available Arabidopsis thaliana DBP1 belongs to the plant-specific family of DNA-binding protein phosphatases. Although recently identified as a novel host factor mediating susceptibility to potyvirus, little is known about DBP1 targets and partners and the molecular mechanisms underlying its function. Analyzing changes in the phosphoproteome of a loss-of-function dbp1 mutant enabled the identification of 14-3-3λ isoform (GRF6, a previously reported DBP1 interactor, and MAP kinase (MAPK MPK11 as components of a small protein network nucleated by DBP1, in which GRF6 stability is modulated by MPK11 through phosphorylation, while DBP1 in turn negatively regulates MPK11 activity. Interestingly, grf6 and mpk11 loss-of-function mutants showed altered response to infection by the potyvirus Plum pox virus (PPV, and the described molecular mechanism controlling GRF6 stability was recapitulated upon PPV infection. These results not only contribute to a better knowledge of the biology of DBP factors, but also of MAPK signalling in plants, with the identification of GRF6 as a likely MPK11 substrate and of DBP1 as a protein phosphatase regulating MPK11 activity, and unveils the implication of this protein module in the response to PPV infection in Arabidopsis.

Biomimetic devices functionalized by membrane channel proteins

Science.gov (United States)

Schmidt, Jacob

2004-03-01

We are developing a new family of active materials which derive their functional properties from membrane proteins. These materials have two primary components: the proteins and the membranes themselves. I will discuss our recent work directed toward development of a generic platform for a "plug-and-play" philosophy of membrane protein engineering. By creating a stable biomimetic polymer membrane a single molecular monolayer thick, we will enable the exploitation of the function of any membrane protein, from pores and pumps to sensors and energy transducers. Our initial work has centered on the creation, study, and characterization of the biomimetic membranes. We are attempting to make large areas of membrane monolayers using Langmuir-Blodgett film formation as well as through arrays of microfabricated black lipid membrane-type septa. A number of techniques allow the insertion of protein into the membranes. As a benchmark, we have been employing a model system of voltage-gated pore proteins, which have electrically controllable porosities. I will report on the progress of this work, the characterization of the membranes, protein insertion processes, and the yield and functionality of the composite.

Improving protein function prediction methods with integrated literature data

Directory of Open Access Journals (Sweden)

Gabow Aaron P

2008-04-01

Full Text Available Abstract Background Determining the function of uncharacterized proteins is a major challenge in the post-genomic era due to the problem's complexity and scale. Identifying a protein's function contributes to an understanding of its role in the involved pathways, its suitability as a drug target, and its potential for protein modifications. Several graph-theoretic approaches predict unidentified functions of proteins by using the functional annotations of better-characterized proteins in protein-protein interaction networks. We systematically consider the use of literature co-occurrence data, introduce a new method for quantifying the reliability of co-occurrence and test how performance differs across species. We also quantify changes in performance as the prediction algorithms annotate with increased specificity. Results We find that including information on the co-occurrence of proteins within an abstract greatly boosts performance in the Functional Flow graph-theoretic function prediction algorithm in yeast, fly and worm. This increase in performance is not simply due to the presence of additional edges since supplementing protein-protein interactions with co-occurrence data outperforms supplementing with a comparably-sized genetic interaction dataset. Through the combination of protein-protein interactions and co-occurrence data, the neighborhood around unknown proteins is quickly connected to well-characterized nodes which global prediction algorithms can exploit. Our method for quantifying co-occurrence reliability shows superior performance to the other methods, particularly at threshold values around 10% which yield the best trade off between coverage and accuracy. In contrast, the traditional way of asserting co-occurrence when at least one abstract mentions both proteins proves to be the worst method for generating co-occurrence data, introducing too many false positives. Annotating the functions with greater specificity is harder

Organ accumulation and subcellular location of Cicer arietinum ST1 protein.

Science.gov (United States)

Albornos, Lucía; Cabrera, Javier; Hernández-Nistal, Josefina; Martín, Ignacio; Labrador, Emilia; Dopico, Berta

2014-07-01

The ST (ShooT Specific) proteins are a new family of proteins characterized by a signal peptide, tandem repeats of 25/26 amino acids, and a domain of unknown function (DUF2775), whose presence is limited to a few families of dicotyledonous plants, mainly Fabaceae and Asteraceae. Their function remains unknown, although involvement in plant growth, fruit morphogenesis or in biotic and abiotic interactions have been suggested. This work is focused on ST1, a Cicer arietinum ST protein. We established the protein accumulation in different tissues and organs of chickpea seedlings and plants and its subcellular localization, which could indicate the possible function of ST1. The raising of specific antibodies against ST1 protein revealed that its accumulation in epicotyls and radicles was related to their elongation rate. Its pattern of tissue location in cotyledons during seed formation and early seed germination, as well as its localization in the perivascular fibres of epicotyls and radicles, indicated a possible involvement in seed germination and seedling growth. ST1 protein appears both inside the cell and in the cell wall. This double subcellular localization was found in every organ in which the ST1 protein was detected: seeds, cotyledons and seedling epicotyls and radicles. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Th1 cytokine-induced syndecan-4 shedding by airway smooth muscle cells is dependent on mitogen-activated protein kinases.

Science.gov (United States)

Tan, Xiahui; Khalil, Najwa; Tesarik, Candice; Vanapalli, Karunasri; Yaputra, Viki; Alkhouri, Hatem; Oliver, Brian G G; Armour, Carol L; Hughes, J Margaret

2012-04-01

In asthma, airway smooth muscle (ASM) chemokine secretion can induce mast cell recruitment into the airways. The functions of the mast cell chemoattractant CXCL10, and other chemokines, are regulated by binding to heparan sulphates such as syndecan-4. This study is the first demonstration that airway smooth muscle cells (ASMC) from people with and without asthma express and shed syndecan-4 under basal conditions. Syndecan-4 shedding was enhanced by stimulation for 24 h with the Th1 cytokines interleukin-1β (IL-1β) or tumor necrosis factor-α (TNF-α), but not interferon-γ (IFNγ), nor the Th2 cytokines IL-4 and IL-13. ASMC stimulation with IL-1β, TNF-α, and IFNγ (cytomix) induced the highest level of syndecan-4 shedding. Nonasthmatic and asthmatic ASM cell-associated syndecan-4 protein expression was also increased by TNF-α or cytomix at 4-8 h, with the highest levels detected in cytomix-stimulated asthmatic cells. Cell-associated syndecan-4 levels were decreased by 24 h, whereas shedding remained elevated at 24 h, consistent with newly synthesized syndecan-4 being shed. Inhibition of ASMC matrix metalloproteinase-2 did not prevent syndecan-4 shedding, whereas inhibition of ERK MAPK activation reduced shedding from cytomix-stimulated ASMC. Although ERK inhibition had no effect on syndecan-4 mRNA levels stimulated by cytomix, it did cause an increase in cell-associated syndecan-4 levels, consistent with the shedding being inhibited. In conclusion, ASMC produce and shed syndecan-4 and although this is increased by the Th1 cytokines, the MAPK ERK only regulates shedding. ASMC syndecan-4 production during Th1 inflammatory conditions may regulate chemokine activity and mast cell recruitment to the ASM in asthma.

The functional interplay between protein kinase CK2 and CCA1 transcriptional activity is essential for clock temperature compensation in Arabidopsis.

Directory of Open Access Journals (Sweden)

Sergi Portolés

2010-11-01

Full Text Available Circadian rhythms are daily biological oscillations driven by an endogenous mechanism known as circadian clock. The protein kinase CK2 is one of the few clock components that is evolutionary conserved among different taxonomic groups. CK2 regulates the stability and nuclear localization of essential clock proteins in mammals, fungi, and insects. Two CK2 regulatory subunits, CKB3 and CKB4, have been also linked with the Arabidopsis thaliana circadian system. However, the biological relevance and the precise mechanisms of CK2 function within the plant clockwork are not known. By using ChIP and Double-ChIP experiments together with in vivo luminescence assays at different temperatures, we were able to identify a temperature-dependent function for CK2 modulating circadian period length. Our study uncovers a previously unpredicted mechanism for CK2 antagonizing the key clock regulator CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1. CK2 activity does not alter protein accumulation or subcellular localization but interferes with CCA1 binding affinity to the promoters of the oscillator genes. High temperatures enhance the CCA1 binding activity, which is precisely counterbalanced by the CK2 opposing function. Altering this balance by over-expression, mutation, or pharmacological inhibition affects the temperature compensation profile, providing a mechanism by which plants regulate circadian period at changing temperatures. Therefore, our study establishes a new model demonstrating that two opposing and temperature-dependent activities (CCA1-CK2 are essential for clock temperature compensation in Arabidopsis.

Characterization of MYG1 gene and protein: subcellular distribution and function

DEFF Research Database (Denmark)

Philips, Mari-Anne; Vikeså, Jonas; Luuk, Hendrik

2009-01-01

. CONCLUSIONS: Taken together, we infer that MYG1 is a ubiquitous nucleo-mitochondrial protein, with differential pattern and level of expression during embryonic development. MYG1 expression in normal adult tissues is stable and our data suggest MYG1 involvement in early developmental processes and also...

Optimization of functionalization conditions for protein analysis by AFM

Energy Technology Data Exchange (ETDEWEB)

Arroyo-Hernández, María, E-mail: maria.arroyo@ctb.upm.es [Centro de Tecnología Biomédica, Universidad Politécnica de Madrid, 28223 Pozuelo de Alarcón, Madrid (Spain); Departamento de Ciencia de Materiales, ETSI Caminos, Canales y Puertos, Universidad Politécnica de Madrid, 28040 Madrid (Spain); Daza, Rafael; Pérez-Rigueiro, Jose; Elices, Manuel; Nieto-Márquez, Jorge; Guinea, Gustavo V. [Centro de Tecnología Biomédica, Universidad Politécnica de Madrid, 28223 Pozuelo de Alarcón, Madrid (Spain); Departamento de Ciencia de Materiales, ETSI Caminos, Canales y Puertos, Universidad Politécnica de Madrid, 28040 Madrid (Spain)

2014-10-30

Highlights: • Highest fluorescence is obtained for central conditions. • Largest primary amine contribution is obtained for central conditions. • RMS roughness is smaller than 1 nm for all functional films. • Selected deposition conditions lead to proper RMS and functionality values. • LDH proteins adsorbed on AVS-films were observed by AFM. - Abstract: Activated vapor silanization (AVS) is used to functionalize silicon surfaces through deposition of amine-containing thin films. AVS combines vapor silanization and chemical vapor deposition techniques and allows the properties of the functionalized layers (thickness, amine concentration and topography) to be controlled by tuning the deposition conditions. An accurate characterization is performed to correlate the deposition conditions and functional-film properties. In particular, it is shown that smooth surfaces with a sufficient surface density of amine groups may be obtained with this technique. These surfaces are suitable for the study of proteins with atomic force microscopy.

Studying Membrane Protein Structure and Function Using Nanodiscs

DEFF Research Database (Denmark)

Huda, Pie

The structure and dynamic of membrane proteins can provide valuable information about general functions, diseases and effects of various drugs. Studying membrane proteins are a challenge as an amphiphilic environment is necessary to stabilise the protein in a functionally and structurally relevant...... form. This is most typically achieved through the use of detergent based reconstitution systems. However, time and again such systems fail to provide a suitable environment causing aggregation and inactivation. Nanodiscs are self-assembled lipoproteins containing two membrane scaffold proteins...... and a lipid bilayer in defined nanometer size, which can act as a stabiliser for membrane proteins. This enables both functional and structural investigation of membrane proteins in a detergent free environment which is closer to the native situation. Understanding the self-assembly of nanodiscs is important...

Functions of the CCCH type zinc finger protein OsGZF1 in regulation of the seed storage protein GluB-1 from rice

NARCIS (Netherlands)

Chen, Y.; Sun, A.; Wang, M.; Zhu, Z.; Ouwerkerk, P.B.F.

2014-01-01

Glutelins are the most abundant storage proteins in rice grain and can make up to 80 % of total protein content. The promoter region of GluB-1, one of the glutelin genes in rice, has been intensively used as a model to understand regulation of seed-storage protein accumulation. In this study, we

Crystal Structure of PAV1-137: A Protein from the Virus PAV1 That Infects Pyrococcus abyssi

Directory of Open Access Journals (Sweden)

N. Leulliot

2013-01-01

Full Text Available Pyrococcus abyssi virus 1 (PAV1 was the first virus particle infecting a hyperthermophilic Euryarchaeota (Pyrococcus abyssi strain GE23 that has been isolated and characterized. It is lemon shaped and is decorated with a short fibered tail. PAV1 morphologically resembles the fusiform members of the family Fuselloviridae or the genus Salterprovirus. The 18 kb dsDNA genome of PAV1 contains 25 predicted genes, most of them of unknown function. To help assigning functions to these proteins, we have initiated structural studies of the PAV1 proteome. We determined the crystal structure of a putative protein of 137 residues (PAV1-137 at a resolution of 2.2 Å. The protein forms dimers both in solution and in the crystal. The fold of PAV1-137 is a four-α-helical bundle analogous to those found in some eukaryotic adhesion proteins such as focal adhesion kinase, suggesting that PAV1-137 is involved in protein-protein interactions.

A comprehensive protein-protein interactome for yeast PAS kinase 1 reveals direct inhibition of respiration through the phosphorylation of Cbf1.

Science.gov (United States)

DeMille, Desiree; Bikman, Benjamin T; Mathis, Andrew D; Prince, John T; Mackay, Jordan T; Sowa, Steven W; Hall, Tacie D; Grose, Julianne H

2014-07-15

Per-Arnt-Sim (PAS) kinase is a sensory protein kinase required for glucose homeostasis in yeast, mice, and humans, yet little is known about the molecular mechanisms of its function. Using both yeast two-hybrid and copurification approaches, we identified the protein-protein interactome for yeast PAS kinase 1 (Psk1), revealing 93 novel putative protein binding partners. Several of the Psk1 binding partners expand the role of PAS kinase in glucose homeostasis, including new pathways involved in mitochondrial metabolism. In addition, the interactome suggests novel roles for PAS kinase in cell growth (gene/protein expression, replication/cell division, and protein modification and degradation), vacuole function, and stress tolerance. In vitro kinase studies using a subset of 25 of these binding partners identified Mot3, Zds1, Utr1, and Cbf1 as substrates. Further evidence is provided for the in vivo phosphorylation of Cbf1 at T211/T212 and for the subsequent inhibition of respiration. This respiratory role of PAS kinase is consistent with the reported hypermetabolism of PAS kinase-deficient mice, identifying a possible molecular mechanism and solidifying the evolutionary importance of PAS kinase in the regulation of glucose homeostasis. © 2014 DeMille et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Lacritin and other new proteins of the lacrimal functional unit.

Science.gov (United States)

McKown, Robert L; Wang, Ningning; Raab, Ronald W; Karnati, Roy; Zhang, Yinghui; Williams, Patricia B; Laurie, Gordon W

2009-05-01

The lacrimal functional unit (LFU) is defined by the 2007 International Dry Eye WorkShop as 'an integrated system comprising the lacrimal glands, ocular surface (cornea, conjunctiva and meibomian glands) and lids, and the sensory and motor nerves that connect them'. The LFU maintains a healthy ocular surface primarily through a properly functioning tear film that provides protection, lubrication, and an environment for corneal epithelial cell renewal. LFU cells express thousands of proteins. Over 200 new LFU proteins have been discovered in the last decade. Lacritin is a new LFU-specific growth factor in human tears that flows through ducts to target corneal epithelial cells on the ocular surface. When applied topically in rabbits, lacritin appears to increase the volume of basal tear secretion. Lacritin is one of only a handful of tear proteins preliminarily reported to be downregulated in blepharitis and in two dry eye syndromes. Computational analysis predicts an ordered C-terminal domain that binds the corneal epithelial cell surface proteoglycan syndecan-1 (SDC1) and is required for lacritin's low nanomolar mitogenic activity. The lacritin-binding site on the N-terminus of SDC1 is exposed by heparanase. Heparanase is constitutively expressed by the corneal epithelium and appears to be a normal constituent of tears. Binding triggers rapid signaling to downstream NFAT and mTOR. A wealth of other new proteins, originally designated as hypothetical when first identified by genomic sequencing, are expressed by the human LFU including: ALS2CL, ARHGEF19, KIAA1109, PLXNA1, POLG, WIPI1 and ZMIZ2. Their demonstrated or implied roles in human genetic disease or basic cellular functions are fuel for new investigation. Addressing topical areas in ocular surface physiology with new LFU proteins may reveal interesting new biological mechanisms and help get to the heart of ocular surface dysfunction.

Structure-based inference of molecular functions of proteins of unknown function from Berkeley Structural Genomics Center

Energy Technology Data Exchange (ETDEWEB)

Kim, Sung-Hou; Shin, Dong Hae; Hou, Jingtong; Chandonia, John-Marc; Das, Debanu; Choi, In-Geol; Kim, Rosalind; Kim, Sung-Hou

2007-09-02

Advances in sequence genomics have resulted in an accumulation of a huge number of protein sequences derived from genome sequences. However, the functions of a large portion of them cannot be inferred based on the current methods of sequence homology detection to proteins of known functions. Three-dimensional structure can have an important impact in providing inference of molecular function (physical and chemical function) of a protein of unknown function. Structural genomics centers worldwide have been determining many 3-D structures of the proteins of unknown functions, and possible molecular functions of them have been inferred based on their structures. Combined with bioinformatics and enzymatic assay tools, the successful acceleration of the process of protein structure determination through high throughput pipelines enables the rapid functional annotation of a large fraction of hypothetical proteins. We present a brief summary of the process we used at the Berkeley Structural Genomics Center to infer molecular functions of proteins of unknown function.

The fission yeast minichromosome maintenance (MCM)-binding protein (MCM-BP), Mcb1, regulates MCM function during prereplicative complex formation in DNA replication.

Science.gov (United States)

Santosa, Venny; Martha, Sabrina; Hirose, Noriaki; Tanaka, Katsunori

2013-03-08

The minichromosome maintenance (MCM) complex is a replicative helicase, which is essential for chromosome DNA replication. In recent years, the identification of a novel MCM-binding protein (MCM-BP) in most eukaryotes has led to numerous studies investigating its function and its relationship to the MCM complex. However, the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood; in addition, the functional role of MCM-BP remains controversial and may vary between model organisms. The present study aims to elucidate the nature and biological function of the MCM-BP ortholog, Mcb1, in fission yeast. The Mcb1 protein continuously interacts with MCM proteins during the cell cycle in vivo and can interact with any individual MCM subunit in vitro. To understand the detailed characteristics of mcb1(+), two temperature-sensitive mcb1 gene mutants (mcb1(ts)) were isolated. Extensive genetic analysis showed that the mcb1(ts) mutants were suppressed by a mcm5(+) multicopy plasmid and displayed synthetic defects with many S-phase-related gene mutants. Moreover, cyclin-dependent kinase modulation by Cig2 repression or Rum1 overproduction suppressed the mcb1(ts) mutants, suggesting the involvement of Mcb1 in pre-RC formation during DNA replication. These data are consistent with the observation that Mcm7 loading onto replication origins is reduced and S-phase progression is delayed in mcb1(ts) mutants. Furthermore, the mcb1(ts) mutation led to the redistribution of MCM subunits to the cytoplasm, and this redistribution was dependent on an active nuclear export system. These results strongly suggest that Mcb1 promotes efficient pre-RC formation during DNA replication by regulating the MCM complex.

The Fission Yeast Minichromosome Maintenance (MCM)-binding Protein (MCM-BP), Mcb1, Regulates MCM Function during Prereplicative Complex Formation in DNA Replication*

Science.gov (United States)

Santosa, Venny; Martha, Sabrina; Hirose, Noriaki; Tanaka, Katsunori

2013-01-01

The minichromosome maintenance (MCM) complex is a replicative helicase, which is essential for chromosome DNA replication. In recent years, the identification of a novel MCM-binding protein (MCM-BP) in most eukaryotes has led to numerous studies investigating its function and its relationship to the MCM complex. However, the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood; in addition, the functional role of MCM-BP remains controversial and may vary between model organisms. The present study aims to elucidate the nature and biological function of the MCM-BP ortholog, Mcb1, in fission yeast. The Mcb1 protein continuously interacts with MCM proteins during the cell cycle in vivo and can interact with any individual MCM subunit in vitro. To understand the detailed characteristics of mcb1+, two temperature-sensitive mcb1 gene mutants (mcb1ts) were isolated. Extensive genetic analysis showed that the mcb1ts mutants were suppressed by a mcm5+ multicopy plasmid and displayed synthetic defects with many S-phase-related gene mutants. Moreover, cyclin-dependent kinase modulation by Cig2 repression or Rum1 overproduction suppressed the mcb1ts mutants, suggesting the involvement of Mcb1 in pre-RC formation during DNA replication. These data are consistent with the observation that Mcm7 loading onto replication origins is reduced and S-phase progression is delayed in mcb1ts mutants. Furthermore, the mcb1ts mutation led to the redistribution of MCM subunits to the cytoplasm, and this redistribution was dependent on an active nuclear export system. These results strongly suggest that Mcb1 promotes efficient pre-RC formation during DNA replication by regulating the MCM complex. PMID:23322785

Functional diversification of hsp40: distinct j-protein functional requirements for two prions allow for chaperone-dependent prion selection.

Science.gov (United States)

Harris, Julia M; Nguyen, Phil P; Patel, Milan J; Sporn, Zachary A; Hines, Justin K

2014-07-01

Yeast prions are heritable amyloid aggregates of functional yeast proteins; their propagation to subsequent cell generations is dependent upon fragmentation of prion protein aggregates by molecular chaperone proteins. Mounting evidence indicates the J-protein Sis1 may act as an amyloid specificity factor, recognizing prion and other amyloid aggregates and enabling Ssa and Hsp104 to act in prion fragmentation. Chaperone interactions with prions, however, can be affected by variations in amyloid-core structure resulting in distinct prion variants or 'strains'. Our genetic analysis revealed that Sis1 domain requirements by distinct variants of [PSI+] are strongly dependent upon overall variant stability. Notably, multiple strong [PSI+] variants can be maintained by a minimal construct of Sis1 consisting of only the J-domain and glycine/phenylalanine-rich (G/F) region that was previously shown to be sufficient for cell viability and [RNQ+] prion propagation. In contrast, weak [PSI+] variants are lost under the same conditions but maintained by the expression of an Sis1 construct that lacks only the G/F region and cannot support [RNQ+] propagation, revealing mutually exclusive requirements for Sis1 function between these two prions. Prion loss is not due to [PSI+]-dependent toxicity or dependent upon a particular yeast genetic background. These observations necessitate that Sis1 must have at least two distinct functional roles that individual prions differentially require for propagation and which are localized to the glycine-rich domains of the Sis1. Based on these distinctions, Sis1 plasmid-shuffling in a [PSI+]/[RNQ+] strain permitted J-protein-dependent prion selection for either prion. We also found that, despite an initial report to the contrary, the human homolog of Sis1, Hdj1, is capable of [PSI+] prion propagation in place of Sis1. This conservation of function is also prion-variant dependent, indicating that only one of the two Sis1-prion functions may have

Mimic Phosphorylation of a βC1 Protein Encoded by TYLCCNB Impairs Its Functions as a Viral Suppressor of RNA Silencing and a Symptom Determinant.

Science.gov (United States)

Zhong, Xueting; Wang, Zhan Qi; Xiao, Ruyuan; Cao, Linge; Wang, Yaqin; Xie, Yan; Zhou, Xueping

2017-08-15

Phosphorylation of the βC1 protein encoded by the betasatellite of tomato yellow leaf curl China virus (TYLCCNB-βC1) by SNF1-related protein kinase 1 (SnRK1) plays a critical role in defense of host plants against geminivirus infection in Nicotiana benthamiana However, how phosphorylation of TYLCCNB-βC1 impacts its pathogenic functions during viral infection remains elusive. In this study, we identified two additional tyrosine residues in TYLCCNB-βC1 that are phosphorylated by SnRK1. The effects of TYLCCNB-βC1 phosphorylation on its functions as a viral suppressor of RNA silencing (VSR) and a symptom determinant were investigated via phosphorylation mimic mutants in N. benthamiana plants. Mutations that mimic phosphorylation of TYLCCNB-βC1 at tyrosine 5 and tyrosine 110 attenuated disease symptoms during viral infection. The phosphorylation mimics weakened the ability of TYLCCNB-βC1 to reverse transcriptional gene silencing and to suppress posttranscriptional gene silencing and abolished its interaction with N. benthamiana ASYMMETRIC LEAVES 1 in N. benthamiana leaves. The mimic phosphorylation of TYLCCNB-βC1 had no impact on its protein stability, subcellular localization, or self-association. Our data establish an inhibitory effect of phosphorylation of TYLCCNB-βC1 on its pathogenic functions as a VSR and a symptom determinant and provide a mechanistic explanation of how SnRK1 functions as a host defense factor. IMPORTANCE Tomato yellow leaf curl China virus (TYLCCNV), which causes a severe yellow leaf curl disease in China, is a monopartite geminivirus associated with the betasatellite (TYLCCNB). TYLCCNB encodes a single pathogenicity protein, βC1 (TYLCCNB-βC1), which functions as both a viral suppressor of RNA silencing (VSR) and a symptom determinant. Here, we show that mimicking phosphorylation of TYLCCNB-βC1 weakens its ability to reverse transcriptional gene silencing, to suppress posttranscriptional gene silencing, and to interact with N

Analysis of substructural variation in families of enzymatic proteins with applications to protein function prediction

Directory of Open Access Journals (Sweden)

Fofanov Viacheslav Y

2010-05-01

Full Text Available Abstract Background Structural variations caused by a wide range of physico-chemical and biological sources directly influence the function of a protein. For enzymatic proteins, the structure and chemistry of the catalytic binding site residues can be loosely defined as a substructure of the protein. Comparative analysis of drug-receptor substructures across and within species has been used for lead evaluation. Substructure-level similarity between the binding sites of functionally similar proteins has also been used to identify instances of convergent evolution among proteins. In functionally homologous protein families, shared chemistry and geometry at catalytic sites provide a common, local point of comparison among proteins that may differ significantly at the sequence, fold, or domain topology levels. Results This paper describes two key results that can be used separately or in combination for protein function analysis. The Family-wise Analysis of SubStructural Templates (FASST method uses all-against-all substructure comparison to determine Substructural Clusters (SCs. SCs characterize the binding site substructural variation within a protein family. In this paper we focus on examples of automatically determined SCs that can be linked to phylogenetic distance between family members, segregation by conformation, and organization by homology among convergent protein lineages. The Motif Ensemble Statistical Hypothesis (MESH framework constructs a representative motif for each protein cluster among the SCs determined by FASST to build motif ensembles that are shown through a series of function prediction experiments to improve the function prediction power of existing motifs. Conclusions FASST contributes a critical feedback and assessment step to existing binding site substructure identification methods and can be used for the thorough investigation of structure-function relationships. The application of MESH allows for an automated

Association of protein structure, protein and carbohydrate subfractions with bioenergy profiles and biodegradation functions in modeled forage

Science.gov (United States)

Ji, Cuiying; Zhang, Xuewei; Yu, Peiqiang

2016-03-01

The objectives of this study were to detect unique aspects and association of forage protein inherent structure, biological compounds, protein and carbohydrate subfractions, bioenergy profiles, and biodegradation features. In this study, common available alfalfa hay from two different sourced-origins (FSO vs. CSO) was used as a modeled forage for inherent structure profile, bioenergy, biodegradation and their association between their structure and bio-functions. The molecular spectral profiles were determined using non-invasive molecular spectroscopy. The parameters included: protein structure amide I group, amide II group and their ratios; protein subfractions (PA1, PA2, PB1, PB2, PC); carbohydrate fractions (CA1, CA2, CA3, CA4, CB1, CB2, CC); biodegradable and undegradable fractions of protein (RDPA2, RDPB1, RDPB2, RDP; RUPA2 RUPB1, RUPB2, RUPC, RUP); biodegradable and undegradable fractions of carbohydrate (RDCA4, RDCB1, RDCB2, RDCB3, RDCHO; RUCA4, RUCB1; RUCB2; RUCB3 RUCC, RUCHO) and bioenergy profiles (tdNDF, tdFA, tdCP, tdNFC, TDN1 ×, DE3 ×, ME3 ×, NEL3 ×; NEm, NEg). The results show differences in protein and carbohydrate (CHO) subfractions in the moderately degradable true protein fraction (PB1: 502 vs. 420 g/kg CP, P = 0.09), slowly degraded true protein fraction (PB2: 45 vs. 96 g/kg CP, P = 0.02), moderately degradable CHO fraction (CB2: 283 vs. 223 g/kg CHO, P = 0.06) and slowly degraded CHO fraction (CB3: 369 vs. 408 g/kg CHO) between the two sourced origins. As to biodegradable (RD) fractions of protein and CHO in rumen, there were differences in RD of PB1 (417 vs. 349 g/kg CP, P = 0.09), RD of PB2 (29 vs. 62 g/kg CP, P = 0.02), RD of CB2 (251 vs. 198 g/kg DM, P = 0.06), RD of CB3 (236 vs. 261 g/kg CHO, P = 0.08). As to bioenergy profile, there were differences in total digestible nutrient (TDN: 551 vs. 537 g/kg DM, P = 0.06), and metabolic bioenergy (P = 0.095). As to protein molecular structure, there were differences in protein structure 1st

Decreased function of survival motor neuron protein impairs endocytic pathways.