Interaction of AIM with insulin-like growth factor-binding protein-4.

PubMed

You, Qiang; Wu, Yan; Yao, Nannan; Shen, Guannan; Zhang, Ying; Xu, Liangguo; Li, Guiying; Ju, Cynthia

2015-09-01

Apoptosis inhibitor of macrophages (AIM/cluster of differentiation 5 antigen-like/soluble protein α) has been shown to inhibit cellular apoptosis; however, the underlying molecular mechanism has not been elucidated. Using yeast two‑hybrid screening, the present study uncovered that AIM binds to insulin‑like growth factor binding protein‑4 (IGFBP‑4). AIM interaction with IGFBP‑4, as well as IGFBP‑2 and ‑3, but not with IGFBP‑1, ‑5 and ‑6, was further confirmed by co‑immunoprecipitation (co‑IP) using 293 cells. The binding activity and affinity between AIM and IGFBP‑4 in vitro were analyzed by co‑IP and biolayer interferometry. Serum depletion‑induced cellular apoptosis was attenuated by insulin‑like growth factor‑I (IGF‑I), and this effect was abrogated by IGFBP‑4. Of note, in the presence of AIM, the inhibitory effect of IGFBP‑4 on the anti‑apoptosis function of IGF‑I was attenuated, possibly through binding of AIM with IGFBP‑4. In conclusion, to the best of our knowledge, the present study provides the first evidence that AIM binds to IGFBP‑2, ‑3 and ‑4. The data suggest that this interaction may contribute to the mechanism of AIM-mediated anti-apoptosis function.

Mutant protein of recombinant human granulocyte colony-stimulating factor for receptor binding assay.

PubMed

Watanabe, M; Fukamachi, H; Uzumaki, H; Kabaya, K; Tsumura, H; Ishikawa, M; Matsuki, S; Kusaka, M

1991-05-15

A new mutant protein of recombinant human granulocyte colony-stimulating factor (rhG-CSF) was produced for the studies on receptors for human G-CSF. The mutant protein [(Tyr1, Tyr3]rhG-CSF), the biological activity of which was almost equal to that of rhG-CSF, was prepared by the replacement of threonine-1 and leucine-3 of rhG-CSF with tyrosine. The radioiodinated preparation of the mutant protein showed high specific radioactivity and retained full biological activity for at least 3 weeks. The binding capacity of the radioiodinated ligand was compared with that of [35S]rhG-CSF. Both radiolabeled ligands showed specific binding to murine bone marrow cells. Unlabeled rhG-CSF and human G-CSF purified from the culture supernatant of the human bladder carcinoma cell line 5637 equally competed for the binding of labeled rhG-CSFs in a dose-dependent manner, demonstrating that the sugar moiety of human G-CSF made no contribution to the binding of human G-CSF to target cells. In contrast, all other colony-stimulating factors and lymphokines examined did not affect the binding. Scatchard analysis of the specific binding of both labeled ligands revealed a single class of binding site with an apparent dissociation constant (Kd) of 20-30 pM and 100-200 maximal binding sites per cell. These data indicate that the radioiodinated preparation of the mutant protein binds the same specific receptor with the same affinity as [35S]rhG-CSF. The labeled mutant protein also showed specific binding to human circulating neutrophils.(ABSTRACT TRUNCATED AT 250 WORDS)

Competition between Ski and CREB-binding protein for binding to Smad proteins in transforming growth factor-beta signaling.

PubMed

Chen, Weijun; Lam, Suvana S; Srinath, Hema; Schiffer, Celia A; Royer, William E; Lin, Kai

2007-04-13

The family of Smad proteins mediates transforming growth factor-beta (TGF-beta) signaling in cell growth and differentiation. Smads repress or activate TGF-beta signaling by interacting with corepressors (e.g. Ski) or coactivators (e.g. CREB-binding protein (CBP)), respectively. Specifically, Ski has been shown to interfere with the interaction between Smad3 and CBP. However, it is unclear whether Ski competes with CBP for binding to Smads and whether they can interact with Smad3 at the same binding surface on Smad3. We investigated the interactions among purified constructs of Smad, Ski, and CBP in vitro by size-exclusion chromatography, isothermal titration calorimetry, and mutational studies. Here, we show that Ski-(16-192) interacted directly with a homotrimer of receptor-regulated Smad protein (R-Smad), e.g. Smad2 or Smad3, to form a hexamer; Ski-(16-192) interacted with an R-Smad.Smad4 heterotrimer to form a pentamer. CBP-(1941-1992) was also found to interact directly with an R-Smad homotrimer to form a hexamer and with an R-Smad.Smad4 heterotrimer to form a pentamer. Moreover, these domains of Ski and CBP competed with each other for binding to Smad3. Our mutational studies revealed that domains of Ski and CBP interacted with Smad3 at a portion of the binding surface of the Smad anchor for receptor activation. Our results suggest that Ski negatively regulates TGF-beta signaling by replacing CBP in R-Smad complexes. Our working model suggests that Smad protein activity is delicately balanced by Ski and CBP in the TGF-beta pathway.

Sperm and Spermatids Contain Different Proteins and Bind Distinct Egg Factors

PubMed Central

Teperek, Marta; Miyamoto, Kei; Simeone, Angela; Feret, Renata; Deery, Michael J.; Gurdon, John B.; Jullien, Jerome

2014-01-01

Spermatozoa are more efficient at supporting normal embryonic development than spermatids, their immature, immediate precursors. This suggests that the sperm acquires the ability to support embryonic development during spermiogenesis (spermatid to sperm maturation). Here, using Xenopus laevis as a model organism, we performed 2-D Fluorescence Difference Gel Electrophoresis (2D-DIGE) and mass spectrometry analysis of differentially expressed proteins between sperm and spermatids in order to identify factors that could be responsible for the efficiency of the sperm to support embryonic development. Furthermore, benefiting from the availability of egg extracts in Xenopus, we also tested whether the chromatin of sperm could attract different egg factors compared to the chromatin of spermatids. Our analysis identified: (1) several proteins which were present exclusively in sperm; but not in spermatid nuclei and (2) numerous egg proteins binding to the sperm (but not to the spermatid chromatin) after incubation in egg extracts. Amongst these factors we identified many chromatin-associated proteins and transcriptional repressors. Presence of transcriptional repressors binding specifically to sperm chromatin could suggest its preparation for the early embryonic cell cycles, during which no transcription is observed and suggests that sperm chromatin has a unique protein composition, which facilitates the recruitment of egg chromatin remodelling factors. It is therefore likely that the acquisition of these sperm-specific factors during spermiogenesis makes the sperm chromatin suitable to interact with the maternal factors and, as a consequence, to support efficient embryonic development. PMID:25244019

The Fn14 cytoplasmic tail binds tumour-necrosis-factor-receptor-associated factors 1, 2, 3 and 5 and mediates nuclear factor-kappaB activation.

PubMed Central

Brown, Sharron A N; Richards, Christine M; Hanscom, Heather N; Feng, Sheau-Line Y; Winkles, Jeffrey A

2003-01-01

Fn14 is a growth-factor-inducible immediate-early-response gene encoding a 102-amino-acid type I transmembrane protein. The human Fn14 protein was recently identified as a cell-surface receptor for the tumour necrosis factor (TNF) superfamily member named TWEAK (TNF-like weak inducer of apoptosis). In the present paper, we report that the human TWEAK extracellular domain can also bind the murine Fn14 protein. Furthermore, site-specific mutagenesis and directed yeast two-hybrid interaction assays revealed that the TNFR-associated factor (TRAF) 1, 2, 3 and 5 adaptor molecules bind the murine Fn14 cytoplasmic tail at an overlapping, but non-identical, amino acid sequence motif. We also found that TWEAK treatment of quiescent NIH 3T3 cells stimulates inhibitory kappaBalpha phosphorylation and transcriptional activation of a nuclear factor-kappaB (NF-kappaB) enhancer/luciferase reporter construct. Fn14 overexpression in transiently transfected NIH 3T3 cells also promotes NF-kappaB activation, and this cellular response requires an intact TRAF binding site. These results indicate that Fn14 is a functional TWEAK receptor that can associate with four distinct TRAF family members and stimulate the NF-kappaB transcription factor signalling pathway. PMID:12529173

Ethylene-responsive element-binding factor 5, ERF5, is involved in chitin-induced innate immunity response.

PubMed

Son, Geon Hui; Wan, Jinrong; Kim, Hye Jin; Nguyen, Xuan Canh; Chung, Woo Sik; Hong, Jong Chan; Stacey, Gary

2012-01-01

Our recent work demonstrated that chitin treatment modulated the expression of 118 transcription factor (TF) genes in Arabidopsis. To investigate the potential roles of these TF in chitin signaling and plant defense, we initiated an interaction study among these TF proteins, as well as two chitin-activated mitogen-activated protein kinases (MPK3 and MPK6), using a yeast two-hybrid system. This study revealed interactions among the following proteins: three ethylene-responsive element-binding factors (ERF), five WRKY transcription factors, one scarecrow-like (SCL), and the two MPK, in addition to many other interactions, reflecting a complex TF interaction network. Most of these interactions were subsequently validated by other methods, such as pull-down and in planta bimolecular fluorescence complementation assays. The key node ERF5 was shown to interact with multiple proteins in the network, such as ERF6, ERF8, and SCL13, as well as MPK3 and MPK6. Interestingly, ERF5 appeared to negatively regulate chitin signaling and plant defense against the fungal pathogen Alternaria brassicicola and positively regulate salicylic acid signaling and plant defense against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Therefore, ERF5 may play an important role in plant innate immunity, likely through coordinating chitin and other defense pathways in plants in response to different pathogens.

RNA-binding Protein Immunoprecipitation (RIP) to Examine AUF1 Binding to Senescence-Associated Secretory Phenotype (SASP) Factor mRNA

PubMed Central

Alspach, Elise; Stewart, Sheila A.

2016-01-01

Immunoprecipitation and subsequent isolation of nucleic acids allows for the investigation of protein:nucleic acid interactions. RNA-binding protein immunoprecipitation (RIP) is used for the analysis of protein interactions with mRNA. Combining RIP with quantitative real-time PCR (qRT-PCR) further enhances the RIP technique by allowing for the quantitative assessment of RNA-binding protein interactions with their target mRNAs, and how these interactions change in different cellular settings. Here, we describe the immunoprecipitation of the RNA-binding protein AUF1 with several different factors associated with the senescence-associated secretory phenotype (SASP) (Alspach and Stewart, 2013), specifically IL6 and IL8. This protocol was originally published in Alspach et al. (2014). PMID:27453911

Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

PubMed

In, K H; Asano, K; Beier, D; Grobholz, J; Finn, P W; Silverman, E K; Silverman, E S; Collins, T; Fischer, A R; Keith, T P; Serino, K; Kim, S W; De Sanctis, G T; Yandava, C; Pillari, A; Rubin, P; Kemp, J; Israel, E; Busse, W; Ledford, D; Murray, J J; Segal, A; Tinkleman, D; Drazen, J M

1997-03-01

Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation.

The cancer-promoting gene fatty acid-binding protein 5 (FABP5) is epigenetically regulated during human prostate carcinogenesis.

PubMed

Kawaguchi, Koichiro; Kinameri, Ayumi; Suzuki, Shunsuke; Senga, Shogo; Ke, Youqiang; Fujii, Hiroshi

2016-02-15

FABPs (fatty-acid-binding proteins) are a family of low-molecular-mass intracellular lipid-binding proteins consisting of ten isoforms. FABPs are involved in binding and storing hydrophobic ligands such as long-chain fatty acids, as well as transporting these ligands to the appropriate compartments in the cell. FABP5 is overexpressed in multiple types of tumours. Furthermore, up-regulation of FABP5 is strongly associated with poor survival in triple-negative breast cancer. However, the mechanisms underlying the specific up-regulation of the FABP5 gene in these cancers remain poorly characterized. In the present study, we determined that FABP5 has a typical CpG island around its promoter region. The DNA methylation status of the CpG island in the FABP5 promoter of benign prostate cells (PNT2), prostate cancer cells (PC-3, DU-145, 22Rv1 and LNCaP) and human normal or tumour tissue was assessed by bisulfite sequencing analysis, and then confirmed by COBRA (combined bisulfite restriction analysis) and qAMP (quantitative analysis of DNA methylation using real-time PCR). These results demonstrated that overexpression of FABP5 in prostate cancer cells can be attributed to hypomethylation of the CpG island in its promoter region, along with up-regulation of the direct trans-acting factors Sp1 (specificity protein 1) and c-Myc. Together, these mechanisms result in the transcriptional activation of FABP5 expression during human prostate carcinogenesis. Importantly, silencing of Sp1, c-Myc or FABP5 expression led to a significant decrease in cell proliferation, indicating that up-regulation of FABP5 expression by Sp1 and c-Myc is critical for the proliferation of prostate cancer cells. © 2016 Authors; published by Portland Press Limited.

The Dishevelled-binding protein CXXC5 negatively regulates cutaneous wound healing

PubMed Central

Lee, Soung-Hoon; Kim, Mi-Yeon; Kim, Hyun-Yi; Lee, Young-Mi; Kim, Heesu; Nam, Kyoung Ae; Roh, Mi Ryung; Min, Do Sik; Chung, Kee Yang

2015-01-01

Wnt/β-catenin signaling plays important roles in cutaneous wound healing and dermal fibrosis. However, its regulatory mechanism has not been fully elucidated, and a commercially available wound-healing agent targeting this pathway is desirable but currently unavailable. We found that CXXC-type zinc finger protein 5 (CXXC5) serves as a negative feedback regulator of the Wnt/β-catenin pathway by interacting with the Dishevelled (Dvl) protein. In humans, CXXC5 protein levels were reduced in epidermal keratinocytes and dermal fibroblasts of acute wounds. A differential regulation of β-catenin, α-smooth muscle actin (α-SMA), and collagen I by overexpression and silencing of CXXC5 in vitro indicated a critical role for this factor in myofibroblast differentiation and collagen production. In addition, CXXC5−/− mice exhibited accelerated cutaneous wound healing, as well as enhanced keratin 14 and collagen synthesis. Protein transduction domain (PTD)–Dvl-binding motif (DBM), a competitor peptide blocking CXXC5-Dvl interactions, disrupted this negative feedback loop and activated β-catenin and collagen production in vitro. Co-treatment of skin wounds with PTD-DBM and valproic acid (VPA), a glycogen synthase kinase 3β (GSK3β) inhibitor which activates the Wnt/β-catenin pathway, synergistically accelerated cutaneous wound healing in mice. Together, these data suggest that CXXC5 would represent a potential target for future therapies aimed at improving wound healing. PMID:26056233

Binding Mode Analysis of Zerumbone to Key Signal Proteins in the Tumor Necrosis Factor Pathway

PubMed Central

Fatima, Ayesha; Abdul, Ahmad Bustamam Hj.; Abdullah, Rasedee; Karjiban, Roghayeh Abedi; Lee, Vannajan Sanghiran

2015-01-01

Breast cancer is the second most common cancer among women worldwide. Several signaling pathways have been implicated as causative and progression agents. The tumor necrosis factor (TNF) α protein plays a dual role in promoting and inhibiting cancer depending largely on the pathway initiated by the binding of the protein to its receptor. Zerumbone, an active constituent of Zingiber zerumbet, Smith, is known to act on the tumor necrosis factor pathway upregulating tumour necrosis factor related apoptosis inducing ligand (TRAIL) death receptors and inducing apoptosis in cancer cells. Zerumbone is a sesquiterpene that is able to penetrate into the hydrophobic pockets of proteins to exert its inhibiting activity with several proteins. We found a good binding with the tumor necrosis factor, kinase κB (IKKβ) and the Nuclear factor κB (NF-κB) component proteins along the TNF pathway. Our results suggest that zerumbone can exert its apoptotic activities by inhibiting the cytoplasmic proteins. It inhibits the IKKβ kinase that activates the NF-κB and also binds to the NF-κB complex in the TNF pathway. Blocking both proteins can lead to inhibition of cell proliferating proteins to be downregulated and possibly ultimate induction of apoptosis. PMID:25629232

Promoter-dependent and -independent activation of insulin-like growth factor binding protein-5 gene expression by prostaglandin E2 in primary rat osteoblasts

NASA Technical Reports Server (NTRS)

McCarthy, T. L.; Casinghino, S.; Mittanck, D. W.; Ji, C. H.; Centrella, M.; Rotwein, P.

1996-01-01

Insulin-like growth factor (IGF) action is mediated by high affinity cell surface IGF receptors and modulated by a family of secreted IGF binding proteins (IGFBPs). IGFBP-5, the most conserved of six IGFBPs characterized to date, uniquely potentiates the anabolic actions of IGF-I for skeletal cells. In osteoblasts, IGFBP-5 production is stimulated by prostaglandin E2 (PGE2), a local factor that mediates certain effects induced by parathyroid hormone, cytokines such as interleukin-1 and transforming growth factor-beta, and mechanical strain. In this study, we show that transcriptional and post-transcriptional events initiated by PGE2 collaborate to enhance IGFBP-5 gene expression in primary fetal rat osteoblast cultures. PGE2 treatment stimulated up to a 7-fold rise in steady-state levels of IGFBP-5 mRNA throughout 32 h of incubation. Analysis of nascent IGFBP-5 mRNA suggested that PGE2 had only a modest stimulatory effect on IGFBP-5 gene transcription, and transient transfection studies with IGFBP-5 promoter-reporter genes confirmed that PGE2 enhanced promoter activity by approximately 2-fold. Similar stimulatory effects were seen with forskolin. A DNA fragment with only 51 base pairs of the 5'-flanking sequence retained hormonal responsiveness, which may be mediated by a binding site for transcription factor AP-2 located at positions -44 to -36 in the proximal IGFBP-5 promoter. Incubation of osteoblasts with the mRNA transcriptional inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole demonstrated that PGE2 enhanced IGFBP-5 mRNA stability by 2-fold, increasing the t1/2 from 9 to 18 h. The effects of PGE2 on steady-state IGFBP-5 transcripts were abrogated by preincubating cells with cycloheximide, indicating that the effects of PGE2 on both gene transcription and mRNA stability required ongoing protein synthesis. Therefore, both promoter-dependent and -independent pathways converge to enhance IGFBP-5 gene expression in response to PGE2 in osteoblasts.

Naturally occurring mutations in the human 5-lipoxygenase gene promoter that modify transcription factor binding and reporter gene transcription.

PubMed Central

In, K H; Asano, K; Beier, D; Grobholz, J; Finn, P W; Silverman, E K; Silverman, E S; Collins, T; Fischer, A R; Keith, T P; Serino, K; Kim, S W; De Sanctis, G T; Yandava, C; Pillari, A; Rubin, P; Kemp, J; Israel, E; Busse, W; Ledford, D; Murray, J J; Segal, A; Tinkleman, D; Drazen, J M

1997-01-01

Five lipoxygenase (5-LO) is the first committed enzyme in the metabolic pathway leading to the synthesis of the leukotrienes. We examined genomic DNA isolated from 25 normal subjects and 31 patients with asthma (6 of whom had aspirin-sensitive asthma) for mutations in the known transcription factor binding regions and the protein encoding region of the 5-LO gene. A family of mutations in the G + C-rich transcription factor binding region was identified consisting of the deletion of one, deletion of two, or addition of one zinc finger (Sp1/Egr-1) binding sites in the region 176 to 147 bp upstream from the ATG translation start site where there are normally 5 Sp1 binding motifs in tandem. Reporter gene activity directed by any of the mutant forms of the transcription factor binding region was significantly (P < 0.05) less effective than the activity driven by the wild type transcription factor binding region. Electrophoretic mobility shift assays (EMSAs) demonstrated the capacity of wild type and mutant transcription factor binding regions to bind nuclear extracts from human umbilical vein endothelial cells (HUVECs). These data are consistent with a family of mutations in the 5-LO gene that can modify reporter gene transcription possibly through differences in Sp1 and Egr-1 transactivation. PMID:9062372

Cell proteins bind to multiple sites within the 5' untranslated region of poliovirus RNA.

PubMed Central

del Angel, R M; Papavassiliou, A G; Fernández-Tomás, C; Silverstein, S J; Racaniello, V R

1989-01-01

The 5' noncoding region of poliovirus RNA contains sequences necessary for translation and replication. These functions are probably carried out by recognition of poliovirus RNA by cellular and/or viral proteins. Using a mobility-shift electrophoresis assay and 1,10-phenanthroline/Cu+ footprinting, we demonstrate specific binding of cytoplasmic factors with a sequence from nucleotides 510-629 within the 5' untranslated region (UTR). Complex formation was also observed with a second sequence (nucleotides 97-182) within the 5' UTR. These two regions of the 5' UTR appear to be recognized by distinct cell factors as determined by competition analysis and the effects of ionic strength on complex formation. However, both complexes contain eukaryotic initiation factor 2 alpha, as revealed by their reaction with specific antibody. Images PMID:2554308

Acute handling disturbance modulates plasma insulin-like growth factor binding proteins in rainbow trout (Oncorhynchus mykiss)

USDA-ARS?s Scientific Manuscript database

The effects of acute stressor exposure on proximal (growth hormone; GH) and distal (insulin-like growth factor-I; IGF-I and IGF-binding proteins) components of the somatotropic axis are poorly understood in finfish. We exposed rainbow trout (Oncorhynchus mykiss) to a 5-minute handling disturbance to...

Structural motif screening reveals a novel, conserved carbohydrate-binding surface in the pathogenesis-related protein PR-5d.

PubMed

Doxey, Andrew C; Cheng, Zhenyu; Moffatt, Barbara A; McConkey, Brendan J

2010-08-03

Aromatic amino acids play a critical role in protein-glycan interactions. Clusters of surface aromatic residues and their features may therefore be useful in distinguishing glycan-binding sites as well as predicting novel glycan-binding proteins. In this work, a structural bioinformatics approach was used to screen the Protein Data Bank (PDB) for coplanar aromatic motifs similar to those found in known glycan-binding proteins. The proteins identified in the screen were significantly associated with carbohydrate-related functions according to gene ontology (GO) enrichment analysis, and predicted motifs were found frequently within novel folds and glycan-binding sites not included in the training set. In addition to numerous binding sites predicted in structural genomics proteins of unknown function, one novel prediction was a surface motif (W34/W36/W192) in the tobacco pathogenesis-related protein, PR-5d. Phylogenetic analysis revealed that the surface motif is exclusive to a subfamily of PR-5 proteins from the Solanaceae family of plants, and is absent completely in more distant homologs. To confirm PR-5d's insoluble-polysaccharide binding activity, a cellulose-pulldown assay of tobacco proteins was performed and PR-5d was identified in the cellulose-binding fraction by mass spectrometry. Based on the combined results, we propose that the putative binding site in PR-5d may be an evolutionary adaptation of Solanaceae plants including potato, tomato, and tobacco, towards defense against cellulose-containing pathogens such as species of the deadly oomycete genus, Phytophthora. More generally, the results demonstrate that coplanar aromatic clusters on protein surfaces are a structural signature of glycan-binding proteins, and can be used to computationally predict novel glycan-binding proteins from 3 D structure.

Analysis of Cry8Ka5-binding proteins from Anthonomus grandis (Coleoptera: Curculionidae) midgut.

PubMed

Nakasu, Erich Y T; Firmino, Alexandre A P; Dias, Simoni C; Rocha, Thales L; Ramos, Hudson B; Oliveira, Gustavo R; Lucena, Wagner; Carlini, Célia R; Grossi-de-Sá, Maria Fátima

2010-07-01

Biotech crops expressing Bacillus thuringiensis Cry toxins present a valuable approach for insect control. Cry8Ka5, which is highly toxic to the cotton boll weevil (Anthonomus grandis), was used as a model to study toxin-ligand interactions. Three Cry-binding proteins were detected after toxin overlay assays. Following de novo sequencing, a heat-shock cognate protein and a V-ATPase were identified, whilst a approximately 120 kDa protein remained unknown. Additional Cry8Ka5-binding proteins were visualized by two-dimensional gel electrophoresis ligand blots. (c) 2010 Elsevier Inc. All rights reserved.

The complexity of minocycline serum protein binding.

PubMed

Zhou, Jian; Tran, Brian T; Tam, Vincent H

2017-06-01

Serum protein binding is critical for understanding the pharmacology of antimicrobial agents. Tigecycline and eravacycline were previously reported to have atypical non-linear protein binding; the percentage of free fraction decreased with increasing total concentration. In this study, we extended the investigation to other tetracyclines and examined the factors that might impact protein binding. Different minocycline concentrations (0.5-50 mg/L) and perfusion media (saline, 0.1 M HEPES buffer and 0.1 and 1 M PBS) were examined by in vitro microdialysis. After equilibration, two dialysate samples were taken from each experiment and the respective antimicrobial agent concentrations were analysed by validated LC-MS/MS methods. For comparison, the serum protein bindings of doxycycline and levofloxacin were also determined. The free fraction of minocycline decreased with increasing total concentration, and the results depended on the perfusion media used. The trends of minocycline protein binding in mouse and human sera were similar. In addition, serum protein binding of doxycycline showed the same concentration-dependent trend as minocycline, while the results of levofloxacin were concentration independent. The serum protein bindings of minocycline and doxycycline are negatively correlated with their total concentrations. It is possible that all tetracyclines share the same pharmacological property. Moreover, the specific perfusion media used could also impact the results of microdialysis. Additional studies are warranted to understand the mechanism(s) and clinical implications of serum protein binding of tetracyclines. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Insulin-like growth factor-binding protein-5 (IGFBP-5) inhibits TNF-{alpha}-induced NF-{kappa}B activity by binding to TNFR1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hwang, Jae Ryoung; Huh, Jae Ho; Lee, Yoonna

2011-02-25

Research highlights: {yields} Binding assays demonstrated that secreted- and cellular-IGFBP-5 interacted with TNFR1. {yields} The interaction between IGFBP-5 and TNFR1 was inhibited by TNF-{alpha} and was blocked TNF-{alpha}-activated NF-{kappa}B activity. {yields} IGFBP-5 interacted with TNFR1 through its N- and L-domains but the binding of L-domain to TNFR1 was blocked by TNF-{alpha}. {yields} Competition between the L-domain of IGFBP-5 and TNF-{alpha} blocked TNF-{alpha}-induced NF-{kappa}B activity. {yields} This study suggests that the L-domain of IGFBP-5 is a novel TNFR1 ligand that functions as a competitive TNF-{alpha} inhibitor. -- Abstract: IGFBP-5 is known to be involved in various cell phenomena such as proliferation,more » differentiation, and apoptosis. However, the exact mechanisms by which IGFBP-5 exerts its functions are unclear. In this study, we demonstrate for the first time that IGFBP-5 is a TNFR1-interacting protein. We found that ectopic expression of IGFBP-5 induced TNFR1 gene expression, and that IGFBP-5 interacted with TNFR1 in both an in vivo and an in vitro system. Secreted IGFBP-5 interacted with GST-TNFR1 and this interaction was blocked by TNF-{alpha}, demonstrating that IGFBP-5 might be a TNFR1 ligand. Furthermore, conditioned media containing secreted IGFBP-5 inhibited PMA-induced NF-{kappa}B activity and IL-6 expression in U-937 cells. Coimmunoprecipitation assays of TNFR1 and IGFBP-5 wild-type and truncation mutants revealed that IGFBP-5 interacts with TNFR1 through its N- and L-domains. However, only the interaction between the L-domain of IGFBP-5 and TNFR1 was blocked by TNF-{alpha} in a dose-dependent manner, suggesting that the L-domain of IGFBP-5 can function as a TNFR1 ligand. Competition between the L-domain of IGFBP-5 and TNF-{alpha} resulted in inhibition of TNF-{alpha}-induced NF-{kappa}{Beta} activity. Taken together, our results suggest that the L-domain of IGFBP-5 is a novel TNFR1 ligand that functions as a

Structural basis for the ligand-binding specificity of fatty acid-binding proteins (pFABP4 and pFABP5) in gentoo penguin.

PubMed

Lee, Chang Woo; Kim, Jung Eun; Do, Hackwon; Kim, Ryeo-Ok; Lee, Sung Gu; Park, Hyun Ho; Chang, Jeong Ho; Yim, Joung Han; Park, Hyun; Kim, Il-Chan; Lee, Jun Hyuck

2015-09-11

Fatty acid-binding proteins (FABPs) are involved in transporting hydrophobic fatty acids between various aqueous compartments of the cell by directly binding ligands inside their β-barrel cavities. Here, we report the crystal structures of ligand-unbound pFABP4, linoleate-bound pFABP4, and palmitate-bound pFABP5, obtained from gentoo penguin (Pygoscelis papua), at a resolution of 2.1 Å, 2.2 Å, and 2.3 Å, respectively. The pFABP4 and pFABP5 proteins have a canonical β-barrel structure with two short α-helices that form a cap region and fatty acid ligand binding sites in the hydrophobic cavity within the β-barrel structure. Linoleate-bound pFABP4 and palmitate-bound pFABP5 possess different ligand-binding modes and a unique ligand-binding pocket due to several sequence dissimilarities (A76/L78, T30/M32, underlining indicates pFABP4 residues) between the two proteins. Structural comparison revealed significantly different conformational changes in the β3-β4 loop region (residues 57-62) as well as the flipped Phe60 residue of pFABP5 than that in pFABP4 (the corresponding residue is Phe58). A ligand-binding study using fluorophore displacement assays shows that pFABP4 has a relatively strong affinity for linoleate as compared to pFABP5. In contrast, pFABP5 exhibits higher affinity for palmitate than that for pFABP4. In conclusion, our high-resolution structures and ligand-binding studies provide useful insights into the ligand-binding preferences of pFABPs based on key protein-ligand interactions. Copyright © 2015 Elsevier Inc. All rights reserved.

Dynamic factors affecting gaseous ligand binding in an artificial oxygen transport protein.

PubMed

Zhang, Lei; Andersen, Eskil M E; Khajo, Abdelahad; Magliozzo, Richard S; Koder, Ronald L

2013-01-22

We report the functional analysis of an artificial hexacoordinate oxygen transport protein, HP7, which operates via a mechanism similar to that of human neuroglobin and cytoglobin: the destabilization of one of two heme-ligating histidine residues. In the case of HP7, this is the result of the coupling of histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Here we compare gaseous ligand binding, including rates, affinities, and oxyferrous state lifetimes, of both heme binding sites in HP7. We find that despite the identical sequence of helices in both binding sites, there are differences in oxygen affinity and oxyferrous state lifetime that may be the result of differences in the freedom of motion imposed by the candelabra fold on the two sites of the protein. We further examine the effect of mutational removal of the buried glutamates on function. Heme iron in the ferrous state of this mutant is rapidly oxidized when exposed to oxygen. Compared to that of HP7, the distal histidine affinity is increased by a 22-fold decrease in the histidine ligand off rate. Electron paramagnetic resonance comparison of these ferric hemoproteins demonstrates that the mutation increases the level of disorder at the heme binding site. Nuclear magnetic resonance-detected deuterium exchange demonstrates that the mutation greatly increases the degree of penetration of water into the protein core. The inability of the mutant protein to bind oxygen may be due to an increased level of water penetration, the large decrease in binding rate caused by the increase in distal histidine affinity, or a combination of the two factors. Together, these data underline the importance of the control of protein dynamics in the design of functional artificial proteins.

Tyrosine sulfation in N-terminal domain of human C5a receptor is necessary for binding of chemotaxis inhibitory protein of Staphylococcus aureus

PubMed Central

Liu, Zhen-jia; Yang, Yan-juan; Jiang, Lei; Xu, Ying-chun; Wang, Ai-xia; Du, Guan-hua; Gao, Jin-ming

2011-01-01

Aim: Staphylococcus aureus evades host defense through releasing several virulence proteins, such as chemotaxis inhibitory protein of staphylococcus aureus (CHIPS). It has been shown that extracellular N terminus of C5a receptor (C5aR) forms the binding domain for CHIPS, and tyrosine sulfation is emerging as a key factor in determining protein-protein interaction. The aim of this study was to evaluate the role of tyrosine sulfation of N-terminal of C5aR in its binding with CHIPS. Methods: Expression plasmids encoding C5aR and its mutants were prepared using PCR and site-directed mutagenesis and were used to transfect HEK 293T cells using calcium phosphate. Recombinant CHIPS protein was purified. Western blotting was used to examine the binding efficiency of CHIPS to C5aR or its mutants. Results: CHIPS exclusively binds to C5aR, but not to C5L2 or C3aR. A nonspecific sulfation inhibitor, sodium chlorate (50 nmol/L), diminishes the binding ability of C5aR with CHIPS. Blocking sulfation by mutation of tyrosine to phenylalanine at positions 11 and 14 of C5aR N terminus, which blocked sulfation, completely abrogates CHIPS binding. When tyrosine 14 alone was mutated to phenylalanine, the binding efficiency of recombinant CHIPS was substantially decreased. Conclusion: The results demonstrate a structural basis of C5aR-CHIPS association, in which tyrosine sulfation of N-terminal C5aR plays an important role. Our data may have potential significance in development of novel drugs for therapeutic intervention. PMID:21706042

[Insulin-like growth factor-binding protein-1: a new biochemical marker of nonalcoholic fatty liver disease?].

PubMed

Graffigna, Mabel Nora; Belli, Susana H; de Larrañaga, Gabriela; Fainboim, Hugo; Estepo, Claudio; Peres, Silvia; García, Natalia; Levalle, Oscar

2009-03-01

to assess the presence of nonalcoholic fatty liver disease in patients with risk factors for this pathology (obesity, dyslipidemia, metabolic syndrome and diabetes type 2) and to determine the role of insulin, HOMA index, insulin-like growth factor-binding protein-1, sex hormone-binding globulin and plasminogen activator inhibitor type 1, as biochemical markers. Ninety-one patients with risk factors for nonalcoholic fatty liver disease were evaluated. Serum transaminases, insulin, sex hormone-binding globulin, insulin-like growth factor-binding protein-1 and plasminogen activator inhibitor type 1 were measured. The diagnosis of fatty liver was performed by ultrasonography and liver biopsies were performed to 31 subjects who had steatosis by ultrasonography and high alanine aminotransferase. Nonalcoholic fatty liver disease was present in 65 out of 91 patients (71,4%). Liver biopsy performed to 31 subjects confirmed nonalcoholic steatohepatitis. Twenty-five patients had different degrees of fibrosis. Those individuals with fatty liver had higher waist circumference, serum levels of triglycerides, insulin and HOMA index, and lower serum insulin-like growth factor-binding protein-1 concentration. The degree ofhepatic steatosis by ultrasonography was positively correlated to waist circumference, triglycerides, insulin and HOMA index (p<0,003; p<0,003; p<0,002 and p<0,001, respectively), and was negatively correlated to HDL-cholesterol and insulin-like growth factor-binding protein-1 (p<0,025 and p<0,018, respectively). We found a high prevalence of NAFLD in patients with risk factors, most of them overweight or obese. Although SHBG and PAI-1 have a closely relationship to insulin resistance, they did not show to be markers of NAFLD. Regardless of low IGFBP-1 levels associated with NAFLD, serum IGFBP-1 measure is less accessible than insulin and triglycerides levels, HOMA index and waist circumference. Moreover, it is not a better marker for NAFLD than the above

Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins.

PubMed

Nishiyama, Keita; Nakamata, Koichi; Ueno, Shintaro; Terao, Akari; Aryantini, Ni Putu Desy; Sujaya, I Nengah; Fukuda, Kenji; Urashima, Tadasu; Yamamoto, Yuji; Mukai, Takao

2015-01-01

We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding.

Dissecting the protein architecture of DNA-binding transcription factors in bacteria and archaea.

PubMed

Rivera-Gómez, Nancy; Martínez-Núñez, Mario Alberto; Pastor, Nina; Rodriguez-Vazquez, Katya; Perez-Rueda, Ernesto

2017-08-01

Gene regulation at the transcriptional level is a central process in all organisms where DNA-binding transcription factors play a fundamental role. This class of proteins binds specifically at DNA sequences, activating or repressing gene expression as a function of the cell's metabolic status, operator context and ligand-binding status, among other factors, through the DNA-binding domain (DBD). In addition, TFs may contain partner domains (PaDos), which are involved in ligand binding and protein-protein interactions. In this work, we systematically evaluated the distribution, abundance and domain organization of DNA-binding TFs in 799 non-redundant bacterial and archaeal genomes. We found that the distributions of the DBDs and their corresponding PaDos correlated with the size of the genome. We also identified specific combinations between the DBDs and their corresponding PaDos. Within each class of DBDs there are differences in the actual angle formed at the dimerization interface, responding to the presence/absence of ligands and/or crystallization conditions, setting the orientation of the resulting helices and wings facing the DNA. Our results highlight the importance of PaDos as central elements that enhance the diversity of regulatory functions in all bacterial and archaeal organisms, and our results also demonstrate the role of PaDos in sensing diverse signal compounds. The highly specific interactions between DBDs and PaDos observed in this work, together with our structural analysis highlighting the difficulty in predicting both inter-domain geometry and quaternary structure, suggest that these systems appeared once and evolved with diverse duplication events in all the analysed organisms.

Epstein-Barr virus-encoded EBNA-5 binds to Epstein-Barr virus-induced Fte1/S3a protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kashuba, Elena; Yurchenko, Mariya; Szirak, Krisztina

Epstein-Barr virus (EBV) transforms resting human B cells into immortalized immunoblasts. EBV-encoded nuclear antigens EBNA-5 (also called EBNA-LP) is one of the earliest viral proteins expressed in freshly infected B cells. We have recently shown that EBNA-5 binds p14ARF, a nucleolar protein that regulates the p53 pathway. Here, we report the identification of another protein with partially nucleolar localization, the v-fos transformation effector Fte-1 (Fte-1/S3a), as an EBNA-5 binding partner. In transfected cells, Fte-1/S3a and EBNA-5 proteins showed high levels of colocalization in extranucleolar inclusions. Fte-1/S3a has multiple biological functions. It enhances v-fos-mediated cellular transformation and is part of themore » small ribosomal subunit. It also interacts with the transcriptional factor CHOP and apoptosis regulator poly(ADP-ribose) polymerase (PARP). Fte-1/S3a is regularly expressed at high levels in both tumors and cancer cell lines. Its high expression favors the maintenance of malignant phenotype and undifferentiated state, whereas its down-regulation is associated with cellular differentiation and growth arrest. Here, we show that EBV-induced B cell transformation leads to the up-regulation of Fte-1/S3a. We suggest that EBNA-5 through binding may influence the growth promoting, differentiation inhibiting, or apoptosis regulating functions of Fte-1/S3a.« less

pH modulates the binding of early growth response protein 1 transcription factor to DNA.

PubMed

Mikles, David C; Bhat, Vikas; Schuchardt, Brett J; Deegan, Brian J; Seldeen, Kenneth L; McDonald, Caleb B; Farooq, Amjad

2013-08-01

The transcription factor early growth response protein (EGR)1 orchestrates a plethora of signaling cascades involved in cellular homeostasis, and its downregulation has been implicated in the development of prostate cancer. Herein, using a battery of biophysical tools, we show that the binding of EGR1 to DNA is tightly regulated by solution pH. Importantly, the binding affinity undergoes an enhancement of more than an order of magnitude with an increase in pH from 5 to 8, implying that the deprotonation of an ionizable residue accounts for such behavior. This ionizable residue is identified as His382 by virtue of the fact that its replacement by nonionizable residues abolishes the pH dependence of the binding of EGR1 to DNA. Notably, His382 inserts into the major groove of DNA, and stabilizes the EGR1-DNA interaction via both hydrogen bonding and van der Waals contacts. Remarkably, His382 is mainly conserved across other members of the EGR family, implying that histidine protonation-deprotonation may serve as a molecular switch for modulating the protein-DNA interactions that are central to this family of transcription factors. Collectively, our findings reveal an unexpected but a key step in the molecular recognition of the EGR family of transcription factors, and suggest that they may act as sensors of pH within the intracellular environment. © 2013 FEBS.

Insulin-Like Growth Factor Binding Proteins Increase Intracellular Calcium Levels in Two Different Cell Lines

PubMed Central

Seurin, Danielle; Lombet, Alain; Babajko, Sylvie; Godeau, François; Ricort, Jean-Marc

2013-01-01

Background Insulin-like growth factor binding proteins (IGFBPs) are six related secreted proteins that share IGF-dependent and -independent functions. If the former functions begin to be well described, the latter are somewhat more difficult to investigate and to characterize. At the cellular level, IGFBPs were shown to modulate numerous processes including cell growth, differentiation and apoptosis. However, the molecular mechanisms implicated remain largely unknown. We previously demonstrated that IGFBP-3, but not IGFBP-1 or IGFBP-5, increase intracellular calcium concentration in MCF-7 cells (Ricort J-M et al. (2002) FEBS lett 527: 293–297). Methodology/Principal Findings We perform a global analysis in which we studied, by two different approaches, the binding of each IGFBP isoform (i.e., IGFBP-1 to -6) to the surface of two different cellular models, MCF-7 breast adenocarcinoma cells and C2 myoblast proliferative cells, as well as the IGFBP-induced increase of intracellular calcium concentration. Using both confocal fluorescence microscopy and flow cytometry analysis, we showed that all IGFBPs bind to MCF-7 cell surface. By contrast, only four IGFBPs can bind to C2 cell surface since neither IGFBP-2 nor IGFBP-4 were detected. Among the six IGFBPs tested, only IGFBP-1 did not increased intracellular calcium concentration whatever the cellular model studied. By contrast, IGFBP-2, -3, -4 and -6, in MCF-7 cells, and IGFBP-3, -5 and -6, in C2 proliferative cells, induce a rapid and transient increase in intracellular free calcium concentration. Moreover, IGFBP-2 and -3 (in MCF-7 cells) and IGFBP-5 (in C2 cells) increase intracellular free calcium concentration by a pertussis toxin sensitive signaling pathway. Conclusions Our results demonstrate that IGFBPs are able to bind to cell surface and increase intracellular calcium concentration. By characterizing the IGFBPs-induced cell responses and intracellular couplings, we highlight the cellular specificity and

Cloning of Human Tumor Necrosis Factor (TNF) Receptor cDNA and Expression of Recombinant Soluble TNF-Binding Protein

NASA Astrophysics Data System (ADS)

Gray, Patrick W.; Barrett, Kathy; Chantry, David; Turner, Martin; Feldmann, Marc

1990-10-01

The cDNA for one of the receptors for human tumor necrosis factor (TNF) has been isolated. This cDNA encodes a protein of 455 amino acids that is divided into an extracellular domain of 171 residues and a cytoplasmic domain of 221 residues. The extracellular domain has been engineered for expression in mammalian cells, and this recombinant derivative binds TNFα with high affinity and inhibits its cytotoxic activity in vitro. The TNF receptor exhibits similarity with a family of cell surface proteins that includes the nerve growth factor receptor, the human B-cell surface antigen CD40, and the rat T-cell surface antigen OX40. The TNF receptor contains four cysteine-rich subdomains in the extra-cellular portion. Mammalian cells transfected with the entire TNF receptor cDNA bind radiolabeled TNFα with an affinity of 2.5 x 10-9 M. This binding can be competitively inhibited with unlabeled TNFα or lymphotoxin (TNFβ).

Insulin-like growth factor binding protein 5 suppresses tumor growth and metastasis of human osteosarcoma.

PubMed

Su, Y; Wagner, E R; Luo, Q; Huang, J; Chen, L; He, B-C; Zuo, G-W; Shi, Q; Zhang, B-Q; Zhu, G; Bi, Y; Luo, J; Luo, X; Kim, S H; Shen, J; Rastegar, F; Huang, E; Gao, Y; Gao, J-L; Yang, K; Wietholt, C; Li, M; Qin, J; Haydon, R C; He, T-C; Luu, H H

2011-09-15

Osteosarcoma (OS) is the most common primary malignancy of bone. There is a critical need to identify the events that lead to the poorly understood mechanism of OS development and metastasis. The goal of this investigation is to identify and characterize a novel marker of OS progression. We have established and characterized a highly metastatic OS subline that is derived from the less metastatic human MG63 line through serial passages in nude mice via intratibial injections. Microarray analysis of the parental MG63, the highly metastatic MG63.2 subline, as well as the corresponding primary tumors and pulmonary metastases revealed insulin-like growth factor binding protein 5 (IGFBP5) to be one of the significantly downregulated genes in the metastatic subline. Confirmatory quantitative RT-PCR on 20 genes of interest demonstrated IGFBP5 to be the most differentially expressed and was therefore chosen to be one of the genes for further investigation. Adenoviral mediated overexpression and knockdown of IGFBP5 in the MG63 and MG63.2 cell lines, as well as other OS lines (143B and MNNG/HOS) that are independent of our MG63 lines, were employed to examine the role of IGFBP5. We found that overexpression of IGFBP5 inhibited in vitro cell proliferation, migration and invasion of OS cells. Additionally, IGFBP5 overexpression promoted apoptosis and cell cycle arrest in the G1 phase. In an orthotopic xenograft animal model, overexpression of IGFBP5 inhibited OS tumor growth and pulmonary metastases. Conversely, siRNA-mediated knockdown of IGFBP5 promoted OS tumor growth and pulmonary metastases in vivo. Immunohistochemical staining of patient-matched primary and metastatic OS samples demonstrated decreased IGFBP5 expression in the metastases. These results suggest 1) a role for IGFBP5 as a novel marker that has an important role in the pathogenesis of OS, and 2) that the loss of IGFBP5 function may contribute to more metastatic phenotypes in OS.

Collagen and Stretch Modulate Autocrine Secretion of Insulin-like Growth Factor-1 and Insulin-like Growth Factor Binding Proteins from Differentiated Skeletal Muscle Cells

NASA Technical Reports Server (NTRS)

Perrone, Carmen E.; Fenwick-Smith, Daniela; Vandenburgh, Herman H.

1995-01-01

Stretch-induced skeletal muscle growth may involve increased autocrine secretion of insulin-like growth factor-1 (IGF-1) since IGF-1 is a potent growth factor for skeletal muscle hypertrophy, and stretch elevates IGF-1 mRNA levels in vivo. In tissue cultures of differentiated avian pectoralis skeletal muscle cells, nanomolar concentrations of exogenous IGF-1 stimulated growth in mechanically stretched but not static cultures. These cultures released up to 100 pg of endogenously produced IGF-1/micro-g of protein/day, as well as three major IGF binding proteins of 31, 36, and 43 kilodaltons (kDa). IGF-1 was secreted from both myofibers and fibroblasts coexisting in the muscle cultures. Repetitive stretch/relaxation of the differentiated skeletal muscle cells stimulated the acute release of IGF-1 during the first 4 h after initiating mechanical activity, but caused no increase in the long-term secretion over 24-72 h of IGF-1, or its binding proteins. Varying the intensity and frequency of stretch had no effect on the long-term efflux of IGF-1. In contrast to stretch, embedding the differentiated muscle cells in a three-dimensional collagen (Type I) matrix resulted in a 2-5-fold increase in long-term IGF-1 efflux over 24-72 h. Collagen also caused a 2-5-fold increase in the release of the IGF binding proteins. Thus, both the extracellular matrix protein type I collagen and stretch stimulate the autocrine secretion of IGF-1, but with different time kinetics. This endogenously produced growth factor may be important for the growth response of skeletal myofibers to both types of external stimuli.

COUP-TF (chicken ovalbumin upstream promoter transcription factor)-interacting protein 1 (CTIP1) is a sequence-specific DNA binding protein.

PubMed Central

Avram, Dorina; Fields, Andrew; Senawong, Thanaset; Topark-Ngarm, Acharawan; Leid, Mark

2002-01-01

Chicken ovalbumin upstream promoter transcription factor (COUP-TF)-interacting proteins 1 and 2 [CTIP1/Evi9/B cell leukaemia (Bcl) l1a and CTIP2/Bcl11b respectively] are highly related C(2)H(2) zinc finger proteins that are abundantly expressed in brain and the immune system, and are associated with immune system malignancies. A selection procedure was employed to isolate high-affinity DNA binding sites for CTIP1. The core binding site on DNA identified in these studies, 5'-GGCCGG-3' (upper strand), is highly related to the canonical GC box and was bound by a CTIP1 oligomeric complex(es) in vitro. Furthermore, both CTIP1 and CTIP2 repressed transcription of a reporter gene harbouring a multimerized CTIP binding site, and this repression was neither reversed by trichostatin A (an inhibitor of known class I and II histone deacetylases) nor stimulated by co-transfection of a COUP-TF family member. These results demonstrate that CTIP1 is a sequence-specific DNA binding protein and a bona fide transcriptional repressor that is capable of functioning independently of COUP-TF family members. These findings may be relevant to the physiological and/or pathological action(s) of CTIPs in cells that do not express COUP-TF family members, such as cells of the haematopoietic and immune systems. PMID:12196208

Establishment of a robust dengue virus NS3-NS5 binding assay for identification of protein-protein interaction inhibitors.

PubMed

Takahashi, Hirotaka; Takahashi, Chikako; Moreland, Nicole J; Chang, Young-Tae; Sawasaki, Tatsuya; Ryo, Akihide; Vasudevan, Subhash G; Suzuki, Youichi; Yamamoto, Naoki

2012-12-01

Whereas the dengue virus (DENV) non-structural (NS) proteins NS3 and NS5 have been shown to interact in vitro and in vivo, the biological relevance of this interaction in viral replication has not been fully clarified. Here, we first applied a simple and robust in vitro assay based on AlphaScreen technology in combination with the wheat-germ cell-free protein production system to detect the DENV-2 NS3-NS5 interaction in a 384-well plate. The cell-free-synthesized NS3 and NS5 recombinant proteins were soluble and in possession of their respective enzymatic activities in vitro. In addition, AlphaScreen assays using the recombinant proteins detected a specific interaction between NS3 and NS5 with a robust Z' factor of 0.71. By employing the AlphaScreen assay, we found that both the N-terminal protease and C-terminal helicase domains of NS3 are required for its association with NS5. Furthermore, a competition assay revealed that the binding of full-length NS3 to NS5 was significantly inhibited by the addition of an excess of NS3 protease or helicase domains. Our results demonstrate that the AlphaScreen assay can be used to discover novel antiviral agents targeting the interactions between DENV NS proteins. Copyright © 2012 Elsevier B.V. All rights reserved.

The Apc5 Subunit of the Anaphase-Promoting Complex/Cyclosome Interacts with Poly(A) Binding Protein and Represses Internal Ribosome Entry Site-Mediated Translation

PubMed Central

Koloteva-Levine, Nadejda; Pinchasi, Dalia; Pereman, Idan; Zur, Amit; Brandeis, Michael; Elroy-Stein, Orna

2004-01-01

The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin ligase that mediates the proteolysis of cell cycle proteins in mitosis and G1. We used a yeast three-hybrid screen to identify proteins that interact with the internal ribosome entry site (IRES) of platelet-derived growth factor 2 mRNA. Surprisingly, this screen identified Apc5, although it does not harbor a classical RNA binding domain. We found that Apc5 binds the poly(A) binding protein (PABP), which directly binds the IRES element. PABP was found to enhance IRES-mediated translation, whereas Apc5 overexpression counteracted this effect. In addition to its association with the APC/C complex, Apc5 binds much heavier complexes and cosediments with the ribosomal fraction. In contrast to Apc3, which is associated only with the APC/C and remains intact during differentiation, Apc5 is degraded upon megakaryocytic differentiation in correlation with IRES activation. Expression of Apc5 in differentiated cells abolished IRES activation. This is the first report implying an additional role for an APC/C subunit, apart from its being part of the APC/C complex. PMID:15082755

CHEMOSENSITIZATION BY A NON-APOPTOGENIC HEAT SHOCK PROTEIN 70-BINDING APOPTOSIS INDUCING FACTOR MUTANT

EPA Science Inventory

Chemosensitization by a non-apoptogenic heat shock protein 70-binding apoptosis inducing factor mutant

Abstract
HSP70 inhibits apoptosis by neutralizing the caspase activator Apaf-1 and by interacting with apoptosis inducing factor (AIF), a mitochondrial flavoprotein wh...

The C-module-binding factor supports amplification of TRE5-A retrotransposons in the Dictyostelium discoideum genome.

PubMed

Bilzer, Annika; Dölz, Heike; Reinhardt, Alexander; Schmith, Anika; Siol, Oliver; Winckler, Thomas

2011-01-01

Retrotransposable elements are molecular parasites that have invaded the genomes of virtually all organisms. Although retrotransposons encode essential proteins to mediate their amplification, they also require assistance by host cell-encoded machineries that perform functions such as DNA transcription and repair. The retrotransposon TRE5-A of the social amoeba Dictyostelium discoideum generates a notable amount of both sense and antisense RNAs, which are generated from element-internal promoters, located in the A module and the C module, respectively. We observed that TRE5-A retrotransposons depend on the C-module-binding factor (CbfA) to maintain high steady-state levels of TRE5-A transcripts and that CbfA supports the retrotransposition activity of TRE5-A elements. The carboxy-terminal domain of CbfA was found to be required and sufficient to mediate the accumulation of TRE5-A transcripts, but it did not support productive retrotransposition of TRE5-A. This result suggests different roles for CbfA protein domains in the regulation of TRE5-A retrotransposition frequency in D. discoideum cells. Although CbfA binds to the C module in vitro, the factor regulates neither C-module nor A-module promoter activity in vivo. We speculate that CbfA supports the amplification of TRE5-A retrotransposons by suppressing the expression of an as yet unidentified component of the cellular posttranscriptional gene silencing machinery.

Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase.

PubMed

Kudlow, J E; Leung, Y

1984-06-15

Epidermal growth factor (EGF), after binding to its receptor, activates a tyrosine-specific protein kinase which phosphorylates several substrates, including the EGF receptor itself. The effects of a photoaffinity analogue of ATP, 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5'-triphosphate (arylazido-beta-alanyl-ATP) on the EGF-dependent protein kinase in A431 human tumour cell plasma membrane vesicles was investigated. This analogue was capable of inactivating the EGF-receptor kinase in a photodependent manner. Partial inactivation occurred at an analogue concentration of 1 microM and complete inactivation occurred at 10 microM when a 2 min light exposure was used. Arylazido-beta-alanine at 100 microM and ATP at 100 microM were incapable of inactivating the enzyme with 2 min of light exposure. The photodependent inactivation of the enzyme by the analogue could be partially blocked by 20 mM-ATP and more effectively blocked by either 20 mM-adenosine 5'-[beta gamma-imido]triphosphate or 20 mM-guanosine 5'-[beta gamma-imido]triphosphate, indicating nucleotide-binding site specificity. Arylazido-beta-alanyl-[alpha-32P]ATP was capable of labelling membrane proteins in a photodependent manner. Numerous proteins were labelled, the most prominent of which ran with an apparent Mr of 53000 on polyacrylamide-gel electrophoresis. A band of minor intensity was seen of Mr corresponding to the EGF receptor (170000). Immunoprecipitation of affinity-labelled and solubilized membranes with an anti-(EGF receptor) monoclonal antibody demonstrated that the Mr 170000 receptor protein was photoaffinity labelled by the analogue. The Mr 53000 peptide was not specifically bound by the anti-receptor antibody. The affinity labelling of the receptor was not enhanced by EGF, suggesting that EGF stimulation of the kinase activity does not result from changes in the affinity of the kinase for ATP. These studies demonstrate that arylazido-beta-alanyl-ATP interacts with the ATP-binding

Discovery of binding proteins for a protein target using protein-protein docking-based virtual screening.

PubMed

Zhang, Changsheng; Tang, Bo; Wang, Qian; Lai, Luhua

2014-10-01

Target structure-based virtual screening, which employs protein-small molecule docking to identify potential ligands, has been widely used in small-molecule drug discovery. In the present study, we used a protein-protein docking program to identify proteins that bind to a specific target protein. In the testing phase, an all-to-all protein-protein docking run on a large dataset was performed. The three-dimensional rigid docking program SDOCK was used to examine protein-protein docking on all protein pairs in the dataset. Both the binding affinity and features of the binding energy landscape were considered in the scoring function in order to distinguish positive binding pairs from negative binding pairs. Thus, the lowest docking score, the average Z-score, and convergency of the low-score solutions were incorporated in the analysis. The hybrid scoring function was optimized in the all-to-all docking test. The docking method and the hybrid scoring function were then used to screen for proteins that bind to tumor necrosis factor-α (TNFα), which is a well-known therapeutic target for rheumatoid arthritis and other autoimmune diseases. A protein library containing 677 proteins was used for the screen. Proteins with scores among the top 20% were further examined. Sixteen proteins from the top-ranking 67 proteins were selected for experimental study. Two of these proteins showed significant binding to TNFα in an in vitro binding study. The results of the present study demonstrate the power and potential application of protein-protein docking for the discovery of novel binding proteins for specific protein targets. © 2014 Wiley Periodicals, Inc.

Factor H binds to the hypervariable region of many Streptococcus pyogenes M proteins but does not promote phagocytosis resistance or acute virulence.

PubMed

Gustafsson, Mattias C U; Lannergård, Jonas; Nilsson, O Rickard; Kristensen, Bodil M; Olsen, John E; Harris, Claire L; Ufret-Vincenty, Rafael L; Stålhammar-Carlemalm, Margaretha; Lindahl, Gunnar

2013-01-01

Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems.

Factor H Binds to the Hypervariable Region of Many Streptococcus pyogenes M Proteins but Does Not Promote Phagocytosis Resistance or Acute Virulence

PubMed Central

Kristensen, Bodil M.; Olsen, John E.; Harris, Claire L.; Ufret-Vincenty, Rafael L.; Stålhammar-Carlemalm, Margaretha; Lindahl, Gunnar

2013-01-01

Many pathogens express a surface protein that binds the human complement regulator factor H (FH), as first described for Streptococcus pyogenes and the antiphagocytic M6 protein. It is commonly assumed that FH recruited to an M protein enhances virulence by protecting the bacteria against complement deposition and phagocytosis, but the role of FH-binding in S. pyogenes pathogenesis has remained unclear and controversial. Here, we studied seven purified M proteins for ability to bind FH and found that FH binds to the M5, M6 and M18 proteins but not the M1, M3, M4 and M22 proteins. Extensive immunochemical analysis indicated that FH binds solely to the hypervariable region (HVR) of an M protein, suggesting that selection has favored the ability of certain HVRs to bind FH. These FH-binding HVRs could be studied as isolated polypeptides that retain ability to bind FH, implying that an FH-binding HVR represents a distinct ligand-binding domain. The isolated HVRs specifically interacted with FH among all human serum proteins, interacted with the same region in FH and showed species specificity, but exhibited little or no antigenic cross-reactivity. Although these findings suggested that FH recruited to an M protein promotes virulence, studies in transgenic mice did not demonstrate a role for bound FH during acute infection. Moreover, phagocytosis tests indicated that ability to bind FH is neither sufficient nor necessary for S. pyogenes to resist killing in whole human blood. While these data shed new light on the HVR of M proteins, they suggest that FH-binding may affect S. pyogenes virulence by mechanisms not assessed in currently used model systems. PMID:23637608

Spatial control of translation repression and polarized growth by conserved NDR kinase Orb6 and RNA-binding protein Sts5.

PubMed

Nuñez, Illyce; Rodriguez Pino, Marbelys; Wiley, David J; Das, Maitreyi E; Chen, Chuan; Goshima, Tetsuya; Kume, Kazunori; Hirata, Dai; Toda, Takashi; Verde, Fulvia

2016-07-30

RNA-binding proteins contribute to the formation of ribonucleoprotein (RNP) granules by phase transition, but regulatory mechanisms are not fully understood. Conserved fission yeast NDR (Nuclear Dbf2-Related) kinase Orb6 governs cell morphogenesis in part by spatially controlling Cdc42 GTPase. Here we describe a novel, independent function for Orb6 kinase in negatively regulating the recruitment of RNA-binding protein Sts5 into RNPs to promote polarized cell growth. We find that Orb6 kinase inhibits Sts5 recruitment into granules, its association with processing (P) bodies, and degradation of Sts5-bound mRNAs by promoting Sts5 interaction with 14-3-3 protein Rad24. Many Sts5-bound mRNAs encode essential factors for polarized cell growth, and Orb6 kinase spatially and temporally controls the extent of Sts5 granule formation. Disruption of this control system affects cell morphology and alters the pattern of polarized cell growth, revealing a role for Orb6 kinase in the spatial control of translational repression that enables normal cell morphogenesis.

The antimicrobial peptide derived from insulin-like growth factor-binding protein 5, AMP-IBP5, regulates keratinocyte functions through Mas-related gene X receptors.

PubMed

Chieosilapatham, Panjit; Niyonsaba, François; Kiatsurayanon, Chanisa; Okumura, Ko; Ikeda, Shigaku; Ogawa, Hideoki

2017-10-01

In addition to their microbicidal properties, host defense peptides (HDPs) display various immunomodulatory functions, including keratinocyte production of cytokines/chemokines, proliferation, migration and wound healing. Recently, a novel HDP named AMP-IBP5 (antimicrobial peptide derived from insulin-like growth factor-binding protein 5) was shown to exhibit antimicrobial activity against numerous pathogens, even at concentrations comparable to those of human β-defensins and LL-37. However, the immunomodulatory role of AMP-IBP5 in cutaneous tissue remains unknown. To investigate whether AMP-IBP5 triggers keratinocyte activation and to clarify its mechanism. Production of cytokines/chemokines and growth factors was determined by appropriate ELISA kits. Cell migration was assessed by in vitro wound closure assay, whereas cell proliferation was analyzed using BrdU incorporation assay complimented with XTT assay. MAPK and NF-κB activation was determined by Western blotting. Intracellular cAMP levels were assessed using cAMP enzyme immunoassay kit. Among various cytokines/chemokines and growth factors tested, AMP-IBP5 selectively increased the production of IL-8 and VEGF. Moreover, AMP-IBP5 markedly enhanced keratinocyte migration and proliferation. AMP-IBP5-induced keratinocyte activation was mediated by Mrg X1-X4 receptors with MAPK and NF-κB pathways working downstream, as evidenced by the inhibitory effects of MrgX1-X4 siRNAs and ERK-, JNK-, p38- and NF-κB-specific inhibitors. We confirmed that AMP-IBP5 indeed induced MAPK and NF-κB activation. Furthermore, AMP-IBP5-induced VEGF but not IL-8 production correlated with an increase in intracellular cAMP. Our findings suggest that in addition to its antimicrobial function, AMP-IBP5 might contribute to wound healing process through activation of keratinocytes. Copyright © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.

Clinical relevance of drug binding to plasma proteins

NASA Astrophysics Data System (ADS)

Ascenzi, Paolo; Fanali, Gabriella; Fasano, Mauro; Pallottini, Valentina; Trezza, Viviana

2014-12-01

Binding to plasma proteins highly influences drug efficacy, distribution, and disposition. Serum albumin, the most abundant protein in plasma, is a monomeric multi-domain macromolecule that displays an extraordinary ligand binding capacity, providing a depot and carrier for many endogenous and exogenous compounds, such as fatty acids and most acidic drugs. α-1-Acid glycoprotein, the second main plasma protein, is a glycoprotein physiologically involved in the acute phase reaction and is the main carrier for basic and neutral drugs. High- and low-density lipoproteins play a limited role in drug binding and are natural drug delivery system only for few lipophilic drugs or lipid-based formulations. Several factors influence drug binding to plasma proteins, such as pathological conditions, concurrent administration of drugs, sex, and age. Any of these factors, in turn, influences drug efficacy and toxicity. Here, biochemical, biomedical, and biotechnological aspects of drug binding to plasma proteins are reviewed.

Substitution of blood coagulation factor X-binding to Ad5 by position-specific PEGylation: Preventing vector clearance and preserving infectivity.

PubMed

Krutzke, L; Prill, J M; Engler, T; Schmidt, C Q; Xu, Z; Byrnes, A P; Simmet, T; Kreppel, F

2016-08-10

The biodistribution of adenovirus type 5 (Ad5) vector particles is heavily influenced by interaction of the particles with plasma proteins, including coagulation factor X (FX), which binds specifically to the major Ad5 capsid protein hexon. FX mediates hepatocyte transduction by intravenously-injected Ad5 vectors and shields vector particles from neutralization by natural antibodies and complement. In mice, mutant Ad5 vectors that are ablated for FX-binding become detargeted from hepatocytes, which is desirable for certain applications, but unfortunately such FX-nonbinding vectors also become sensitive to neutralization by mouse plasma proteins. To improve the properties of Ad5 vectors for systemic delivery, we developed a strategy to replace the natural FX shield by a site-specific chemical polyethylene glycol shield. Coupling of polyethylene glycol to a specific site in hexon hypervariable region 1 yielded vector particles that were protected from neutralization by natural antibodies and complement although they were unable to bind FX. These vector particles evaded macrophages in vitro and showed significantly improved pharmacokinetics and hepatocyte transduction in vivo. Thus, site-specific shielding of Ad5 vectors with polyethylene glycol rendered vectors FX-independent and greatly improved their properties for systemic gene therapy. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Structure and Function of Lipopolysaccharide Binding Protein

NASA Astrophysics Data System (ADS)

Schumann, Ralf R.; Leong, Steven R.; Flaggs, Gail W.; Gray, Patrick W.; Wright, Samuel D.; Mathison, John C.; Tobias, Peter S.; Ulevitch, Richard J.

1990-09-01

The primary structure of lipopolysaccharide binding protein (LBP), a trace plasma protein that binds to the lipid A moiety of bacterial lipopolysaccharides (LPSs), was deduced by sequencing cloned complementary DNA. LBP shares sequence identity with another LPS binding protein found in granulocytes, bactericidal/permeability-increasing protein, and with cholesterol ester transport protein of the plasma. LBP may control the response to LPS under physiologic conditions by forming high-affinity complexes with LPS that bind to monocytes and macrophages, which then secrete tumor necrosis factor. The identification of this pathway for LPS-induced monocyte stimulation may aid in the development of treatments for diseases in which Gram-negative sepsis or endotoxemia are involved.

Molecular recognition of avirulence protein (avrxa5) by eukaryotic transcription factor xa5 of rice (Oryza sativa L.): insights from molecular dynamics simulations.

PubMed

Dehury, Budheswar; Maharana, Jitendra; Sahoo, Bikash Ranjan; Sahu, Jagajjit; Sen, Priyabrata; Modi, Mahendra Kumar; Barooah, Madhumita

2015-04-01

The avirulence gene avrxa5 of bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) recognized by the resistant rice lines having corresponding resistance (xa5) gene in a gene-for-gene manner. We used a combinatorial approach involving protein-protein docking, molecular dynamics (MD) simulations and binding free energy calculations to gain novel insights into the gene-for-gene mechanism that governs the direct interaction of R-Avr protein. From the best three binding poses predicted by molecular docking, MD simulations were performed to explore the dynamic binding mechanism of xa5 and avrxa5. Molecular Mechanics/Poisson Boltzmann Surface Area (MM/PBSA) techniques were employed to calculate the binding free energy and to uncover the thriving force behind the molecular recognition of avrxa5 by eukaryotic transcription factor xa5. Binding free energy analysis revealed van der Waals term as the most constructive component that favors the xa5 and avrxa5 interaction. In addition, hydrogen bonds (H-bonds) and essential electrostatic interactions analysis highlighted amino acid residues Lys54/Asp870, Lys56/Ala868, Lys56/Ala866, Lys56/Glu871, Ile59/His862, Gly61/Phe858, His62/Arg841, His62/Leu856, Ser101/Ala872 and Ser105/Asp870 plays pivotal role for the energetically stability of the R-Avr complex. Insights gained from the present study are expected to unveil the molecular mechanisms that define the transcriptional activator mediated transcriptome modification in host plants. Copyright © 2015 Elsevier Inc. All rights reserved.

The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence.

PubMed

Lahr, Roni M; Mack, Seshat M; Héroux, Annie; Blagden, Sarah P; Bousquet-Antonelli, Cécile; Deragon, Jean-Marc; Berman, Andrea J

2015-09-18

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. A putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. These studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5'TOP sequence

DOE PAGES

Lahr, Roni M.; Mack, Seshat M.; Heroux, Annie; ...

2015-07-22

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5'TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5' UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5'TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. Amore » putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. Ultimately, these studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signalling to ribosome biogenesis.« less

Interaction entropy for protein-protein binding

NASA Astrophysics Data System (ADS)

Sun, Zhaoxi; Yan, Yu N.; Yang, Maoyou; Zhang, John Z. H.

2017-03-01

Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interaction entropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interaction entropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.

Interaction entropy for protein-protein binding.

PubMed

Sun, Zhaoxi; Yan, Yu N; Yang, Maoyou; Zhang, John Z H

2017-03-28

Protein-protein interactions are at the heart of signal transduction and are central to the function of protein machine in biology. The highly specific protein-protein binding is quantitatively characterized by the binding free energy whose accurate calculation from the first principle is a grand challenge in computational biology. In this paper, we show how the interactionentropy approach, which was recently proposed for protein-ligand binding free energy calculation, can be applied to computing the entropic contribution to the protein-protein binding free energy. Explicit theoretical derivation of the interactionentropy approach for protein-protein interaction system is given in detail from the basic definition. Extensive computational studies for a dozen realistic protein-protein interaction systems are carried out using the present approach and comparisons of the results for these protein-protein systems with those from the standard normal mode method are presented. Analysis of the present method for application in protein-protein binding as well as the limitation of the method in numerical computation is discussed. Our study and analysis of the results provided useful information for extracting correct entropic contribution in protein-protein binding from molecular dynamics simulations.

Predicting protein-binding RNA nucleotides with consideration of binding partners.

PubMed

Tuvshinjargal, Narankhuu; Lee, Wook; Park, Byungkyu; Han, Kyungsook

2015-06-01

In recent years several computational methods have been developed to predict RNA-binding sites in protein. Most of these methods do not consider interacting partners of a protein, so they predict the same RNA-binding sites for a given protein sequence even if the protein binds to different RNAs. Unlike the problem of predicting RNA-binding sites in protein, the problem of predicting protein-binding sites in RNA has received little attention mainly because it is much more difficult and shows a lower accuracy on average. In our previous study, we developed a method that predicts protein-binding nucleotides from an RNA sequence. In an effort to improve the prediction accuracy and usefulness of the previous method, we developed a new method that uses both RNA and protein sequence data. In this study, we identified effective features of RNA and protein molecules and developed a new support vector machine (SVM) model to predict protein-binding nucleotides from RNA and protein sequence data. The new model that used both protein and RNA sequence data achieved a sensitivity of 86.5%, a specificity of 86.2%, a positive predictive value (PPV) of 72.6%, a negative predictive value (NPV) of 93.8% and Matthews correlation coefficient (MCC) of 0.69 in a 10-fold cross validation; it achieved a sensitivity of 58.8%, a specificity of 87.4%, a PPV of 65.1%, a NPV of 84.2% and MCC of 0.48 in independent testing. For comparative purpose, we built another prediction model that used RNA sequence data alone and ran it on the same dataset. In a 10 fold-cross validation it achieved a sensitivity of 85.7%, a specificity of 80.5%, a PPV of 67.7%, a NPV of 92.2% and MCC of 0.63; in independent testing it achieved a sensitivity of 67.7%, a specificity of 78.8%, a PPV of 57.6%, a NPV of 85.2% and MCC of 0.45. In both cross-validations and independent testing, the new model that used both RNA and protein sequences showed a better performance than the model that used RNA sequence data alone in

The C-Module-Binding Factor Supports Amplification of TRE5-A Retrotransposons in the Dictyostelium discoideum Genome ▿

PubMed Central

Bilzer, Annika; Dölz, Heike; Reinhardt, Alexander; Schmith, Anika; Siol, Oliver; Winckler, Thomas

2011-01-01

Retrotransposable elements are molecular parasites that have invaded the genomes of virtually all organisms. Although retrotransposons encode essential proteins to mediate their amplification, they also require assistance by host cell-encoded machineries that perform functions such as DNA transcription and repair. The retrotransposon TRE5-A of the social amoeba Dictyostelium discoideum generates a notable amount of both sense and antisense RNAs, which are generated from element-internal promoters, located in the A module and the C module, respectively. We observed that TRE5-A retrotransposons depend on the C-module-binding factor (CbfA) to maintain high steady-state levels of TRE5-A transcripts and that CbfA supports the retrotransposition activity of TRE5-A elements. The carboxy-terminal domain of CbfA was found to be required and sufficient to mediate the accumulation of TRE5-A transcripts, but it did not support productive retrotransposition of TRE5-A. This result suggests different roles for CbfA protein domains in the regulation of TRE5-A retrotransposition frequency in D. discoideum cells. Although CbfA binds to the C module in vitro, the factor regulates neither C-module nor A-module promoter activity in vivo. We speculate that CbfA supports the amplification of TRE5-A retrotransposons by suppressing the expression of an as yet unidentified component of the cellular posttranscriptional gene silencing machinery. PMID:21076008

Membrane Binding of HIV-1 Matrix Protein: Dependence on Bilayer Composition and Protein Lipidation

PubMed Central

Barros, Marilia; Nanda, Hirsh

2016-01-01

ABSTRACT By assembling in a protein lattice on the host's plasma membrane, the retroviral Gag polyprotein triggers formation of the viral protein/membrane shell. The MA domain of Gag employs multiple signals—electrostatic, hydrophobic, and lipid-specific—to bring the protein to the plasma membrane, thereby complementing protein-protein interactions, located in full-length Gag, in lattice formation. We report the interaction of myristoylated and unmyristoylated HIV-1 Gag MA domains with bilayers composed of purified lipid components to dissect these complex membrane signals and quantify their contributions to the overall interaction. Surface plasmon resonance on well-defined planar membrane models is used to quantify binding affinities and amounts of protein and yields free binding energy contributions, ΔG, of the various signals. Charge-charge interactions in the absence of the phosphatidylinositide PI(4,5)P2 attract the protein to acidic membrane surfaces, and myristoylation increases the affinity by a factor of 10; thus, our data do not provide evidence for a PI(4,5)P2 trigger of myristate exposure. Lipid-specific interactions with PI(4,5)P2, the major signal lipid in the inner plasma membrane, increase membrane attraction at a level similar to that of protein lipidation. While cholesterol does not directly engage in interactions, it augments protein affinity strongly by facilitating efficient myristate insertion and PI(4,5)P2 binding. We thus observe that the isolated MA protein, in the absence of protein-protein interaction conferred by the full-length Gag, binds the membrane with submicromolar affinities. IMPORTANCE Like other retroviral species, the Gag polyprotein of HIV-1 contains three major domains: the N-terminal, myristoylated MA domain that targets the protein to the plasma membrane of the host; a central capsid-forming domain; and the C-terminal, genome-binding nucleocapsid domain. These domains act in concert to condense Gag into a membrane

Substrate-Triggered Exosite Binding: Synergistic Dendrimer/Folic Acid Action for Achieving Specific, Tight-Binding to Folate Binding Protein.

PubMed

Chen, Junjie; van Dongen, Mallory A; Merzel, Rachel L; Dougherty, Casey A; Orr, Bradford G; Kanduluru, Ananda Kumar; Low, Philip S; Marsh, E Neil G; Banaszak Holl, Mark M

2016-03-14

Polymer-ligand conjugates are designed to bind proteins for applications as drugs, imaging agents, and transport scaffolds. In this work, we demonstrate a folic acid (FA)-triggered exosite binding of a generation five poly(amidoamine) (G5 PAMAM) dendrimer scaffold to bovine folate binding protein (bFBP). The protein exosite is a secondary binding site on the protein surface, separate from the FA binding pocket, to which the dendrimer binds. Exosite binding is required to achieve the greatly enhanced binding constants and protein structural change observed in this study. The G5Ac-COG-FA1.0 conjugate bound tightly to bFBP, was not displaced by a 28-fold excess of FA, and quenched roughly 80% of the initial fluorescence. Two-step binding kinetics were measured using the intrinsic fluorescence of the FBP tryptophan residues to give a KD in the low nanomolar range for formation of the initial G5Ac-COG-FA1.0/FBP* complex, and a slow conversion to the tight complex formed between the dendrimer and the FBP exosite. The extent of quenching was sensitive to the choice of FA-dendrimer linker chemistry. Direct amide conjugation of FA to G5-PAMAM resulted in roughly 50% fluorescence quenching of the FBP. The G5Ac-COG-FA, which has a longer linker containing a 1,2,3-triazole ring, exhibited an ∼80% fluorescence quenching. The binding of the G5Ac-COG-FA1.0 conjugate was compared to poly(ethylene glycol) (PEG) conjugates of FA (PEGn-FA). PEG2k-FA had a binding strength similar to that of FA, whereas other PEG conjugates with higher molecular weight showed weaker binding. However, no PEG conjugates gave an increased degree of total fluorescence quenching.

Nuclear actions of insulin-like growth factor binding protein-3.

PubMed

Baxter, Robert C

2015-09-10

In addition to its actions outside the cell, cellular uptake and nuclear import of insulin-like growth factor binding protein-3 (IGFBP-3) has been recognized for almost two decades, but knowledge of its nuclear actions has been slow to emerge. IGFBP-3 has a functional nuclear localization signal and interacts with the nuclear transport protein importin-β. Within the nucleus IGFBP-3 appears to have a role in transcriptional regulation. It can bind to the nuclear receptor, retinoid X receptor-α and several of its dimerization partners, including retinoic acid receptor, vitamin D receptor (VDR), and peroxisome proliferator-activated receptor-γ (PPARγ). These interactions modulate the functions of these receptors, for example inhibiting VDR-dependent transcription in osteoblasts and PPARγ-dependent transcription in adipocytes. Nuclear IGFBP-3 can be detected by immunohistochemistry in cancer and other tissues, and its presence in the nucleus has been shown in many cell culture studies to be necessary for its pro-apoptotic effect, which may also involve interaction with the nuclear receptor Nur77, and export from the nucleus. IGFBP-3 is p53-inducible and in response to DNA damage, forms a complex with the epidermal growth factor receptor (EGFR), translocating to the nucleus to interact with DNA-dependent protein kinase. Inhibition of EGFR kinase activity or downregulation of IGFBP-3 can inhibit DNA double strand-break repair by nonhomologous end joining. IGFBP-3 thus has the ability to influence many cell functions through its interactions with intranuclear pathways, but the importance of these interactions in vivo, and their potential to be targeted for therapeutic benefit, require further investigation. Copyright © 2015 Elsevier B.V. All rights reserved.

Clinical role of protein binding of quinolones.

PubMed

Bergogne-Bérézin, Eugénie

2002-01-01

Protein binding of antibacterials in plasma and tissues has long been considered a component of their pharmacokinetic parameters, playing a potential role in distribution, excretion and therapeutic effectiveness. Since the beginning of the 'antibacterial era', this factor has been extensively analysed for all antibacterial classes, showing that wide variations of the degree of protein binding occur even in the same antibacterial class, as with beta-lactams. As the understanding of protein binding grew, the complexity of the binding system was increasingly perceived and its dynamic character described. Studies of protein binding of the fluoroquinolones have shown that the great majority of these drugs exhibit low protein binding, ranging from approximately 20 to 40% in plasma, and that they are bound predominantly to albumin. The potential role in pharmacokinetics-pharmacodynamics of binding of fluoroquinolones to plasma, tissue and intracellular proteins has been analysed, but it has not been established that protein binding has any significant direct or indirect impact on therapeutic effectiveness. Regarding the factors influencing the tissue distribution of antibacterials, physicochemical characteristics and the small molecular size of fluoroquinolones permit a rapid penetration into extravascular sites and intracellularly, with a rapid equilibrium being established between intravascular and extravascular compartments. The high concentrations of these drugs achieved in tissues, body fluids and intracellularly, in addition to their wide antibacterial spectrum, mean that fluoroquinolones have therapeutic effectiveness in a large variety of infections. The tolerability of quinolones has generally been reported as good, based upon long experience in using pefloxacin, ciprofloxacin and ofloxacin in clinical practice. Among more recently developed molecules, good tolerability has been reported for levofloxacin, moxifloxacin and gatifloxacin, but certain other new

Localization of serotoni (5-hydroxytryptamine, 5-HT) with partial purification and characterization of a serotonin binding protein in the intestinal tissue of the nematode Ascaris suum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Martin, R.E.

1989-01-01

An intracellular 5-HT binding protein (SBP) from intestinal tissue was partially purified and characterized. Binding of ({sup 3}H) 5-HT to the protein appeared to be Fe{sup +2}-sensitive and maximal (20.8pmol/mg protein) at 5 {times} 10{sup {minus}4}M Fe{sup +2} and 10{sup {minus}7}M ({sup 3}H) 5-HT. There were two 5-HT binding sites present at optimum Fe{sup +2} concentrations. The Bmax values of these sites were more sensitive to Fe{sup +2} than Kd values. Sulfhydryl reducing agents, cation chelators, Fe{sup +3}, Ca{sup +2} and antagonists of 5-HT uptake and storage inhibited binding of 5-HT to SBP. Gel exclusion chromatography indicated the presence ofmore » a 45Kda SBP that in 5 {times} 10{sup {minus}5}M Fe{sup +2} may form aggregates ranging in size from approximately 80 to >1000Kda. The data indicate these in vitro aggregates may correspond to the electron-opaque patches observed in situ. Ascaris suum may provide a model system to further elucidate the physiological role of analogous serotonin binding proteins that have been identified in mammalian systems.« less

Insulin-like growth factor (IGF) binding protein from human decidua inhibits the binding and biological action of IGF-I in cultured choriocarcinoma cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ritvos, O.; Ranta, T.; Jalkanen, J.

1988-05-01

The placenta expresses genes for insulin-like growth factors (IGFs) and possesses IGF-receptors, suggesting that placental growth is regulated by IGFs in an autocrine manner. We have previously shown that human decidua, but not placenta, synthesizes and secretes a 34 K IGF-binding protein (34 K IGF-BP) called placental protein 12. We now used human choriocarcinoma JEG-3 cell monolayer cultures and recombinant (Thr59)IGF-I as a model to study whether the decidual 34 K IGF-BP is able to modulate the receptor binding and biological activity of IGFs in trophoblasts. JEG-3 cells, which possess type I IGF receptors, were unable to produce IGF-BPs. Purifiedmore » 34 K IGF-BP specifically bound (125I)iodo-(Thr59)IGF-I. Multiplication-stimulating activity had 2.5% the potency of (Thr59)IGF-I, and insulin had no effect on the binding of (125I) iodo-(Thr59)IGF-I. 34 K IGF-BP inhibited the binding of (125I) iodo-(Thr59)IGF-I to JEG-3 monolayers in a concentration-dependent manner by forming with the tracer a soluble complex that could not bind to the cell surface as demonstrated by competitive binding and cross-linking experiments. After incubating the cell monolayers with (125I)iodo-(Thr59)IGF-I in the presence of purified binding protein, followed by cross-linking, no affinity labeled bands were seen on autoradiography. In contrast, an intensely labeled band at 40 K was detected when the incubation medium was analyzed, suggesting that (Thr59)IGF-I and 34 K IGF-BP formed a complex in a 1:1 molar ratio. Also, 34 K IGF-BP inhibited both basal and IGF-I-stimulated uptake of alpha-(3H)aminoisobutyric acid in JEG-3 cells. RNA analysis revealed that IGF-II is expressed in JEG-3 cells.« less

Identification of AOSC-binding proteins in neurons

NASA Astrophysics Data System (ADS)

Liu, Ming; Nie, Qin; Xin, Xianliang; Geng, Meiyu

2008-11-01

Acidic oligosaccharide sugar chain (AOSC), a D-mannuronic acid oligosaccharide, derived from brown algae polysaccharide, has been completed Phase I clinical trial in China as an anti-Alzheimer’s Disease (AD) drug candidate. The identification of AOSC-binding protein(s) in neurons is very important for understanding its action mechanism. To determine the binding protein(s) of AOSC in neurons mediating its anti-AD activities, confocal microscopy, affinity chromatography, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were used. Confocal microscopy analysis shows that AOSC binds to SH-SY5Y cells in concentration-, time-, and temperature-dependent fashions. The AOSC binding proteins were purified by affinity chromatography and identified by LC-MS/MS analysis. The results showed that there are 349 proteins binding AOSC, including clathrin, adaptor protein-2 (AP-2) and amyloid precursor protein (APP). These results suggest that the binding/entrance of AOSC to neurons is probably responsible for anti-AD activities.

Phosphatidylinositol 3,4,5-trisphosphate activity probes for the labeling and proteomic characterization of protein binding partners.

PubMed

Rowland, Meng M; Bostic, Heidi E; Gong, Denghuang; Speers, Anna E; Lucas, Nathan; Cho, Wonhwa; Cravatt, Benjamin F; Best, Michael D

2011-12-27

Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃], regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P₃ that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins and a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by in-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P₃ headgroup analogue as well as through protein denaturation, indicating specific labeling. In addition, probes featuring linkers of different lengths between the PI(3,4,5)P₃ headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts; labeled proteins were observed by in-gel detection and characterized using postlabeling with biotin, affinity chromatography, and identification via tandem mass spectrometry. These studies yielded a total of 265

Functions of Intracellular Retinoid Binding-Proteins.

PubMed

Napoli, Joseph L

Multiple binding and transport proteins facilitate many aspects of retinoid biology through effects on retinoid transport, cellular uptake, metabolism, and nuclear delivery. These include the serum retinol binding protein sRBP (aka Rbp4), the plasma membrane sRBP receptor Stra6, and the intracellular retinoid binding-proteins such as cellular retinol-binding proteins (CRBP) and cellular retinoic acid binding-proteins (CRABP). sRBP transports the highly lipophilic retinol through an aqueous medium. The major intracellular retinol-binding protein, CRBP1, likely enhances efficient retinoid use by providing a sink to facilitate retinol uptake from sRBP through the plasma membrane or via Stra6, delivering retinol or retinal to select enzymes that generate retinyl esters or retinoic acid, and protecting retinol/retinal from excess catabolism or opportunistic metabolism. Intracellular retinoic acid binding-proteins (CRABP1 and 2, and FABP5) seem to have more diverse functions distinctive to each, such as directing retinoic acid to catabolism, delivering retinoic acid to specific nuclear receptors, and generating non-canonical actions. Gene ablation of intracellular retinoid binding-proteins does not cause embryonic lethality or gross morphological defects. Metabolic and functional defects manifested in knockouts of CRBP1, CRBP2 and CRBP3, however, illustrate their essentiality to health, and in the case of CRBP2, to survival during limited dietary vitamin A. Future studies should continue to address the specific molecular interactions that occur between retinoid binding-proteins and their targets and their precise physiologic contributions to retinoid homeostasis and function.

Modeling Ionization Events iduced by Protein Protein Binding

NASA Astrophysics Data System (ADS)

Mitra, Rooplekha; Shyam, Radhey; Alexov, Emil

2009-11-01

The association of two or more biological macromolecules dramatically change the environment of the amino acids situated at binding interface and could change ionization states of titratable groups. The change of ionization due to the binding results in proton uptake/release and causes pH-dependence of the binding free energy. We apply computational method, as implemented in Multi Conformation Continuum Electrostatics (MCCE) algorithm, to study protonation evens on a large set of protein-protein complexes. Our results indicate that proton uptake/release is a common phenomena in protein binding since in vast majority of the cases (70%) the binding caused at least 0.5 units proton change. The proton uptake/release was further investigated with respect to interfacial area and charges of the monomers and it was found that macroscopic characteristics are not important determinants. Instead, charge complementarity across the interface and the number of unpaired ionizable groups at the interface are the primary source of proton uptake/release.

Bacterial periplasmic sialic acid-binding proteins exhibit a conserved binding site

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gangi Setty, Thanuja; Cho, Christine; Govindappa, Sowmya

2014-07-01

Structure–function studies of sialic acid-binding proteins from F. nucleatum, P. multocida, V. cholerae and H. influenzae reveal a conserved network of hydrogen bonds involved in conformational change on ligand binding. Sialic acids are a family of related nine-carbon sugar acids that play important roles in both eukaryotes and prokaryotes. These sialic acids are incorporated/decorated onto lipooligosaccharides as terminal sugars in multiple bacteria to evade the host immune system. Many pathogenic bacteria scavenge sialic acids from their host and use them for molecular mimicry. The first step of this process is the transport of sialic acid to the cytoplasm, which oftenmore » takes place using a tripartite ATP-independent transport system consisting of a periplasmic binding protein and a membrane transporter. In this paper, the structural characterization of periplasmic binding proteins from the pathogenic bacteria Fusobacterium nucleatum, Pasteurella multocida and Vibrio cholerae and their thermodynamic characterization are reported. The binding affinities of several mutations in the Neu5Ac binding site of the Haemophilus influenzae protein are also reported. The structure and the thermodynamics of the binding of sugars suggest that all of these proteins have a very well conserved binding pocket and similar binding affinities. A significant conformational change occurs when these proteins bind the sugar. While the C1 carboxylate has been identified as the primary binding site, a second conserved hydrogen-bonding network is involved in the initiation and stabilization of the conformational states.« less

Role of transcription factor Sp1 and RNA binding protein HuR in the down-regulation of Dr+ Escherichia coli receptor protein Decay Accelerating Factor (DAF or CD55) by Nitric oxide

PubMed Central

Banadakoppa, Manu; Liebenthal, Daniel; Nowak, David E; Urvil, Petri; Yallampalli, Uma; Wilson, Gerald M; Kishor, Aparna; Yallampalli, Chandra

2012-01-01

We previously reported that nitric oxide (NO) reduces the rate of bacteremia and maternal mortality in pregnant rats with uterine infection by Escherichia coli expressing the Dr Fimbria (Dr+). The epithelial invasion of Dr+ E. coli is dependent on the expression level of its cellular receptor decay accelerating factor (DAF). NO reduces the rate of bacteremia by down-regulating the expression of DAF. In this study, we elucidated the role of transcription factor Sp1 and RNA binding protein HuR in the down-regulation of human DAF by NO. We generated a series of deletion mutant constructs of DAF gene 5′-untranslated region and mapped NO-response region upstream to the core promoter region of the DAF gene. One of the several Sp1 binding sites in the DAF 5′-untranslated region was located within the NO-response region. The binding of Sp1 to this site was inhibited by NO. Furthermore, NO also promoted the degradation of DAF mRNA. The 3′-untranslated region of DAF harbors an AU-rich element and this element destabilized the mRNA transcript. The NO promoted the rapid degradation of DAF mRNA by inhibiting the binding of mRNA stabilizing protein HuR to this AU-rich region. The inhibition of binding of HuR to AU-rich region was due to the S-nitrosylation of one or more cysteine residues by NO. Thus, these data reveal the molecular mediators of transcriptional and post-transcriptional regulation of DAF by NO with implications in pathophysiology related to DAF. PMID:23176121

Tumor Necrosis Factor Receptor-Associated Factor 5 Interacts with the NS3 Protein and Promotes Classical Swine Fever Virus Replication.

PubMed

Lv, Huifang; Dong, Wang; Guo, Kangkang; Jin, Mingxing; Li, Xiaomeng; Li, Cunfa; Zhang, Yanming

2018-06-05

Classical swine fever, caused by classical swine fever virus (CSFV), is a highly contagious and high-mortality viral disease, causing huge economic losses in the swine industry worldwide. CSFV non-structural protein 3 (NS3), a multifunctional protein, plays crucial roles in viral replication. However, how NS3 exactly exerts these functions is currently unknown. Here, we identified tumor necrosis factor receptor-associated factor 5 (TRAF5) as a novel binding partner of the NS3 protein via yeast two-hybrid, co-immunoprecipitation and glutathione S -transferase pull-down assays. Furthermore, we observed that TRAF5 promoted CSFV replication in porcine alveolar macrophages (PAMs). Additionally, CSFV infection or NS3 expression upregulated TRAF5 expression, implying that CSFV may exploit TRAF5 via NS3 for better growth. Moreover, CSFV infection and TRAF5 expression activated p38 mitogen activated protein kinase (MAPK) activity, and inhibition of p38 MAPK activation by the SB203580 inhibitor suppressed CSFV replication. Notably, TRAF5 overexpression did not promote CSFV replication following inhibition of p38 MAPK activation. Our findings reveal that TRAF5 promotes CSFV replication via p38 MAPK activation. This work provides a novel insight into the role of TRAF5 in CSFV replication capacity.

Structural Determination of Functional Domains in Early B-cell Factor (EBF) Family of Transcription Factors Reveals Similarities to Rel DNA-binding Proteins and a Novel Dimerization Motif*

PubMed Central

Siponen, Marina I.; Wisniewska, Magdalena; Lehtiö, Lari; Johansson, Ida; Svensson, Linda; Raszewski, Grzegorz; Nilsson, Lennart; Sigvardsson, Mikael; Berglund, Helena

2010-01-01

The early B-cell factor (EBF) transcription factors are central regulators of development in several organs and tissues. This protein family shows low sequence similarity to other protein families, which is why structural information for the functional domains of these proteins is crucial to understand their biochemical features. We have used a modular approach to determine the crystal structures of the structured domains in the EBF family. The DNA binding domain reveals a striking resemblance to the DNA binding domains of the Rel homology superfamily of transcription factors but contains a unique zinc binding structure, termed zinc knuckle. Further the EBF proteins contain an IPT/TIG domain and an atypical helix-loop-helix domain with a novel type of dimerization motif. The data presented here provide insights into unique structural features of the EBF proteins and open possibilities for detailed molecular investigations of this important transcription factor family. PMID:20592035

A Single Rainbow Trout Cobalamin-binding Protein Stands in for Three Human Binders

PubMed Central

Greibe, Eva; Fedosov, Sergey; Sorensen, Boe S.; Højrup, Peter; Poulsen, Steen S.; Nexo, Ebba

2012-01-01

Cobalamin uptake and transport in mammals are mediated by three cobalamin-binding proteins: haptocorrin, intrinsic factor, and transcobalamin. The nature of cobalamin-binding proteins in lower vertebrates remains to be elucidated. The aim of this study was to characterize the cobalamin-binding proteins of the rainbow trout (Oncorhynchus mykiss) and to compare their properties with those of the three human cobalamin-binding proteins. High cobalamin-binding capacity was found in trout stomach (210 pmol/g), roe (400 pmol/g), roe fluid (390 nmol/liter), and plasma (2500 nmol/liter). In all cases, it appeared to be the same protein based on analysis of partial sequences and immunological responses. The trout cobalamin-binding protein was purified from roe fluid, sequenced, and further characterized. Like haptocorrin, the trout cobalamin-binding protein was stable at low pH and had a high binding affinity for the cobalamin analog cobinamide. Like haptocorrin and transcobalamin, the trout cobalamin-binding protein was present in plasma and recognized ligands with altered nucleotide moiety. Like intrinsic factors, the trout cobalamin-binding protein was present in the stomach and resisted degradation by trypsin and chymotrypsin. It also resembled intrinsic factor in the composition of conserved residues in the primary cobalamin-binding site in the C terminus. The trout cobalamin-binding protein was glycosylated and displayed spectral properties comparable with those of haptocorrin and intrinsic factor. In conclusion, only one soluble cobalamin-binding protein was identified in the rainbow trout, a protein that structurally behaves like an intermediate between the three human cobalamin-binding proteins. PMID:22872637

Insulin-like growth factor binding protein-2: contributions of the C-terminal domain to insulin-like growth factor-1 binding.

PubMed

Kibbey, Megan M; Jameson, Mark J; Eaton, Erin M; Rosenzweig, Steven A

2006-03-01

Signaling by the insulin-like growth factor (IGF)-1 receptor (IGF-1R) has been implicated in the promotion and aggressiveness of breast, prostate, colorectal, and lung cancers. The IGF binding proteins (IGFBPs) represent a class of natural IGF antagonists that bind to and sequester IGF-1/2 from the IGF-1R, making them attractive candidates as therapeutics for cancer prevention and control. Recombinant human IGFBP-2 significantly attenuated IGF-1-stimulated MCF-7 cell proliferation with coaddition of 20 or 100 nM IGFBP-2 (50 or 80% inhibition, respectively). We previously identified IGF-1 contact sites both upstream and downstream of the CWCV motif (residues 247-250) in human IGFBP-2 (J Biol Chem 276:2880-2889, 2001). To further test their contributions to IGFBP-2 function, the single tryptophan in human IGFBP-2, Trp-248, was selectively cleaved with 2-(2'nitrophenylsulfenyl)-3-methyl-3 bromoindolenine (BNPS-skatole) and the BNPS-skatole products IGFBP-2(1-248) and IGFBP-2(249-289) as well as IGFBP-2(1-190) were expressed as glutathione S-transferase-fusion proteins and purified. Based on competition binding analysis, deletion of residues 249 to 289 caused an approximately 20-fold decrease in IGF-1 binding affinity (IGFBP-2 EC50 = 0.35 nM and IGFBP-2(1-248) = 7 nM). Removal of the remainder of the C-terminal domain had no further effect on affinity (IGFBP-2(1-190) EC50 = 9.2 nM). In kinetic assays, IGFBP-2(1-248) and IGFBP-2(1-190) exhibited more rapid association and dissociation rates than full-length IGFBP-2. These results confirm that regions upstream and downstream of the CWCV motif participate in IGF-1 binding. They further support the development of full-length IGFBP-2 as a cancer therapeutic.

Role of transcription factor Sp1 and RNA binding protein HuR in the downregulation of Dr+ Escherichia coli receptor protein decay accelerating factor (DAF or CD55) by nitric oxide.

PubMed

Banadakoppa, Manu; Liebenthal, Daniel; Nowak, David E; Urvil, Petri; Yallampalli, Uma; Wilson, Gerald M; Kishor, Aparna; Yallampalli, Chandra

2013-02-01

We previously reported that nitric oxide (NO) reduces the rate of bacteremia and maternal mortality in pregnant rats with uterine infection by Escherichia coli expressing the Dr Fimbria (Dr(+) ). The epithelial invasion of Dr(+) E. coli is dependent on the expression level of its cellular receptor decay accelerating factor (DAF). NO reduces the rate of bacteremia by downregulating the expression of DAF. In this study, we elucidated the role of transcription factor Sp1 and RNA binding protein HuR in the downregulation of human DAF by NO. We generated a series of deletion mutant constructs of DAF gene 5'-untranslated region and mapped the NO-response region upstream to the core promoter region of the DAF gene. One of the several Sp1 binding sites in the DAF 5'-untranslated region was located within the NO-response region. The binding of Sp1 to this site was inhibited by NO. Furthermore, NO also promoted the degradation of DAF mRNA. The 3'-untranslated region of DAF harbors an AU-rich element and this element destabilized the mRNA transcript. NO promoted the rapid degradation of DAF mRNA by inhibiting the binding of mRNA stabilizing protein HuR to this AU-rich region. The inhibition of binding of HuR to the AU-rich region was due to the S-nitrosylation of one or more cysteine residues by NO. Thus, these data reveal the molecular mediators of transcriptional and post-transcriptional regulation of DAF by NO with implications in pathophysiology related to DAF. © 2012 The Authors Journal compilation © 2012 FEBS.

Production of functional human insulin-like growth factor binding proteins (IGFBPs) using recombinant expression in HEK293 cells.

PubMed

Wanscher, Anne Sofie Molsted; Williamson, Michael; Ebersole, Tasja Wainani; Streicher, Werner; Wikström, Mats; Cazzamali, Giuseppe

2015-04-01

Insulin-like growth factor binding proteins (IGFBPs) display many functions in humans including regulation of the insulin-like growth factor (IGF) signaling pathway. The various roles of human IGFBPs make them attractive protein candidates in drug discovery. Structural and functional knowledge on human proteins with therapeutic relevance is needed to design and process the next generation of protein therapeutics. In order to conduct structural and functional investigations large quantities of recombinant proteins are needed. However, finding a suitable recombinant production system for proteins such as full-length human IGFBPs, still remains a challenge. Here we present a mammalian HEK293 expression method suitable for over-expression of secretory full-length human IGFBP-1 to -7. Protein purification of full-length human IGFBP-1, -2, -3 and -5 was conducted using a two-step chromatography procedure and the final protein yields were between 1 and 12mg protein per liter culture media. The recombinant IGFBPs contained PTMs and exhibited high-affinity interactions with their natural ligands IGF-1 and IGF-2. Copyright © 2014 Elsevier Inc. All rights reserved.

Lead inhibition of DNA-binding mechanism of Cys(2)His(2) zinc finger proteins.

PubMed

Hanas, J S; Rodgers, J S; Bantle, J A; Cheng, Y G

1999-11-01

The association of lead with chromatin in cells suggests that deleterious metal effects may in part be mediated through alterations in gene function. To elucidate if and how lead may alter DNA binding of cysteine-rich zinc finger proteins, lead ions were analyzed for their ability to alter the DNA binding mechanism of the Cys(2)His(2) zinc finger protein transcription factor IIIA (TFIIIA). As assayed by DNase I protection, the interaction of TFIIIA with the 50-bp internal control region of the 5S ribosomal gene was partially inhibited by 5 microM lead ions and completely inhibited by 10 to 20 microM lead ions. Preincubation of free TFIIIA with lead resulted in DNA-binding inhibition, whereas preincubation of a TFIIIA/5S RNA complex with lead did not result in DNA-binding inhibition. Because 5S RNA binds TFIIIA zinc fingers, this result is consistent with an inhibition mechanism via lead binding to zinc fingers. The complete loss of DNase I protection on the 5S gene indicates the mechanism of inhibition minimally involves the N-terminal fingers of TFIIIA. Inhibition was not readily reversible and occurred in the presence of an excess of beta-mercaptoethanol. Inhibition kinetics were fast, progressing to completion in approximately 5 min. Millimolar concentrations of sulfhydryl-specific arsenic ions were not inhibitory for TFIIIA binding. Micromolar concentrations of lead inhibited DNA binding by Sp1, another Cys(2)His(2) finger protein, but not by the nonfinger protein AP2. Inhibition of Cys(2)His(2) zinc finger transcription factors by lead ions at concentrations near those known to have deleterious physiological effects points to new molecular mechanisms for lead toxicity in promoting disease.

Evidence for a G protein-coupled diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) receptor binding site in lung membranes from rat.

PubMed

Laubinger, W; Reiser, G

1999-01-29

Nucleotide receptors are of considerable importance in the treatment of lung diseases, such as cystic fibrosis. Because diadenosine polyphosphates may also be of significance as signalling molecules in lung, as they are in a variety of tissues, in the present work we investigated the binding sites for [3H]diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) in plasma membranes from rat lung and studied their possible coupling to G proteins. We present evidence for a single high-affinity binding site for [3H]Ap4A with similar affinity for other diadenosine polyphosphates ApnA (n = 2 to 6). Displacement studies with different nucleotides revealed that the [3H]Ap4A binding site was different from P2X and P2Y2 receptor binding sites. Pretreatment of lung membranes with GTPgammaS or GTP in the presence of Mg2+ increased the Ki for Ap4A from 91 nM to 5.1 microM, which is indicative of G protein coupling. The putative coupling to G proteins was further confirmed by the enhancement of [35S]GTPgammaS binding (to Galpha proteins) to lung membranes by Ap4A (63% increase over basal) in a concentration-dependent manner. Therefore, our data for the first time provide evidence of a G protein-coupled Ap4A binding site in lung membranes.

Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding.

PubMed

Nakashima, T; Yao, M; Kawamura, S; Iwasaki, K; Kimura, M; Tanaka, I

2001-05-01

Ribosomal protein L5 is a 5S rRNA binding protein in the large subunit and plays an essential role in the promotion of a particular conformation of 5S rRNA. The crystal structure of the ribosomal protein L5 from Bacillus stearothermophilus has been determined at 1.8 A resolution. The molecule consists of a five-stranded antiparallel beta-sheet and four alpha-helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. The molecular shape and electrostatic representation suggest that the concave surface and loop regions are involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurring in the beta-strands and loop regions. The mutations of Asn37 at the beta1-strand and Gln63 at the loop between helix 2 and beta3-strand as well as that of Phe77 at the tip of the loop structure between the beta2- and beta3-strands caused a significant reduction in 5S rRNA binding. In addition, the mutations of Thr90 on the beta3-strand and Ile141 and Asp144 at the loop between beta4- and beta5-strands moderately reduced the 5S rRNA-binding affinity. Comparison of these results with the more recently analyzed structure of the 50S subunit from Haloarcula marismortui suggests that there are significant differences in the structure at N- and C-terminal regions and probably in the 5S rRNA binding.

Ribosomal protein L5 has a highly twisted concave surface and flexible arms responsible for rRNA binding.

PubMed Central

Nakashima, T; Yao, M; Kawamura, S; Iwasaki, K; Kimura, M; Tanaka, I

2001-01-01

Ribosomal protein L5 is a 5S rRNA binding protein in the large subunit and plays an essential role in the promotion of a particular conformation of 5S rRNA. The crystal structure of the ribosomal protein L5 from Bacillus stearothermophilus has been determined at 1.8 A resolution. The molecule consists of a five-stranded antiparallel beta-sheet and four alpha-helices, which fold in a way that is topologically similar to the ribonucleoprotein (RNP) domain. The molecular shape and electrostatic representation suggest that the concave surface and loop regions are involved in 5S rRNA binding. To identify amino acid residues responsible for 5S rRNA binding, we made use of Ala-scanning mutagenesis of evolutionarily conserved amino acids occurring in the beta-strands and loop regions. The mutations of Asn37 at the beta1-strand and Gln63 at the loop between helix 2 and beta3-strand as well as that of Phe77 at the tip of the loop structure between the beta2- and beta3-strands caused a significant reduction in 5S rRNA binding. In addition, the mutations of Thr90 on the beta3-strand and Ile141 and Asp144 at the loop between beta4- and beta5-strands moderately reduced the 5S rRNA-binding affinity. Comparison of these results with the more recently analyzed structure of the 50S subunit from Haloarcula marismortui suggests that there are significant differences in the structure at N- and C-terminal regions and probably in the 5S rRNA binding. PMID:11350033

The peritumoural adipose tissue microenvironment and cancer. The roles of fatty acid binding protein 4 and fatty acid binding protein 5.

PubMed

Guaita-Esteruelas, S; Gumà, J; Masana, L; Borràs, J

2018-02-15

The adipose tissue microenvironment plays a key role in tumour initiation and progression because it provides fatty acids and adipokines to tumour cells. The fatty acid-binding protein (FABP) family is a group of small proteins that act as intracellular fatty acid transporters. Adipose-derived FABPs include FABP4 and FABP5. Both have an important role in lipid-related metabolic processes and overexpressed in many cancers, such as breast, prostate, colorectal and ovarian. Moreover, their expression in peritumoural adipose tissue is deregulated, and their circulating levels are upregulated in some tumours. In this review, we discuss the role of the peritumoural adipose tissue and the related adipokines FABP4 and FABP5 in cancer initiation and progression and the possible pathways implicated in these processes. Copyright © 2017 Elsevier B.V. All rights reserved.

Phosphatidylinositol (3,4,5)-Trisphosphate Activity Probes for the Labeling and Proteomic Characterization of Protein Binding Partners

PubMed Central

Rowland, Meng M.; Bostic, Heidi E.; Gong, Denghuang; Speers, Anna E.; Lucas, Nathan; Cho, Wonhwa; Cravatt, Benjamin F.; Best, Michael D.

2013-01-01

Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3), regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane-association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P3 that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins as well as a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by on-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P3 headgroup analog as well as through protein denaturation, indicating specific labeling. In addition, probes featuring different linker lengths between the PI(3,4,5)P3 headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts, labeled proteins were observed by in-gel detection and characterized using post-labeling with biotin, affinity chromatography and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins

A Multiprotein Binding Interface in an Intrinsically Disordered Region of the Tumor Suppressor Protein Interferon Regulatory Factor-1*

PubMed Central

Narayan, Vikram; Halada, Petr; Hernychová, Lenka; Chong, Yuh Ping; Žáková, Jitka; Hupp, Ted R.; Vojtesek, Borivoj; Ball, Kathryn L.

2011-01-01

The interferon-regulated transcription factor and tumor suppressor protein IRF-1 is predicted to be largely disordered outside of the DNA-binding domain. One of the advantages of intrinsically disordered protein domains is thought to be their ability to take part in multiple, specific but low affinity protein interactions; however, relatively few IRF-1-interacting proteins have been described. The recent identification of a functional binding interface for the E3-ubiquitin ligase CHIP within the major disordered domain of IRF-1 led us to ask whether this region might be employed more widely by regulators of IRF-1 function. Here we describe the use of peptide aptamer-based affinity chromatography coupled with mass spectrometry to define a multiprotein binding interface on IRF-1 (Mf2 domain; amino acids 106–140) and to identify Mf2-binding proteins from A375 cells. Based on their function as known transcriptional regulators, a selection of the Mf2 domain-binding proteins (NPM1, TRIM28, and YB-1) have been validated using in vitro and cell-based assays. Interestingly, although NPM1, TRIM28, and YB-1 all bind to the Mf2 domain, they have differing amino acid specificities, demonstrating the degree of combinatorial diversity and specificity available through linear interaction motifs. PMID:21245151

A multiprotein binding interface in an intrinsically disordered region of the tumor suppressor protein interferon regulatory factor-1.

PubMed

Narayan, Vikram; Halada, Petr; Hernychová, Lenka; Chong, Yuh Ping; Žáková, Jitka; Hupp, Ted R; Vojtesek, Borivoj; Ball, Kathryn L

2011-04-22

The interferon-regulated transcription factor and tumor suppressor protein IRF-1 is predicted to be largely disordered outside of the DNA-binding domain. One of the advantages of intrinsically disordered protein domains is thought to be their ability to take part in multiple, specific but low affinity protein interactions; however, relatively few IRF-1-interacting proteins have been described. The recent identification of a functional binding interface for the E3-ubiquitin ligase CHIP within the major disordered domain of IRF-1 led us to ask whether this region might be employed more widely by regulators of IRF-1 function. Here we describe the use of peptide aptamer-based affinity chromatography coupled with mass spectrometry to define a multiprotein binding interface on IRF-1 (Mf2 domain; amino acids 106-140) and to identify Mf2-binding proteins from A375 cells. Based on their function as known transcriptional regulators, a selection of the Mf2 domain-binding proteins (NPM1, TRIM28, and YB-1) have been validated using in vitro and cell-based assays. Interestingly, although NPM1, TRIM28, and YB-1 all bind to the Mf2 domain, they have differing amino acid specificities, demonstrating the degree of combinatorial diversity and specificity available through linear interaction motifs.

Disulfide bridge regulates ligand-binding site selectivity in liver bile acid-binding proteins.

PubMed

Cogliati, Clelia; Tomaselli, Simona; Assfalg, Michael; Pedò, Massimo; Ferranti, Pasquale; Zetta, Lucia; Molinari, Henriette; Ragona, Laura

2009-10-01

Bile acid-binding proteins (BABPs) are cytosolic lipid chaperones that play central roles in driving bile flow, as well as in the adaptation to various pathological conditions, contributing to the maintenance of bile acid homeostasis and functional distribution within the cell. Understanding the mode of binding of bile acids with their cytoplasmic transporters is a key issue in providing a model for the mechanism of their transfer from the cytoplasm to the nucleus, for delivery to nuclear receptors. A number of factors have been shown to modulate bile salt selectivity, stoichiometry, and affinity of binding to BABPs, e.g. chemistry of the ligand, protein plasticity and, possibly, the formation of disulfide bridges. Here, the effects of the presence of a naturally occurring disulfide bridge on liver BABP ligand-binding properties and backbone dynamics have been investigated by NMR. Interestingly, the disulfide bridge does not modify the protein-binding stoichiometry, but has a key role in modulating recognition at both sites, inducing site selectivity for glycocholic and glycochenodeoxycholic acid. Protein conformational changes following the introduction of a disulfide bridge are small and located around the inner binding site, whereas significant changes in backbone motions are observed for several residues distributed over the entire protein, both in the apo form and in the holo form. Site selectivity appears, therefore, to be dependent on protein mobility rather than being governed by steric factors. The detected properties further establish a parallelism with the behaviour of human ileal BABP, substantiating the proposal that BABPs have parallel functions in hepatocytes and enterocytes.

Childhood maltreatment and methylation of FK506 binding protein 5 gene (FKBP5).

PubMed

Tyrka, Audrey R; Ridout, Kathryn K; Parade, Stephanie H; Paquette, Alison; Marsit, Carmen J; Seifer, Ronald

2015-11-01

A growing body of evidence suggests that alterations of the stress response system may be a mechanism by which childhood maltreatment alters risk for psychopathology. FK506 binding protein 51 (FKBP5) binds to the glucocorticoid receptor and alters its ability to respond to stress signaling. The aim of the present study was to examine methylation of the FKBP5 gene (FKBP5), and the role of an FKBP5 genetic variant, in relation to childhood maltreatment in a sample of impoverished preschool-aged children. One hundred seventy-four families participated in this study, including 69 with child welfare documentation of moderate to severe maltreatment in the past 6 months. The children, who ranged in age from 3 to 5 years, were racially and ethnically diverse. Structured record review and interviews in the home were used to assess a history of maltreatment, other traumas, and contextual life stressors; and a composite variable assessed the number exposures to these adversities. Methylation of two sites in intron 7 of FKBP5 was measured via sodium bisulfite pyrosequencing. Maltreated children had significantly lower levels of methylation at both CpG sites (p < .05). Lifetime contextual stress exposure showed a trend for lower levels of methylation at one of the sites, and a trend for an interaction with the FKBP5 polymorphism. A composite adversity variable was associated with lower levels of methylation at one of the sites as well (p < .05). FKBP5 alters glucocorticoid receptor responsiveness, and FKBP5 gene methylation may be a mechanism of the biobehavioral effects of adverse exposures in young children.

Double-Stranded RNA-Binding Protein Regulates Vascular Endothelial Growth Factor mRNA Stability, Translation, and Breast Cancer Angiogenesis▿

PubMed Central

Vumbaca, Frank; Phoenix, Kathryn N.; Rodriguez-Pinto, Daniel; Han, David K.; Claffey, Kevin P.

2008-01-01

Vascular endothelial growth factor (VEGF) is a key angiogenic factor expressed under restricted nutrient and oxygen conditions in most solid tumors. The expression of VEGF under hypoxic conditions requires transcription through activated hypoxia-inducible factor 1 (HIF-1), increased mRNA stability, and facilitated translation. This study identified double-stranded RNA-binding protein 76/NF90 (DRBP76/NF90), a specific isoform of the DRBP family, as a VEGF mRNA-binding protein which plays a key role in VEGF mRNA stability and protein synthesis under hypoxia. The DRBP76/NF90 protein binds to a human VEGF 3′ untranslated mRNA stability element. RNA interference targeting the DRBP76/NF90 isoform limited hypoxia-inducible VEGF mRNA and protein expression with no change in HIF-1-dependent transcriptional activity. Stable repression of DRBP76/NF90 in MDA-MB-435 breast cancer cells demonstrated reduced polysome-associated VEGF mRNA levels under hypoxic conditions and reduced mRNA stability. Transient overexpression of the DRBP76/NF90 protein increased both VEGF mRNA and protein levels synthesized under normoxic and hypoxic conditions. Cells with stable repression of the DRBP76/NF90 isoform showed reduced tumorigenic and angiogenic potential in an orthotopic breast tumor model. These data demonstrate that the DRBP76/NF90 isoform facilitates VEGF expression by promoting VEGF mRNA loading onto polysomes and translation under hypoxic conditions, thus promoting breast cancer growth and angiogenesis in vivo. PMID:18039850

Search for Limiting Factors in the RNAi Pathway in Silkmoth Tissues and the Bm5 Cell Line: The RNA-Binding Proteins R2D2 and Translin

PubMed Central

Swevers, Luc; Liu, Jisheng; Huvenne, Hanneke; Smagghe, Guy

2011-01-01

RNA interference (RNAi), an RNA-dependent gene silencing process that is initiated by double-stranded RNA (dsRNA) molecules, has been applied with variable success in lepidopteran insects, in contrast to the high efficiency achieved in the coleopteran Tribolium castaneum. To gain insight into the factors that determine the efficiency of RNAi, a survey was carried out to check the expression of factors that constitute the machinery of the small interfering RNA (siRNA) and microRNA (miRNA) pathways in different tissues and stages of the silkmoth, Bombyx mori. It was found that the dsRNA-binding protein R2D2, an essential component in the siRNA pathway in Drosophila, was expressed at minimal levels in silkmoth tissues. The silkmoth-derived Bm5 cell line was also deficient in expression of mRNA encoding full-length BmTranslin, an RNA-binding factor that has been shown to stimulate the efficiency of RNAi. However, despite the lack of expression of the RNA-binding proteins, silencing of a luciferase reporter gene was observed by co-transfection of luc dsRNA using a lipophilic reagent. In contrast, gene silencing was not detected when the cells were soaked in culture medium supplemented with dsRNA. The introduction of an expression construct for Tribolium R2D2 (TcR2D2) did not influence the potency of luc dsRNA to silence the luciferase reporter. Immunostaining experiments further showed that both TcR2D2 and BmTranslin accumulated at defined locations within the cytoplasm of transfected cells. Our results offer a first evaluation of the expression of the RNAi machinery in silkmoth tissues and Bm5 cells and provide evidence for a functional RNAi response to intracellular dsRNA in the absence of R2D2 and Translin. The failure of TcR2D2 to stimulate the intracellular RNAi pathway in Bombyx cells is discussed. PMID:21637842

Selective enrichment of metal-binding proteins based on magnetic core/shell microspheres functionalized with metal cations.

PubMed

Fang, Caiyun; Zhang, Lei; Zhang, Xiaoqin; Lu, Haojie

2015-06-21

Metal binding proteins play many important roles in a broad range of biological processes. Characterization of metal binding proteins is important for understanding their structure and biological functions, thus leading to a clear understanding of metal associated diseases. The present study is the first to investigate the effectiveness of magnetic microspheres functionalized with metal cations (Ca(2+), Cu(2+), Zn(2+) and Fe(3+)) as the absorbent matrix in IMAC technology to enrich metal containing/binding proteins. The putative metal binding proteins in rat liver were then globally characterized by using this strategy which is very easy to handle and can capture a number of metal binding proteins effectively. In total, 185 putative metal binding proteins were identified from rat liver including some known less abundant and membrane-bound metal binding proteins such as Plcg1, Acsl5, etc. The identified proteins are involved in many important processes including binding, catalytic activity, translation elongation factor activity, electron carrier activity, and so on.

BindML/BindML+: Detecting Protein-Protein Interaction Interface Propensity from Amino Acid Substitution Patterns.

PubMed

Wei, Qing; La, David; Kihara, Daisuke

2017-01-01

Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .

How proteins bind to DNA: target discrimination and dynamic sequence search by the telomeric protein TRF1

PubMed Central

2017-01-01

Abstract Target search as performed by DNA-binding proteins is a complex process, in which multiple factors contribute to both thermodynamic discrimination of the target sequence from overwhelmingly abundant off-target sites and kinetic acceleration of dynamic sequence interrogation. TRF1, the protein that binds to telomeric tandem repeats, faces an intriguing variant of the search problem where target sites are clustered within short fragments of chromosomal DNA. In this study, we use extensive (>0.5 ms in total) MD simulations to study the dynamical aspects of sequence-specific binding of TRF1 at both telomeric and non-cognate DNA. For the first time, we describe the spontaneous formation of a sequence-specific native protein–DNA complex in atomistic detail, and study the mechanism by which proteins avoid off-target binding while retaining high affinity for target sites. Our calculated free energy landscapes reproduce the thermodynamics of sequence-specific binding, while statistical approaches allow for a comprehensive description of intermediate stages of complex formation. PMID:28633355

New Binding Mode to TNF-Alpha Revealed by Ubiquitin-Based Artificial Binding Protein

PubMed Central

Hoffmann, Andreas; Kovermann, Michael; Lilie, Hauke; Fiedler, Markus; Balbach, Jochen; Rudolph, Rainer; Pfeifer, Sven

2012-01-01

A variety of approaches have been employed to generate binding proteins from non-antibody scaffolds. Utilizing a beta-sheet of the human ubiquitin for paratope creation we obtained binding proteins against tumor necrosis factor (TNF)-alpha. The bioactive form of this validated pharmacological target protein is a non-covalently linked homo-trimer. This structural feature leads to the observation of a certain heterogeneity concerning the binding mode of TNF-alpha binding molecules, for instance in terms of monomer/trimer specificity. We analyzed a ubiquitin-based TNF-alpha binder, selected by ribosome display, with a particular focus on its mode of interaction. Using enzyme-linked immunosorbent assays, specific binding to TNF-alpha with nanomolar affinity was observed. In isothermal titration calorimetry we obtained comparable results regarding the affinity and detected an exothermic reaction with one ubiquitin-derived binding molecule binding one TNF-alpha trimer. Using NMR spectroscopy and other analytical methods the 1∶3 stoichiometry could be confirmed. Detailed binding analysis showed that the interaction is affected by the detergent Tween-20. Previously, this phenomenon was reported only for one other type of alternative scaffold-derived binding proteins – designed ankyrin repeat proteins – without further investigation. As demonstrated by size exclusion chromatography and NMR spectroscopy, the presence of the detergent increases the association rate significantly. Since the special architecture of TNF-alpha is known to be modulated by detergents, the access to the recognized epitope is indicated to be restricted by conformational transitions within the target protein. Our results suggest that the ubiquitin-derived binding protein targets a new epitope on TNF-alpha, which differs from the epitopes recognized by TNF-alpha neutralizing antibodies. PMID:22363609

Organization, chromosomal localization and promoter analysis of the gene encoding human acidic fibroblast growth factor intracellular binding protein.

PubMed Central

Kolpakova, E; Frengen, E; Stokke, T; Olsnes, S

2000-01-01

Acidic fibroblast growth factor (aFGF) intracellular binding protein (FIBP) is a protein found mainly in the nucleus that might be involved in the intracellular function of aFGF. Here we present a comparative analysis of the deduced amino acid sequences of human, murine and Drosophila FIBP analogues and demonstrate that FIBP is an evolutionarily conserved protein. The human gene spans more than 5 kb, comprising ten exons and nine introns, and maps to chromosome 11q13.1. Two slightly different splice variants found in different tissues were isolated and characterized. Sequence analysis of the region surrounding the translation start revealed a CpG island, a classical feature of widely expressed genes. Functional studies of the promoter region with a luciferase reporter system suggested a strong transcriptional activity residing within 600 bp of the 5' flanking region. PMID:11104667

Annexin A5 binds to lipopolysaccharide and reduces its endotoxin activity.

PubMed

Rand, Jacob H; Wu, Xiao-Xuan; Lin, Elaine Y; Griffel, Alexander; Gialanella, Philip; McKitrick, John C

2012-01-01

Annexin A5 (AnxA5) has a high affinity for phosphatidylserine. The protein is widely used to detect apoptotic cells because phosphatidylserine, a phospholipid that is normally present in the inner leaflets of cytoplasmic membranes, becomes translocated to the outer leaflets during programmed cell death. Here we report the novel observation that AnxA5 binds to Gram-negative bacteria via the lipid A domain of lipopolysaccharide (LPS). Binding of AnxA5 to bacteria was measured quantitatively, confirmed by fluorescence microscopy, and found to be inhibited by antibodies against lipid A. AnxA5 also bound to purified dot-blotted LPS and lipid A. Through ellipsometry, we found that the binding of AnxA5 to purified LPS was calcium dependent and rapid and showed a high affinity-characteristics similar to those of AnxA5 binding to phosphatidylserine. Initial functional studies indicated that AnxA5 can affect LPS activities. AnxA5 inhibited LPS-mediated gelation in the Limulus amebocyte lysate assay. Incubation of LPS with the protein reduced the quantity of tumor necrosis factor alpha (TNF-α) released by cultured monocytes compared to that released upon incubation with LPS alone. Initial in vivo experiments indicated that injection of mice with LPS preincubated with AnxA5 produced serum TNF-α levels lower than those seen after injection of LPS alone. These data demonstrate that AnxA5 binds to LPS and open paths to investigation of the potential biological and therapeutic implications of this interaction. AnxA5 is highly expressed in cells that have a barrier function-including, among others, vascular endothelium, placental trophoblasts, and epithelial cells lining bile ducts, renal tubules, mammary ducts, and nasal epithelium. The protein has been well characterized for its binding to phospholipid bilayers that contain phosphatidylserine. This report of a previously unrecognized activity of AnxA5 opens the door to investigation of the possibility that this binding may have

Proteomic analysis identifies insulin-like growth factor-binding protein-related protein-1 as a podocyte product.

PubMed

Matsumoto, Takayuki; Hess, Sonja; Kajiyama, Hiroshi; Sakairi, Toru; Saleem, Moin A; Mathieson, Peter W; Nojima, Yoshihisa; Kopp, Jeffrey B

2010-10-01

The podocyte secretory proteome may influence the phenotype of adjacent podocytes, endothelial cells, parietal epithelial cells, and tubular epithelial cells but has not been systematically characterized. We have initiated studies to characterize this proteome, with the goal of further understanding the podocyte cell biology. We cultured differentiated conditionally immortalized human podocytes and subjected the proteins in conditioned medium to mass spectrometry. At a false discovery rate of <3%, we identified 111 candidates from conditioned medium, including 44 proteins that have signal peptides or are described as secreted proteins in the UniProt database. As validation, we confirmed that one of these proteins, insulin-like growth factor-binding protein-related protein-1 (IGFBP-rP1), was expressed in mRNA and protein of cultured podocytes. In addition, transforming growth factor-β1 stimulation increased IGFBP-rP1 in conditioned medium. We analyzed IGFBP-rP1 glomerular expression in a mouse model of human immunodeficiency virus-associated nephropathy. IGFBP-rP1 was absent from podocytes of normal mice and was expressed in podocytes and pseudocrescents of transgenic mice, where it was coexpressed with desmin, a podocyte injury marker. We conclude that IGFBP-rP1 may be a product of injured podocytes. Further analysis of the podocyte secretory proteome may identify biomarkers of podocyte injury.

Protein Cofactors Are Essential for High-Affinity DNA Binding by the Nuclear Factor κB RelA Subunit.

PubMed

Mulero, Maria Carmen; Shahabi, Shandy; Ko, Myung Soo; Schiffer, Jamie M; Huang, De-Bin; Wang, Vivien Ya-Fan; Amaro, Rommie E; Huxford, Tom; Ghosh, Gourisankar

2018-05-22

Transcription activator proteins typically contain two functional domains: a DNA binding domain (DBD) that binds to DNA with sequence specificity and an activation domain (AD) whose established function is to recruit RNA polymerase. In this report, we show that purified recombinant nuclear factor κB (NF-κB) RelA dimers bind specific κB DNA sites with an affinity significantly lower than that of the same dimers from nuclear extracts of activated cells, suggesting that additional nuclear cofactors might facilitate DNA binding by the RelA dimers. Additionally, recombinant RelA binds DNA with relatively low affinity at a physiological salt concentration in vitro. The addition of p53 or RPS3 (ribosomal protein S3) increases RelA:DNA binding affinity 2- to >50-fold depending on the protein and ionic conditions. These cofactor proteins do not form stable ternary complexes, suggesting that they stabilize the RelA:DNA complex through dynamic interactions. Surprisingly, the RelA-DBD alone fails to bind DNA under the same solution conditions even in the presence of cofactors, suggesting an important role of the RelA-AD in DNA binding. Reduced RelA:DNA binding at a physiological ionic strength suggests that multiple cofactors might be acting simultaneously to mitigate the electrolyte effect and stabilize the RelA:DNA complex in vivo. Overall, our observations suggest that the RelA-AD and multiple cofactor proteins function cooperatively to prime the RelA-DBD and stabilize the RelA:DNA complex in cells. Our study provides a mechanism for nuclear cofactor proteins in NF-κB-dependent gene regulation.

The protein network surrounding the human telomere repeat binding factors TRF1, TRF2, and POT1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Giannone, Richard J; McDonald, W Hayes; Hurst, Gregory

Telomere integrity (including telomere length and capping) is critical in overall genomic stability. Telomere repeat binding factors and their associated proteins play vital roles in telomere length regulation and end protection. In this study, we explore the protein network surrounding telomere repeat binding factors, TRF1, TRF2, and POT1 using dual-tag affinity purification in combination with multidimensional protein identification technology liquid chromatography - tandem mass spectrometry (MudPIT LC-MS/MS). After control subtraction and data filtering, we found that TRF2 and POT1 co-purified all six members of the telomere protein complex, while TRF1 identified five of six components at frequencies that lend evidencemore » towards the currently accepted telomere architecture. Many of the known TRF1 or TRF2 interacting proteins were also identified. Moreover, putative associating partners identified for each of the three core components fell into functional categories such as DNA damage repair, ubiquitination, chromosome cohesion, chromatin modification/remodeling, DNA replication, cell cycle and transcription regulation, nucleotide metabolism, RNA processing, and nuclear transport. These putative protein-protein associations may participate in different biological processes at telomeres or, intriguingly, outside telomeres.« less

POZ domain transcription factor, FBI-1, represses transcription of ADH5/FDH by interacting with the zinc finger and interfering with DNA binding activity of Sp1.

PubMed

Lee, Dong-Kee; Suh, Dongchul; Edenberg, Howard J; Hur, Man-Wook

2002-07-26

The POZ domain is a protein-protein interaction motif that is found in many transcription factors, which are important for development, oncogenesis, apoptosis, and transcription repression. We cloned the POZ domain transcription factor, FBI-1, that recognizes the cis-element (bp -38 to -22) located just upstream of the core Sp1 binding sites (bp -22 to +22) of the ADH5/FDH minimal promoter (bp -38 to +61) in vitro and in vivo, as revealed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. The ADH5/FDH minimal promoter is potently repressed by the FBI-1. Glutathione S-transferase fusion protein pull-down showed that the POZ domains of FBI-1, Plzf, and Bcl-6 directly interact with the zinc finger DNA binding domain of Sp1. DNase I footprinting assays showed that the interaction prevents binding of Sp1 to the GC boxes of the ADH5/FDH promoter. Gal4-POZ domain fusions targeted proximal to the GC boxes repress transcription of the Gal4 upstream activator sequence-Sp1-adenovirus major late promoter. Our data suggest that POZ domain represses transcription by interacting with Sp1 zinc fingers and by interfering with the DNA binding activity of Sp1.

A dye-binding assay for measurement of the binding of Cu(II) to proteins.

PubMed

Wilkinson-White, Lorna E; Easterbrook-Smith, Simon B

2008-10-01

We analysed the theory of the coupled equilibria between a metal ion, a metal ion-binding dye and a metal ion-binding protein in order to develop a procedure for estimating the apparent affinity constant of a metal ion:protein complex. This can be done by analysing from measurements of the change in the concentration of the metal ion:dye complex with variation in the concentration of either the metal ion or the protein. Using experimentally determined values for the affinity constant of Cu(II) for the dye, 2-(5-bromo-2-pyridylaxo)-5-(N-propyl-N-sulfopropylamino) aniline (5-Br-PSAA), this procedure was used to estimate the apparent affinity constants for formation of Cu(II):transthyretin, yielding values which were in agreement with literature values. An apparent affinity constant for Cu(II) binding to alpha-synuclein of approximately 1 x 10(9)M(-1) was obtained from measurements of tyrosine fluorescence quenching by Cu(II). This value was in good agreement with that obtained using 5-Br-PSAA. Our analysis and data therefore show that measurement of changes in the equilibria between Cu(II) and 5-Br-PSAA by Cu(II)-binding proteins provides a general procedure for estimating the affinities of proteins for Cu(II).

Insulin-like growth factor-binding protein 7 alters the sensitivity to interferon-based anticancer therapy in hepatocellular carcinoma cells.

PubMed

Tomimaru, Y; Eguchi, H; Wada, H; Noda, T; Murakami, M; Kobayashi, S; Marubashi, S; Takeda, Y; Tanemura, M; Umeshita, K; Doki, Y; Mori, M; Nagano, H

2010-05-11

A striking efficiency of interferon (IFN)-based anticancer therapy for advanced hepatocellular carcinoma (HCC) has been reported. Because its clinical efficiency greatly depends on each patient's local response, prediction of local response is crucial. Continuous exposure of IFN-alpha to parental PLC/PRF/5 cells (PLC-P) and a limiting dilution method resulted in the establishment of IFN-resistant cell clones (PLC-Rs). Microarray analyses of PLC-P and PLC-Rs identified insulin-like growth factor-binding protein 7 (IGFBP7) as one of the most significantly downregulated genes in PLC-Rs. Changes in anticancer effects of IFN-alpha were examined in HCC cells after genetic manipulation of IGFBP7 expression. The correlation between immunohistochemically determined IGFBP7 expression and the response to IFN-alpha/5-fluorouracil (5-FU) therapy was investigated in surgically resected HCC specimens. PLC-R cells showed a remarkable downregulation of IGFBP7 and resistance to IFN-alpha, compared with PLC-P. Parental PLC/PRF/5 cells transfected with short hairpin RNA against IGFBP7 showed a significant resistance to IFN-alpha relative to control cells (IC(50) fold increase=14.38 times). Insulin-like growth factor-binding protein 7 transfection into PLC-R restored sensitivity to IFN-alpha. In resected specimens, IGFBP7 expression significantly correlated with the response to IFN-alpha/5-FU therapy. IGFBP7 could be a useful predictor of the response to IFN-based therapy in advanced HCC.

Microtubule plus-end tracking of end-binding protein 1 (EB1) is regulated by CDK5 regulatory subunit-associated protein 2

PubMed Central

Fong, Ka-Wing; Au, Franco K. C.; Jia, Yue; Yang, Shaozhong; Zhou, Liying; Qi, Robert Z.

2017-01-01

Microtubules are polar cytoskeleton filaments that extend via growth at their plus ends. Microtubule plus-end-tracking proteins (+TIPs) accumulate at these growing plus ends to control microtubule dynamics and attachment. The +TIP end-binding protein 1 (EB1) and its homologs possess an autonomous plus-end-tracking mechanism and interact with other known +TIPs, which then recruit those +TIPs to the growing plus ends. A major +TIP class contains the SXIP (Ser-X-Ile-Pro, with X denoting any amino acid residue) motif, known to interact with EB1 and its homologs for plus-end tracking, but the role of SXIP in regulating EB1 activities is unclear. We show here that an interaction of EB1 with the SXIP-containing +TIP CDK5 regulatory subunit-associated protein 2 (CDK5RAP2) regulates several EB1 activities, including microtubule plus-end tracking, dynamics at microtubule plus ends, microtubule and α/β-tubulin binding, and microtubule polymerization. The SXIP motif fused with a dimerization domain from CDK5RAP2 significantly enhanced EB1 plus-end-tracking and microtubule-polymerizing and bundling activities, but the SXIP motif alone failed to do so. An SXIP-binding-deficient EB1 mutant displayed significantly lower microtubule plus-end tracking than the wild-type protein in transfected cells. These results suggest that EB1 cooperates with CDK5RAP2 and perhaps other SXIP-containing +TIPs in tracking growing microtubule tips. We also generated plus-end-tracking chimeras of CDK5RAP2 and the adenomatous polyposis coli protein (APC) and found that overexpression of the dimerization domains interfered with microtubule plus-end tracking of their respective SXIP-containing chimeras. Our results suggest that disruption of SXIP dimerization enables detailed investigations of microtubule plus-end-associated functions of individual SXIP-containing +TIPs. PMID:28320860

Characterization of a Novel Association between Two Trypanosome-Specific Proteins and 5S rRNA

PubMed Central

Ciganda, Martin; Williams, Noreen

2012-01-01

P34 and P37 are two previously identified RNA binding proteins in the flagellate protozoan Trypanosoma brucei. RNA interference studies have determined that the proteins are essential and are involved in ribosome biogenesis. Here, we show that these proteins interact in vitro with the 5S rRNA with nearly identical binding characteristics in the absence of other cellular factors. The T. brucei 5S rRNA has a complex secondary structure and presents four accessible loops (A to D) for interactions with RNA-binding proteins. In other eukaryotes, loop C is bound by the L5 ribosomal protein and loop A mainly by TFIIIA. The binding of P34 and P37 to T. brucei 5S rRNA involves the LoopA region of the RNA, but these proteins also protect the L5 binding site located on LoopC. PMID:22253864

A Comparison Study for DNA Motif Modeling on Protein Binding Microarray.

PubMed

Wong, Ka-Chun; Li, Yue; Peng, Chengbin; Wong, Hau-San

2016-01-01

Transcription factor binding sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k = 8∼10). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build TFBS (also known as DNA motif) models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement if choosing di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

Mechanisms of extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway in depressive disorder☆

PubMed Central

Wang, Hongyan; Zhang, Yingquan; Qiao, Mingqi

2013-01-01

The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor signal transduction pathway plays an important role in the mechanism of action of antidepressant drugs and has dominated recent studies on the pathogenesis of depression. In the present review we summarize the known roles of extracellular signal-regulated kinase, cAMP response element-binding protein and brain-derived neurotrophic factor in the pathogenesis of depression and in the mechanism of action of antidepressant medicines. The extracellular signal-regulated kinase/cAMP response element-binding protein/brain-derived neurotrophic factor pathway has potential to be used as a biological index to help diagnose depression, and as such it is considered as an important new target in the treatment of depression. PMID:25206732

Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

PubMed

Gaviglio, Angela L; Knelson, Erik H; Blobe, Gerard C

2017-05-01

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor-like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.-Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation. © FASEB.

Altered binding of /sup 125/I-labeled calmodulin to a 46. 5-kilodalton protein in skin fibroblasts cultured from patients with cystic fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tallant, E.A.; Wallace, R.W.

1987-02-01

The levels of calmodulin and calmodulin-binding proteins have been determined in cultured skin fibroblasts from patients with cystic fibrosis (CF) and age- and sex-matched controls. Calmodulin ranged from 0.20 to 0.76 microgram/mg protein; there was no difference between calmodulin concentration in fibroblasts from CF patients and controls. Calmodulin-binding proteins of 230, 212, 204, 164, 139, 70, 59, 46.5, and 41 kD were identified. A protein with a mobility identical to the 59-kD calmodulin-binding protein was labeled by antiserum against calmodulin-dependent phosphatase. Although Ca/sup 2 +//calmodulin-dependent phosphatase activity was detected, there was no different in activity between control and CF fibroblastsmore » or in the level of phosphatase protein as determined by radioimmunoassay. Lower amounts of /sup 125/I-calmodulin were bound to the 46.5-kD calmodulin-binding protein in CF fibroblasts as compared with controls. The 46.5-kD calmodulin-binding protein may be reduced in CF fibroblasts or its structure may be altered resulting in a reduced binding capacity and/or affinity for calmodulin and perhaps reflecting, either directly or indirectly, the genetic defect responsible for cystic fibrosis.« less

Isolation and characterizations of oxalate-binding proteins in the kidney

DOE Office of Scientific and Technical Information (OSTI.GOV)

Roop-ngam, Piyachat; Chaiyarit, Sakdithep; Pongsakul, Nutkridta

Highlights: Black-Right-Pointing-Pointer The first large-scale characterizations of oxalate-binding kidney proteins. Black-Right-Pointing-Pointer The recently developed oxalate-conjugated EAH Sepharose 4B beads were applied. Black-Right-Pointing-Pointer 38 forms of 26 unique oxalate-binding kidney proteins were identified. Black-Right-Pointing-Pointer 25/26 (96%) of identified proteins had 'L-x(3,5)-R-x(2)-[AGILPV]' domain. -- Abstract: Oxalate-binding proteins are thought to serve as potential modulators of kidney stone formation. However, only few oxalate-binding proteins have been identified from previous studies. Our present study, therefore, aimed for large-scale identification of oxalate-binding proteins in porcine kidney using an oxalate-affinity column containing oxalate-conjugated EAH Sepharose 4B beads for purification followed by two-dimensional gel electrophoresis (2-DE) tomore » resolve the recovered proteins. Comparing with those obtained from the controlled column containing uncoupled EAH-Sepharose 4B (to subtract the background of non-specific bindings), a total of 38 protein spots were defined as oxalate-binding proteins. These protein spots were successfully identified by quadrupole time-of-flight mass spectrometry (MS) and/or tandem MS (MS/MS) as 26 unique proteins, including several nuclear proteins, mitochondrial proteins, oxidative stress regulatory proteins, metabolic enzymes and others. Identification of oxalate-binding domain using the PRATT tool revealed 'L-x(3,5)-R-x(2)-[AGILPV]' as a functional domain responsible for oxalate-binding in 25 of 26 (96%) unique identified proteins. We report herein, for the first time, large-scale identification and characterizations of oxalate-binding proteins in the kidney. The presence of positively charged arginine residue in the middle of this functional domain suggested its significance for binding to the negatively charged oxalate. These data will enhance future stone research, particularly on stone

The Rice Resistance Protein Pair RGA4/RGA5 Recognizes the Magnaporthe oryzae Effectors AVR-Pia and AVR1-CO39 by Direct Binding[W][OA

PubMed Central

Cesari, Stella; Thilliez, Gaëtan; Ribot, Cécile; Chalvon, Véronique; Michel, Corinne; Jauneau, Alain; Rivas, Susana; Alaux, Ludovic; Kanzaki, Hiroyuki; Okuyama, Yudai; Morel, Jean-Benoit; Fournier, Elisabeth; Tharreau, Didier; Terauchi, Ryohei; Kroj, Thomas

2013-01-01

Resistance (R) proteins recognize pathogen avirulence (Avr) proteins by direct or indirect binding and are multidomain proteins generally carrying a nucleotide binding (NB) and a leucine-rich repeat (LRR) domain. Two NB-LRR protein-coding genes from rice (Oryza sativa), RGA4 and RGA5, were found to be required for the recognition of the Magnaporthe oryzae effector AVR1-CO39. RGA4 and RGA5 also mediate recognition of the unrelated M. oryzae effector AVR-Pia, indicating that the corresponding R proteins possess dual recognition specificity. For RGA5, two alternative transcripts, RGA5-A and RGA5-B, were identified. Genetic analysis showed that only RGA5-A confers resistance, while RGA5-B is inactive. Yeast two-hybrid, coimmunoprecipitation, and fluorescence resonance energy transfer–fluorescence lifetime imaging experiments revealed direct binding of AVR-Pia and AVR1-CO39 to RGA5-A, providing evidence for the recognition of multiple Avr proteins by direct binding to a single R protein. Direct binding seems to be required for resistance as an inactive AVR-Pia allele did not bind RGA5-A. A small Avr interaction domain with homology to the Avr recognition domain in the rice R protein Pik-1 was identified in the C terminus of RGA5-A. This reveals a mode of Avr protein recognition through direct binding to a novel, non-LRR interaction domain. PMID:23548743

Phospholipase C-gamma 1 binding to intracellular receptors for activated protein kinase C.

PubMed

Disatnik, M H; Hernandez-Sotomayor, S M; Jones, G; Carpenter, G; Mochly-Rosen, D

1994-01-18

Phospholipase C-gamma 1 (PLC-gamma 1; EC 3.1.4.11) hydrolyzes phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol 1,4,5-trisphosphate and is activated in response to growth factor stimulation and tyrosine phosphorylation. Concomitantly, the enzyme translocates from the cytosol to the particulate cell fraction. A similar process of activation-induced translocation from the cytosol to the cell particulate fraction has also been described for protein kinase C (PKC). We have previously shown that activated PKC binds to specific receptor proteins, receptors for activated C kinase, or RACKs, of approximately 30 kDa. Here, we show that PLC-gamma 1 bound to these RACKs and inhibited subsequent PKC binding to RACKs. However, unlike PKC, the binding of PLC-gamma 1 to RACKs did not require phospholipids and calcium. After epidermal growth factor treatment of intact A-431 cells, the binding of PLC-gamma 1 to RACKs increased as compared with PLC-gamma 1 from control cells. This increase in PLC-gamma 1 binding to RACKs was due to the phosphorylation of PLC-gamma 1. Additional data indicated that PLC-gamma 1 binds to RACKs in solution; epidermal growth factor receptor-dependent PLC-gamma 1 phosphorylation and activation decreased in the presence of RACKs. It is possible that, in vivo, PLC-gamma 1 associates with RACKs or with other PLC-gamma 1-specific anchoring proteins in the particulate cell fraction. Since a PKC C2 homologous region is present in PLC-gamma 1, the C2 region may mediate the activation-induced translocation of the enzyme to the cell particulate fraction and the anchoring protein-PLC-gamma 1 complex may be the active translocated form of PLC-gamma 1.

Sequestration of cAMP response element-binding proteins by transcription factor decoys causes collateral elaboration of regenerating Aplysia motor neuron axons.

PubMed

Dash, P K; Tian, L M; Moore, A N

1998-07-07

Axonal injury increases intracellular Ca2+ and cAMP and has been shown to induce gene expression, which is thought to be a key event for regeneration. Increases in intracellular Ca2+ and/or cAMP can alter gene expression via activation of a family of transcription factors that bind to and modulate the expression of CRE (Ca2+/cAMP response element) sequence-containing genes. We have used Aplysia motor neurons to examine the role of CRE-binding proteins in axonal regeneration after injury. We report that axonal injury increases the binding of proteins to a CRE sequence-containing probe. In addition, Western blot analysis revealed that the level of ApCREB2, a CRE sequence-binding repressor, was enhanced as a result of axonal injury. The sequestration of CRE-binding proteins by microinjection of CRE sequence-containing plasmids enhanced axon collateral formation (both number and length) as compared with control plasmid injections. These findings show that Ca2+/cAMP-mediated gene expression via CRE-binding transcription factors participates in the regeneration of motor neuron axons.

Sequence specificity of single-stranded DNA-binding proteins: a novel DNA microarray approach

PubMed Central

Morgan, Hugh P.; Estibeiro, Peter; Wear, Martin A.; Max, Klaas E.A.; Heinemann, Udo; Cubeddu, Liza; Gallagher, Maurice P.; Sadler, Peter J.; Walkinshaw, Malcolm D.

2007-01-01

We have developed a novel DNA microarray-based approach for identification of the sequence-specificity of single-stranded nucleic-acid-binding proteins (SNABPs). For verification, we have shown that the major cold shock protein (CspB) from Bacillus subtilis binds with high affinity to pyrimidine-rich sequences, with a binding preference for the consensus sequence, 5′-GTCTTTG/T-3′. The sequence was modelled onto the known structure of CspB and a cytosine-binding pocket was identified, which explains the strong preference for a cytosine base at position 3. This microarray method offers a rapid high-throughput approach for determining the specificity and strength of ss DNA–protein interactions. Further screening of this newly emerging family of transcription factors will help provide an insight into their cellular function. PMID:17488853

Unconventional RNA-binding proteins: an uncharted zone in RNA biology.

PubMed

Albihlal, Waleed S; Gerber, André P

2018-06-16

RNA-binding proteins play essential roles in the post-transcriptional regulation of gene expression. While hundreds of RNA-binding proteins can be predicted computationally, the recent introduction of proteome-wide approaches has dramatically expanded the repertoire of proteins interacting with RNA. Besides canonical RNA-binding proteins that contain characteristic RNA-binding domains, many proteins that lack such domains but have other well-characterised cellular functions were identified; including metabolic enzymes, heat shock proteins, kinases, as well as transcription factors and chromatin-associated proteins. In the context of these recently published RNA-protein interactome datasets obtained from yeast, nematodes, flies, plants and mammalian cells, we discuss examples for seemingly evolutionary conserved "unconventional" RNA-binding proteins that act in central carbon metabolism, stress response or regulation of transcription. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Positive feedback between RNA-binding protein HuD and transcription factor SATB1 promotes neurogenesis.

PubMed

Wang, Feifei; Tidei, Joseph J; Polich, Eric D; Gao, Yu; Zhao, Huashan; Perrone-Bizzozero, Nora I; Guo, Weixiang; Zhao, Xinyu

2015-09-08

The mammalian embryonic lethal abnormal vision (ELAV)-like protein HuD is a neuronal RNA-binding protein implicated in neuronal development, plasticity, and diseases. Although HuD has long been associated with neuronal development, the functions of HuD in neural stem cell differentiation and the underlying mechanisms have gone largely unexplored. Here we show that HuD promotes neuronal differentiation of neural stem/progenitor cells (NSCs) in the adult subventricular zone by stabilizing the mRNA of special adenine-thymine (AT)-rich DNA-binding protein 1 (SATB1), a critical transcriptional regulator in neurodevelopment. We find that SATB1 deficiency impairs the neuronal differentiation of NSCs, whereas SATB1 overexpression rescues the neuronal differentiation phenotypes resulting from HuD deficiency. Interestingly, we also discover that SATB1 is a transcriptional activator of HuD during NSC neuronal differentiation. In addition, we demonstrate that NeuroD1, a neuronal master regulator, is a direct downstream target of SATB1. Therefore, HuD and SATB1 form a positive regulatory loop that enhances NeuroD1 transcription and subsequent neuronal differentiation. Our results here reveal a novel positive feedback network between an RNA-binding protein and a transcription factor that plays critical regulatory roles in neurogenesis.

Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation

PubMed Central

Gaviglio, Angela L.; Knelson, Erik H.; Blobe, Gerard C.

2017-01-01

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor–like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.—Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation. PMID:28174207

Fragile X mental retardation protein: A paradigm for translational control by RNA-binding proteins.

PubMed

Chen, Eileen; Joseph, Simpson

2015-07-01

Translational control is a common mechanism used to regulate gene expression and occur in bacteria to mammals. Typically in translational control, an RNA-binding protein binds to a unique sequence in the mRNA to regulate protein synthesis by the ribosomes. Alternatively, a protein may bind to or modify a translation factor to globally regulate protein synthesis by the cell. Here, we review translational control by the fragile X mental retardation protein (FMRP), the absence of which causes the neurological disease, fragile X syndrome (FXS). Copyright © 2015 Elsevier B.V. and Société française de biochimie et biologie Moléculaire (SFBBM). All rights reserved.

Highly Pathogenic Influenza A(H5Nx) Viruses with Altered H5 Receptor-Binding Specificity

PubMed Central

Guo, Hongbo; de Vries, Erik; McBride, Ryan; Dekkers, Jojanneke; Peng, Wenjie; Bouwman, Kim M.; Nycholat, Corwin; Verheije, M. Helene; Paulson, James C.; van Kuppeveld, Frank J.M.

2017-01-01

Emergence and intercontinental spread of highly pathogenic avian influenza A(H5Nx) virus clade 2.3.4.4 is unprecedented. H5N8 and H5N2 viruses have caused major economic losses in the poultry industry in Europe and North America, and lethal human infections with H5N6 virus have occurred in Asia. Knowledge of the evolution of receptor-binding specificity of these viruses, which might affect host range, is urgently needed. We report that emergence of these viruses is accompanied by a change in receptor-binding specificity. In contrast to ancestral clade 2.3.4 H5 proteins, novel clade 2.3.4.4 H5 proteins bind to fucosylated sialosides because of substitutions K222Q and S227R, which are unique for highly pathogenic influenza virus H5 proteins. North American clade 2.3.4.4 virus isolates have retained only the K222Q substitution but still bind fucosylated sialosides. Altered receptor-binding specificity of virus clade 2.3.4.4 H5 proteins might have contributed to emergence and spread of H5Nx viruses. PMID:27869615

Carbene footprinting accurately maps binding sites in protein-ligand and protein-protein interactions

NASA Astrophysics Data System (ADS)

Manzi, Lucio; Barrow, Andrew S.; Scott, Daniel; Layfield, Robert; Wright, Timothy G.; Moses, John E.; Oldham, Neil J.

2016-11-01

Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficient photo-activated probe for use with mass spectrometry. Using this methodology the location of a carbohydrate substrate was accurately mapped to the binding cleft of lysozyme, and in a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5 and a diubiquitin substrate were located to different functional domains. The much improved properties of this probe make carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation.

Identification of C1q as a Binding Protein for Advanced Glycation End Products.

PubMed

Chikazawa, Miho; Shibata, Takahiro; Hatasa, Yukinori; Hirose, Sayumi; Otaki, Natsuki; Nakashima, Fumie; Ito, Mika; Machida, Sachiko; Maruyama, Shoichi; Uchida, Koji

2016-01-26

Advanced glycation end products (AGEs) make up a heterogeneous group of molecules formed from the nonenzymatic reaction of reducing sugars with the free amino groups of proteins. The abundance of AGEs in a variety of age-related diseases, including diabetic complications and atherosclerosis, and their pathophysiological effects suggest the existence of innate defense mechanisms. Here we examined the presence of serum proteins that are capable of binding glycated bovine serum albumin (AGEs-BSA), prepared upon incubation of BSA with dehydroascorbate, and identified complement component C1q subcomponent subunit A as a novel AGE-binding protein in human serum. A molecular interaction analysis showed the specific binding of C1q to the AGEs-BSA. In addition, we identified DNA-binding regions of C1q, including a collagen-like domain, as the AGE-binding site and established that the amount of positive charge on the binding site was the determining factor. C1q indeed recognized several other modified proteins, including acylated proteins, suggesting that the binding specificity of C1q might be ascribed, at least in part, to the electronegative potential of the ligand proteins. We also observed that C1q was involved in the AGEs-BSA-activated deposition of complement proteins, C3b and C4b. In addition, the AGEs-BSA mediated the proteolytic cleavage of complement protein 5 to release C5a. These findings provide the first evidence of AGEs as a new ligand recognized by C1q, stimulating the C1q-dependent classical complement pathway.

RNA–protein binding interface in the telomerase ribonucleoprotein

PubMed Central

Bley, Christopher J.; Qi, Xiaodong; Rand, Dustin P.; Borges, Chad R.; Nelson, Randall W.; Chen, Julian J.-L.

2011-01-01

Telomerase is a specialized reverse transcriptase containing an intrinsic telomerase RNA (TR) which provides the template for telomeric DNA synthesis. Distinct from conventional reverse transcriptases, telomerase has evolved a unique TR-binding domain (TRBD) in the catalytic telomerase reverse transcriptase (TERT) protein, integral for ribonucleoprotein assembly. Two structural elements in the vertebrate TR, the pseudoknot and CR4/5, bind TERT independently and are essential for telomerase enzymatic activity. However, the details of the TR–TERT interaction have remained elusive. In this study, we employed a photoaffinity cross-linking approach to map the CR4/5-TRBD RNA–protein binding interface by identifying RNA and protein residues in close proximity. Photoreactive 5-iodouridines were incorporated into the medaka CR4/5 RNA fragment and UV cross-linked to the medaka TRBD protein fragment. The cross-linking RNA residues were identified by alkaline partial hydrolysis and cross-linked protein residues were identified by mass spectrometry. Three CR4/5 RNA residues (U182, U187, and U205) were found cross-linking to TRBD amino acids Tyr503, Phe355, and Trp477, respectively. This CR4/5 binding pocket is distinct and separate from the previously proposed T pocket in the Tetrahymena TRBD. Based on homologous structural models, our cross-linking data position the essential loop L6.1 adjacent to the TERT C-terminal extension domain. We thus propose that stem-loop 6.1 facilitates proper TERT folding by interacting with both TRBD and C-terminal extension. Revealing the telomerase CR4/5-TRBD binding interface with single-residue resolution provides important insights into telomerase ribonucleoprotein architecture and the function of the essential CR4/5 domain. PMID:22123986

Functional interaction of the DNA-binding transcription factor Sp1 through its DNA-binding domain with the histone chaperone TAF-I.

PubMed

Suzuki, Toru; Muto, Shinsuke; Miyamoto, Saku; Aizawa, Kenichi; Horikoshi, Masami; Nagai, Ryozo

2003-08-01

Transcription involves molecular interactions between general and regulatory transcription factors with further regulation by protein-protein interactions (e.g. transcriptional cofactors). Here we describe functional interaction between DNA-binding transcription factor and histone chaperone. Affinity purification of factors interacting with the DNA-binding domain of the transcription factor Sp1 showed Sp1 to interact with the histone chaperone TAF-I, both alpha and beta isoforms. This interaction was specific as Sp1 did not interact with another histone chaperone CIA nor did other tested DNA-binding regulatory factors (MyoD, NFkappaB, p53) interact with TAF-I. Interaction of Sp1 and TAF-I occurs both in vitro and in vivo. Interaction with TAF-I results in inhibition of DNA-binding, and also likely as a result of such, inhibition of promoter activation by Sp1. Collectively, we describe interaction between DNA-binding transcription factor and histone chaperone which results in negative regulation of the former. This novel regulatory interaction advances our understanding of the mechanisms of eukaryotic transcription through DNA-binding regulatory transcription factors by protein-protein interactions, and also shows the DNA-binding domain to mediate important regulatory interactions.

Acquisition of complement inhibitor serine protease factor I and its cofactors C4b-binding protein and factor H by Prevotella intermedia.

PubMed

Malm, Sven; Jusko, Monika; Eick, Sigrun; Potempa, Jan; Riesbeck, Kristian; Blom, Anna M

2012-01-01

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with (125)I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases.

NF-κB and enhancer-binding CREB protein scaffolded by CREB-binding protein (CBP)/p300 proteins regulate CD59 protein expression to protect cells from complement attack.

PubMed

Du, Yiqun; Teng, Xiaoyan; Wang, Na; Zhang, Xin; Chen, Jianfeng; Ding, Peipei; Qiao, Qian; Wang, Qingkai; Zhang, Long; Yang, Chaoqun; Yang, Zhangmin; Chu, Yiwei; Du, Xiang; Zhou, Xuhui; Hu, Weiguo

2014-01-31

The complement system can be activated spontaneously for immune surveillance or induced to clear invading pathogens, in which the membrane attack complex (MAC, C5b-9) plays a critical role. CD59 is the sole membrane complement regulatory protein (mCRP) that restricts MAC assembly. CD59, therefore, protects innocent host cells from attacks by the complement system, and host cells require the constitutive and inducible expression of CD59 to protect themselves from deleterious destruction by complement. However, the mechanisms that underlie CD59 regulation remain largely unknown. In this study we demonstrate that the widely expressed transcription factor Sp1 may regulate the constitutive expression of CD59, whereas CREB-binding protein (CBP)/p300 bridge NF-κB and CREB, which surprisingly functions as an enhancer-binding protein to induce the up-regulation of CD59 during in lipopolysaccharide (LPS)-triggered complement activation, thus conferring host defense against further MAC-mediated destruction. Moreover, individual treatment with LPS, TNF-α, and the complement activation products (sublytic MAC (SC5b-9) and C5a) could increase the expression of CD59 mainly by activating NF-κB and CREB signaling pathways. Together, our findings identify a novel gene regulation mechanism involving CBP/p300, NF-κB, and CREB; this mechanism suggests potential drug targets for controlling various complement-related human diseases.

Human papillomavirus E5 oncoproteins bind the A4 endoplasmic reticulum protein to regulate proliferative ability upon differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kotnik Halavaty, Katarina; Regan, Jennifer; Mehta, Kavi

2014-03-15

Human papillomaviruses (HPV) infect stratified epithelia and link their life cycles to epithelial differentiation. The HPV E5 protein plays a role in the productive phase of the HPV life cycle but its mechanism of action is still unclear. We identify a new binding partner of E5, A4, using a membrane-associated yeast-two hybrid system. The A4 protein co-localizes with HPV 31 E5 in perinuclear regions and forms complexes with E5 and Bap31. In normal keratinocytes, A4 is found primarily in basal cells while in HPV positive cells high levels of A4 are seen in both undifferentiated and differentiated cells. Reduction ofmore » A4 expression by shRNAs, enhanced HPV genome amplification and increased cell proliferation ability following differentiation but this was not seen in cells lacking E5. Our studies suggest that the A4 protein is an important E5 binding partner that plays a role in regulating cell proliferation ability upon differentiation. - Highlights: • A4 associates with HPV 31 E5 proteins. • A4 is localized to endoplasmic reticulum. • HPV proteins induce A4 expression in suprabasal layers of stratified epithelium. • E5 is important for proliferation ability of differentiating HPV positive cells.« less

Immunotherapy of metastatic breast cancer patients with vitamin D-binding protein-derived macrophage activating factor (GcMAF).

PubMed

Yamamoto, Nobuto; Suyama, Hirofumi; Yamamoto, Nobuyuki; Ushijima, Naofumi

2008-01-15

Serum vitamin D3-binding protein (Gc protein) is the precursor for the principal macrophage activating factor (MAF). The MAF precursor activity of serum Gc protein of breast cancer patients was lost or reduced because Gc protein was deglycosylated by serum alpha-N-acetylgalactosaminidase (Nagalase) secreted from cancerous cells. Patient serum Nagalase activity is proportional to tumor burden. The deglycosylated Gc protein cannot be converted to MAF, resulting in no macrophage activation and immunosuppression. Stepwise incubation of purified Gc protein with immobilized beta-galactosidase and sialidase generated probably the most potent macrophage activating factor (termed GcMAF) ever discovered, which produces no adverse effect in humans. Macrophages treated in vitro with GcMAF (100 pg/ml) are highly tumoricidal to mammary adenocarcinomas. Efficacy of GcMAF for treatment of metastatic breast cancer was investigated with 16 nonanemic patients who received weekly administration of GcMAF (100 ng). As GcMAF therapy progresses, the MAF precursor activity of patient Gc protein increased with a concomitant decrease in serum Nagalase. Because of proportionality of serum Nagalase activity to tumor burden, the time course progress of GcMAF therapy was assessed by serum Nagalase activity as a prognostic index. These patients had the initial Nagalase activities ranging from 2.32 to 6.28 nmole/min/mg protein. After about 16-22 administrations (approximately 3.5-5 months) of GcMAF, these patients had insignificantly low serum enzyme levels equivalent to healthy control enzyme levels, ranging from 0.38 to 0.63 nmole/min/mg protein, indicating eradication of the tumors. This therapeutic procedure resulted in no recurrence for more than 4 years. Copyright 2007 Wiley-Liss, Inc.

Isolation of copper-binding proteins from activated sludge culture.

PubMed

Fukushi, K; Kato, S; Antsuki, T; Omura, T

2001-01-01

Six copper-binding microbial proteins were isolated from activated sludge cultures grown on media containing copper at various concentrations. Molecular weights among isolated proteins were ranged from 1.3k to 1 74k dalton. Isolated proteins were compared for their copper binding capabilities. Proteins isolated from cultures grown in the presence of copper in the growth media exhibited higher copper binding capabilities than those isolated from the culture grown in the absence of copper. The highest metal uptake of 61.23 (mol copper/mol protein) was observed by a protein isolated from a culture grown with copper at a concentration of 0.25 mM. This isolated protein (CBP2) had a molecular weight of 24k dalton. Other protein exhibited copper binding capability of 4.8-32.5 (mol copper/mol protein).

Nucleolin: acharan sulfate–binding protein on the surface of cancer cells

PubMed Central

Joo, Eun Ji; ten Dam, Gerdy B.; van Kuppevelt, Toin H.; Toida, Toshihiko; Linhardt, Robert J.; Kim, Yeong Shik

2005-01-01

Glycosaminoglycans (GAGs) are complex polysaccharides that participate in the regulation of physiological processes through the interactions with a wide variety of proteins. Acharan sulfate (AS), isolated from the giant African snail Achatina fulica, primarily consists of the repeating disaccharide structure α-D-N-acetylglucosaminyl (1→4) 2-sulfoiduronic acid. Exogenous AS was injected subcutaneously near the tumor tissue in C57BL/6 mice that had been implanted with Lewis lung carcinoma cells (LLCs). The location of AS in the tumor was assessed by staining of sectioned tissues with alcian blue and periodic acid–Schiff (PAS) reagent. In vitro assays indicated binding of cells to 50 μg/ml AS (or heparin) after a 5-h incubation. Immunofluorescence assays, using anti-AS antibody, detected AS at the cell surface. The outer-surface of LLCs were next biotinylated to identify the AS-binding proteins. Biotinylated cells were lysed, and the lysates were fractionated on the AS affinity column using a stepwise salt gradient (0, 0.1, 0.3, 0.5, 0.7, 1.0, and 2.0 M). The fractions were analyzed by SDS–PAGE with silver staining and western blotting. We focused on the proteins with high affinity for AS (eluting at 1 M NaCl) and detected only two bands by western blotting. ESI Q-TOF MS analysis of one of these bands, molecular weight ~110 kDa, showed it to be nucleolin. A phosphorylated form of nucleolin on the surface of cells acts as a cell surface receptor for a variety of ligands, including growth factors (i.e., basic fibroblast growth factor) and chemokines (i.e., midkine). These results show that nucleolin is one of several AS-binding proteins and suggest that AS might demonstrate its tumor growth inhibitory activity by binding the nucleolin receptor protein on the surface of cancer cells. PMID:15329357

Characterization of the DNA binding properties of polyomavirus capsid protein

NASA Technical Reports Server (NTRS)

Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1993-01-01

The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

Two-step interrogation then recognition of DNA binding site by Integration Host Factor: an architectural DNA-bending protein.

PubMed

Velmurugu, Yogambigai; Vivas, Paula; Connolly, Mitchell; Kuznetsov, Serguei V; Rice, Phoebe A; Ansari, Anjum

2018-02-28

The dynamics and mechanism of how site-specific DNA-bending proteins initially interrogate potential binding sites prior to recognition have remained elusive for most systems. Here we present these dynamics for Integration Host factor (IHF), a nucleoid-associated architectural protein, using a μs-resolved T-jump approach. Our studies show two distinct DNA-bending steps during site recognition by IHF. While the faster (∼100 μs) step is unaffected by changes in DNA or protein sequence that alter affinity by >100-fold, the slower (1-10 ms) step is accelerated ∼5-fold when mismatches are introduced at DNA sites that are sharply kinked in the specific complex. The amplitudes of the fast phase increase when the specific complex is destabilized and decrease with increasing [salt], which increases specificity. Taken together, these results indicate that the fast phase is non-specific DNA bending while the slow phase, which responds only to changes in DNA flexibility at the kink sites, is specific DNA kinking during site recognition. Notably, the timescales for the fast phase overlap with one-dimensional diffusion times measured for several proteins on DNA, suggesting that these dynamics reflect partial DNA bending during interrogation of potential binding sites by IHF as it scans DNA.

How Proteins Bind Macrocycles

PubMed Central

Villar, Elizabeth A.; Beglov, Dmitri; Chennamadhavuni, Spandan; Porco, John A.; Kozakov, Dima; Vajda, Sandor; Whitty, Adrian

2014-01-01

The potential utility of synthetic macrocycles as drugs, particularly against low druggability targets such as protein-protein interactions, has been widely discussed. There is little information, however, to guide the design of macrocycles for good target protein-binding activity or bioavailability. To address this knowledge gap we analyze the binding modes of a representative set of macrocycle-protein complexes. The results, combined with consideration of the physicochemical properties of approved macrocyclic drugs, allow us to propose specific guidelines for the design of synthetic macrocycles libraries possessing structural and physicochemical features likely to favor strong binding to protein targets and also good bioavailability. We additionally provide evidence that large, natural product derived macrocycles can bind to targets that are not druggable by conventional, drug-like compounds, supporting the notion that natural product inspired synthetic macrocycles can expand the number of proteins that are druggable by synthetic small molecules. PMID:25038790

Sampling protein motion and solvent effect during ligand binding

PubMed Central

Limongelli, Vittorio; Marinelli, Luciana; Cosconati, Sandro; La Motta, Concettina; Sartini, Stefania; Mugnaini, Laura; Da Settimo, Federico; Novellino, Ettore; Parrinello, Michele

2012-01-01

An exhaustive description of the molecular recognition mechanism between a ligand and its biological target is of great value because it provides the opportunity for an exogenous control of the related process. Very often this aim can be pursued using high resolution structures of the complex in combination with inexpensive computational protocols such as docking algorithms. Unfortunately, in many other cases a number of factors, like protein flexibility or solvent effects, increase the degree of complexity of ligand/protein interaction and these standard techniques are no longer sufficient to describe the binding event. We have experienced and tested these limits in the present study in which we have developed and revealed the mechanism of binding of a new series of potent inhibitors of Adenosine Deaminase. We have first performed a large number of docking calculations, which unfortunately failed to yield reliable results due to the dynamical character of the enzyme and the complex role of the solvent. Thus, we have stepped up the computational strategy using a protocol based on metadynamics. Our approach has allowed dealing with protein motion and solvation during ligand binding and finally identifying the lowest energy binding modes of the most potent compound of the series, 4-decyl-pyrazolo[1,5-a]pyrimidin-7-one. PMID:22238423

The eukaryotic translation initiation factor 3 subunit E binds to classical swine fever virus NS5A and facilitates viral replication.

PubMed

Liu, Xiaofeng; Wang, Xiaoyu; Wang, Qian; Luo, Mingyang; Guo, Huancheng; Gong, Wenjie; Tu, Changchun; Sun, Jinfu

2018-02-01

Classical swine fever virus (CSFV) NS5A protein is a multifunctional protein, playing critical roles in viral RNA replication, translation and assembly. To further explore its functions in viral replication, interaction of NS5A with host factors was assayed using a his-tag "pull down" assay coupled with shotgun LC-MS/MS. Host protein translation initiation factor 3 subunit E was identified as a binding partner of NS5A, and confirmed by co-immunoprecipitation and co-localization analysis. Overexpression of eIF3E markedly enhanced CSFV genomic replication, viral protein expression and production of progeny virus, and downregulation of eIF3E by siRNA significantly decreased viral proliferation in PK-15 cells. Luciferase reporter assay showed an enhancement of translational activity of the internal ribosome entry site of CSFV by eIF3E and a decrease in cellular translation by NS5A. These data indicate that eIF3E plays an important role in CSFV replication, thereby identifying it as a potential target for inhibition of the virus. Copyright © 2017 Elsevier Inc. All rights reserved.

Dissecting protein:protein interactions between transcription factors with an RNA aptamer.

PubMed Central

Tian, Y; Adya, N; Wagner, S; Giam, C Z; Green, M R; Ellington, A D

1995-01-01

Nucleic acid aptamers isolated from random sequence pools have generally proven useful at inhibiting the interactions of nucleic acid binding proteins with their cognate nucleic acids. In order to develop reagents that could also be used to study protein:protein interactions, we have used in vitro selection to search for RNA aptamers that could interact with the transactivating protein Tax from human T-cell leukemia virus. Tax does not normally bind to nucleic acids, but instead stimulates transcription by interacting with a variety of cellular transcription factors, including the cyclic AMP-response element binding protein (CREB), NF-kappa B, and the serum response factor (SRF). Starting from a pool of greater than 10(13) different RNAs with a core of 120 random sequence positions, RNAs were selected for their ability to be co-retained on nitrocellulose filters with Tax. After five cycles of selection and amplification, a single nucleic acid species remained. This aptamer was found to bind Tax with high affinity and specificity, and could disrupt complex formation between Tax and NF-kappa B, but not with SRF. The differential effects of our aptamer probe on protein:protein interactions suggest a model for how the transcription factor binding sites on the surface of the Tax protein are organized. This model is consistent with data from a variety of other studies. PMID:7489503

Behind the scenes of vitamin D binding protein: more than vitamin D binding.

PubMed

Delanghe, Joris R; Speeckaert, Reinhart; Speeckaert, Marijn M

2015-10-01

Although being discovered in 1959, the number of published papers in recent years reveals that vitamin D binding protein (DBP), a member of the albuminoid superfamily, is a hot research topic. Besides the three major phenotypes (DBP1F, DBP1S and DBP2), more than 120 unique variants have been described of this polymorphic protein. The presence of DBP has been demonstrated in different body fluids (serum, urine, breast milk, ascitic fluid, cerebrospinal fluid, saliva and seminal fluid) and organs (brain, heart, lungs, kidneys, placenta, spleen, testes and uterus). Although the major function is binding, solubilization and transport of vitamin D and its metabolites, the name of this glycoprotein hides numerous other important biological functions. In this review, we will focus on the analytical aspects of the determination of DBP and discuss in detail the multifunctional capacity [actin scavenging, binding of fatty acids, chemotaxis, binding of endotoxins, influence on T cell response and influence of vitamin D binding protein-macrophage activating factor (DBP-MAF) on bone metabolism and cancer] of this abundant plasma protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

[Determination of plasma protein binding rate of arctiin and arctigenin with ultrafiltration].

PubMed

Han, Xue-Ying; Wang, Wei; Tan, Ri-Qiu; Dou, De-Qiang

2013-02-01

To determine the plasma protein binding rate of arctiin and arctigenin. The ultrafiltration combined with HPLC was employed to determine the plasma protein binding rate of arctiin and arctigenin as well as rat plasma and healthy human plasma proteins. The plasma protein binding rate of arctiin with rat plasma at the concentrations of 64. 29, 32.14, 16.07 mg x L(-1) were (71.2 +/- 2.0)%, (73.4 +/- 0.61)%, (78.2 +/- 1.9)%, respectively; while the plasma protein binding rate of arctiin with healthy human plasma at the above concentrations were (64.8 +/- 3.1)%, (64.5 +/- 2.5)%, (77.5 +/- 1.7)%, respectively. The plasma protein binding rate of arctigenin with rat plasma at the concentrations of 77.42, 38.71, 19.36 mg x L(-1) were (96.7 +/- 0.41)%, (96.8 +/- 1.6)%, (97.3 +/- 0.46)%, respectively; while the plasma protein binding rate of arctigenin with normal human plasma at the above concentrations were (94.7 +/- 3.1)%, (96.8 +/- 1.6)%, (97.9 +/- 1.3)%, respectively. The binding rate of arctiin with rat plasma protein was moderate, which is slightly higher than the binding rate of arctiin with healthy human plasma protein. The plasma protein binding rates of arctigenin with both rat plasma and healthy human plasma are very high.

Annexin A5 Binds to Lipopolysaccharide and Reduces Its Endotoxin Activity

PubMed Central

Rand, Jacob H.; Wu, Xiao-Xuan; Lin, Elaine Y.; Griffel, Alexander; Gialanella, Philip; McKitrick, John C.

2012-01-01

ABSTRACT Annexin A5 (AnxA5) has a high affinity for phosphatidylserine. The protein is widely used to detect apoptotic cells because phosphatidylserine, a phospholipid that is normally present in the inner leaflets of cytoplasmic membranes, becomes translocated to the outer leaflets during programmed cell death. Here we report the novel observation that AnxA5 binds to Gram-negative bacteria via the lipid A domain of lipopolysaccharide (LPS). Binding of AnxA5 to bacteria was measured quantitatively, confirmed by fluorescence microscopy, and found to be inhibited by antibodies against lipid A. AnxA5 also bound to purified dot-blotted LPS and lipid A. Through ellipsometry, we found that the binding of AnxA5 to purified LPS was calcium dependent and rapid and showed a high affinity—characteristics similar to those of AnxA5 binding to phosphatidylserine. Initial functional studies indicated that AnxA5 can affect LPS activities. AnxA5 inhibited LPS-mediated gelation in the Limulus amebocyte lysate assay. Incubation of LPS with the protein reduced the quantity of tumor necrosis factor alpha (TNF-α) released by cultured monocytes compared to that released upon incubation with LPS alone. Initial in vivo experiments indicated that injection of mice with LPS preincubated with AnxA5 produced serum TNF-α levels lower than those seen after injection of LPS alone. These data demonstrate that AnxA5 binds to LPS and open paths to investigation of the potential biological and therapeutic implications of this interaction. PMID:22415004

Structural basis for the inhibition of insulin-like growth factors by insulin-like growth factor-binding proteins

PubMed Central

Sitar, Tomasz; Popowicz, Grzegorz M.; Siwanowicz, Igor; Huber, Robert; Holak, Tad A.

2006-01-01

Insulin-like growth factor-binding proteins (IGFBPs) control bioavailability, activity, and distribution of insulin-like growth factor (IGF)1 and -2 through high-affinity IGFBP/IGF complexes. IGF-binding sites are found on N- and C-terminal fragments of IGFBPs, the two conserved domains of IGFBPs. The relative contributions of these domains to IGFBP/IGF complexation has been difficult to analyze, in part, because of the lack of appropriate three-dimensional structures. To analyze the effects of N- and C-terminal domain interactions, we determined several x-ray structures: first, of a ternary complex of N- and C-terminal domain fragments of IGFBP4 and IGF1 and second, of a “hybrid” ternary complex using the C-terminal domain fragment of IGFBP1 instead of IGFBP4. We also solved the binary complex of the N-terminal domains of IGFBP4 and IGF1, again to analyze C- and N-terminal domain interactions by comparison with the ternary complexes. The structures reveal the mechanisms of IGF signaling regulation via IGFBP binding. This finding supports research into the design of IGFBP variants as therapeutic IGF inhibitors for diseases of IGF disregulation. In IGFBP4, residues 1–38 form a rigid disulphide bond ladder-like structure, and the first five N-terminal residues bind to IGF and partially mask IGF residues responsible for the type 1 IGF receptor binding. A high-affinity IGF1-binding site is located in a globular structure between residues 39 and 82. Although the C-terminal domains do not form stable binary complexes with either IGF1 or the N-terminal domain of IGFBP4, in the ternary complex, the C-terminal domain contacts both and contributes to blocking of the IGF1 receptor-binding region of IGF1. PMID:16924115

Productive interaction between transmembrane mutants of the bovine papillomavirus E5 protein and the platelet-derived growth factor beta receptor.

PubMed

Lai, Char-Chang; Edwards, Anne P B; DiMaio, Daniel

2005-02-01

The bovine papillomavirus E5 protein is a 44-amino-acid transmembrane protein that transforms cells by binding to the transmembrane region of the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in sustained receptor signaling. However, there are published reports that certain mutants with amino acid substitutions in the membrane-spanning segment of the E5 protein transform cells without activating the PDGF beta receptor. We re-examined several of these transmembrane mutants, and here we present five lines of evidence that these mutants do in fact activate the PDGF beta receptor, resulting in cellular signaling and transformation.

Detecting cis-regulatory binding sites for cooperatively binding proteins

PubMed Central

van Oeffelen, Liesbeth; Cornelis, Pierre; Van Delm, Wouter; De Ridder, Fedor; De Moor, Bart; Moreau, Yves

2008-01-01

Several methods are available to predict cis-regulatory modules in DNA based on position weight matrices. However, the performance of these methods generally depends on a number of additional parameters that cannot be derived from sequences and are difficult to estimate because they have no physical meaning. As the best way to detect cis-regulatory modules is the way in which the proteins recognize them, we developed a new scoring method that utilizes the underlying physical binding model. This method requires no additional parameter to account for multiple binding sites; and the only necessary parameters to model homotypic cooperative interactions are the distances between adjacent protein binding sites in basepairs, and the corresponding cooperative binding constants. The heterotypic cooperative binding model requires one more parameter per cooperatively binding protein, which is the concentration multiplied by the partition function of this protein. In a case study on the bacterial ferric uptake regulator, we show that our scoring method for homotypic cooperatively binding proteins significantly outperforms other PWM-based methods where biophysical cooperativity is not taken into account. PMID:18400778

Characterization of a unique motif in LIM mineralization protein-1 that interacts with jun activation-domain-binding protein 1.

PubMed

Sangadala, Sreedhara; Yoshioka, Katsuhito; Enyo, Yoshio; Liu, Yunshan; Titus, Louisa; Boden, Scott D

2014-01-01

Development and repair of the skeletal system and other organs are highly dependent on precise regulation of the bone morphogenetic protein (BMP) pathway. The use of BMPs clinically to induce bone formation has been limited in part by the requirement of much higher doses of recombinant proteins in primates than were needed in cell culture or rodents. Therefore, increasing cellular responsiveness to BMPs has become our focus. We determined that an osteogenic LIM mineralization protein, LMP-1 interacts with Smurf1 (Smad ubiquitin regulatory factor 1) and prevents ubiquitination of Smads resulting in potentiation of BMP activity. In the region of LMP-1 responsible for bone formation, there is a motif that directly interacts with the Smurf1 WW2 domain and thus effectively competes for binding with Smad1 and Smad5, key signaling proteins in the BMP pathway. Here we show that the same region also contains a motif that interacts with Jun activation-domain-binding protein 1 (Jab1) which targets a common Smad, Smad4, shared by both the BMP and transforming growth factor-β (TGF-β) pathways, for proteasomal degradation. Jab1 was first identified as a coactivator of the transcription factor c-Jun. Jab1 binds to Smad4, Smad5, and Smad7, key intracellular signaling molecules of the TGF-β superfamily, and causes ubiquitination and/or degradation of these Smads. We confirmed a direct interaction of Jab1 with LMP-1 using recombinantly expressed wild-type and mutant proteins in slot-blot-binding assays. We hypothesized that LMP-1 binding to Jab1 prevents the binding and subsequent degradation of these Smads causing increased accumulation of osteogenic Smads in cells. We identified a sequence motif in LMP-1 that was predicted to interact with Jab1 based on the MAME/MAST sequence analysis of several cellular signaling molecules that are known to interact with Jab-1. We further mutated the potential key interacting residues in LMP-1 and showed loss of binding to Jab1 in binding

Differential compartmentalization of Streptococcus pyogenes virulence factors and host protein binding properties as a mechanism for host adaptation.

PubMed

Kilsgård, Ola; Karlsson, Christofer; Malmström, Erik; Malmström, Johan

2016-11-01

Streptococcus pyogenes is an important human pathogen responsible for substantial morbidity and mortality worldwide. Although S. pyogenes is a strictly human pathogen with no other known animal reservoir, several murine infection models exist to explore different aspects of the bacterial pathogenesis. Inoculating mice with wild-type S. pyogenes strains can result in the generation of new bacterial phenotypes that are hypervirulent compared to the original inoculum. In this study, we used a serial mass spectrometry based proteomics strategy to investigate if these hypervirulent strains have an altered distribution of virulence proteins across the intracellular, surface associated and secreted bacterial compartments and if any change in compartmentalization can alter the protein-protein interaction network between bacteria and host proteins. Quantitative analysis of the S. pyogenes surface and secreted proteomes revealed that animal passaged strains are associated with significantly higher amount of virulence factors on the bacterial surface and in the media. This altered virulence factor compartmentalization results in increased binding of several mouse plasma proteins to the bacterial surface, a trend that was consistent for mouse plasma from several different mouse strains. In general, both the wild-type strain and animal passaged strain were capable of binding high amounts of human plasma proteins. However, compared to the non-passaged strains, the animal passaged strains displayed an increased ability to bind mouse plasma proteins, in particular for M protein binders, indicating that the increased affinity for mouse blood plasma proteins is a consequence of host adaptation of this pathogen to a new host. In conclusion, plotting the total amount of virulence factors against the total amount of plasma proteins associated to the bacterial surface could clearly separate out animal passaged strains from wild type strains indicating a virulence model that could

Salt modulates the stability and lipid binding affinity of the adipocyte lipid-binding proteins

NASA Technical Reports Server (NTRS)

Schoeffler, Allyn J.; Ruiz, Carmen R.; Joubert, Allison M.; Yang, Xuemei; LiCata, Vince J.

2003-01-01

Adipocyte lipid-binding protein (ALBP or aP2) is an intracellular fatty acid-binding protein that is found in adipocytes and macrophages and binds a large variety of intracellular lipids with high affinity. Although intracellular lipids are frequently charged, biochemical studies of lipid-binding proteins and their interactions often focus most heavily on the hydrophobic aspects of these proteins and their interactions. In this study, we have characterized the effects of KCl on the stability and lipid binding properties of ALBP. We find that added salt dramatically stabilizes ALBP, increasing its Delta G of unfolding by 3-5 kcal/mol. At 37 degrees C salt can more than double the stability of the protein. At the same time, salt inhibits the binding of the fluorescent lipid 1-anilinonaphthalene-8-sulfonate (ANS) to the protein and induces direct displacement of the lipid from the protein. Thermodynamic linkage analysis of the salt inhibition of ANS binding shows a nearly 1:1 reciprocal linkage: i.e. one ion is released from ALBP when ANS binds, and vice versa. Kinetic experiments show that salt reduces the rate of association between ANS and ALBP while simultaneously increasing the dissociation rate of ANS from the protein. We depict and discuss the thermodynamic linkages among stability, lipid binding, and salt effects for ALBP, including the use of these linkages to calculate the affinity of ANS for the denatured state of ALBP and its dependence on salt concentration. We also discuss the potential molecular origins and potential intracellular consequences of the demonstrated salt linkages to stability and lipid binding in ALBP.

A Novel Association between Two Trypanosome-Specific Factors and the Conserved L5-5S rRNA Complex

PubMed Central

Ciganda, Martin; Prohaska, Kimberly; Hellman, Kristina; Williams, Noreen

2012-01-01

P34 and P37 are two previously identified RNA binding proteins in the flagellate protozoan Trypanosoma brucei. RNA interference studies have determined that the proteins are involved in and essential for ribosome biogenesis. The proteins interact with the 5S rRNA with nearly identical binding characteristics. We have shown that this interaction is achieved mainly through the LoopA region of the RNA, but P34 and P37 also protect the L5 binding site located on LoopC. We now provide evidence to show that these factors form a novel pre-ribosomal particle through interactions with both 5S rRNA and the L5 ribosomal protein. Further in silico and in vitro analysis of T. brucei L5 indicates a lower affinity for 5S rRNA than expected, based on other eukaryotic L5 proteins. We hypothesize that P34 and P37 complement L5 and bridge the interaction with 5S rRNA, stabilizing it and aiding in the early steps of ribosome biogenesis. PMID:22859981

ON THE MECHANISM OF ACTION OF ADRENOCORTICOTROPIC HORMONE: THE BINDING OF CYCLIC-3′,5′-ADENOSINE MONOPHOSPHATE TO AN ADRENAL CORTICAL PROTEIN*

PubMed Central

Gill, Gordon N.; Garren, Leonard D.

1969-01-01

The binding of cyclic 3′,5′-adenosine monophosphate (cyclic AMP) within the adrenal cortical cell was studied. Cyclic AMP binds specifically to a protein which is associated predominantly with the microsomal fraction of the cell. The binding protein was purified approximately 100-fold. PMID:4308274

Lactoferrin-binding proteins in Shigella flexneri.

PubMed Central

Tigyi, Z; Kishore, A R; Maeland, J A; Forsgren, A; Naidu, A S

1992-01-01

The ability of Shigella flexneri to interact with lactoferrin (Lf) was examined with a 125I-labeled protein-binding assay. The percent binding of human lactoferrin (HLf) and bovine lactoferrin (BLf) to 45 S. flexneri strains was 19 +/- 3 and 21 +/- 3 (mean +/- standard error of the mean), respectively. 125I-labeled HLf and BLf binding to strain M90T reached an equilibrium within 2 h. Unlabeled HLf and BLf displaced the 125I-HLf-bacteria interaction in a dose-dependent manner. The Lf-bacterium complex was uncoupled by KSCN or urea, but not by NaCl. The interaction was specific, and approximately 4,800 HLf binding sites (affinity constant [Ka], 690 nM) or approximately 5,700 BLf binding sites (Ka, 104 nM) per cell were estimated in strain M90T by a Scatchard plot analysis. The native cell envelope (CE) and outer membrane (OM) did not reveal Lf-binding components in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. However, after being boiled, the CE and OM preparations showed three distinct horseradish peroxidase-Lf reactive bands of about 39, 22, and 16 kDa. The 39-kDa component was also reactive to a monoclonal antibody specific for porin (PoI) proteins of members of the family Enterobacteriaceae. The Lf-binding protein pattern was similar with BLf or HLf, for Crb+ and Crb- strains. The protein-Lf complex was dissociable by KSCN or urea and was stable after treatment with NaCl. Variation (loss) in the O chain of lipopolysaccharide (LPS) markedly enhanced the Lf-binding capacity in the isogenic rough strain SFL1070-15 compared with its smooth parent strain, SFL1070. These data establish that Lf binds to specific components in the bacterial OM; the heat-modifiable, anti-PoI-reactive, and LPS-associated properties suggested that the Lf-binding proteins are porins in S. flexneri. Images PMID:1319403

Multivalent DNA-binding properties of the HMG-1 proteins.

PubMed Central

Maher, J F; Nathans, D

1996-01-01

HMG-I proteins are DNA-binding proteins thought to affect the formation and function of transcription complexes. Each protein contains three DNA-binding motifs, known as AT-hooks, that bind in the minor groove of AT tracts in DNA. Multiple AT-hooks within a polypeptide chain should contact multiple AT tracts, but the rules governing these interactions have not been defined. In this study, we demonstrate that high-affinity binding uses two or three appropriately spaced AT tracts as a single multivalent binding site. These principles have implications for binding to regulatory elements such as the interferon beta enhancer, TATA boxes, and serum response elements. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8692884

Type I human T cell leukemia virus tax protein transforms rat fibroblasts through the cyclic adenosine monophosphate response element binding protein/activating transcription factor pathway.

PubMed Central

Smith, M R; Greene, W C

1991-01-01

The Tax oncoprotein of the type I human T cell leukemia virus (HTLV-I) activates transcription of cellular and viral genes through at least two different transcription factor pathways. Tax activates transcription of the c-fos proto-oncogene by a mechanism that appears to involve members of the cAMP response element binding protein (CREB) and activating transcription factor (ATF) family of DNA-binding proteins. Tax also induces the nuclear expression of the NF-kappa B family of rel oncogene-related enhancer-binding proteins. We have investigated the potential role of these CREB/ATF and NF-kappa B/Rel transcription factors in Tax-mediated transformation by analyzing the oncogenic potential of Tax mutants that functionally segregate these two pathways of transactivation. Rat fibroblasts (Rat2) stably expressing either the wild-type Tax protein or a Tax mutant selectively deficient in the ability to induce NF-kappa B/Rel demonstrated marked changes in morphology and growth characteristics including the ability to form tumors in athymic mice. In contrast, Rat2 cells stably expressing a Tax mutant selectively deficient in the ability to activate transcription through CREB/ATF demonstrated no detectable changes in morphology or growth characteristics. These results suggest that transcriptional activation through the CREB/ATF pathway may play an important role in Tax-mediated cellular transformation. Images PMID:1832173

Acquisition of Complement Inhibitor Serine Protease Factor I and Its Cofactors C4b-Binding Protein and Factor H by Prevotella intermedia

PubMed Central

Malm, Sven; Jusko, Monika; Eick, Sigrun; Potempa, Jan; Riesbeck, Kristian; Blom, Anna M.

2012-01-01

Infection with the Gram-negative pathogen Prevotella intermedia gives rise to periodontitis and a growing number of studies implies an association of P. intermedia with rheumatoid arthritis. The serine protease Factor I (FI) is the central inhibitor of complement degrading complement components C3b and C4b in the presence of cofactors such as C4b-binding protein (C4BP) and Factor H (FH). Yet, the significance of complement inhibitor acquisition in P. intermedia infection and FI binding by Gram-negative pathogens has not been addressed. Here we show that P. intermedia isolates bound purified FI as well as FI directly from heat-inactivated human serum. FI bound to bacteria retained its serine protease activity as shown in degradation experiments with 125I-labeled C4b. Since FI requires cofactors for its activity we also investigated the binding of purified cofactors C4BP and FH and found acquisition of both proteins, which retained their activity in FI mediated degradation of C3b and C4b. We propose that FI binding by P. intermedia represents a new mechanism contributing to complement evasion by a Gram-negative bacterial pathogen associated with chronic diseases. PMID:22514678

Imparting albumin-binding affinity to a human protein by mimicking the contact surface of a bacterial binding protein.

PubMed

Oshiro, Satoshi; Honda, Shinya

2014-04-18

Attachment of a bacterial albumin-binding protein module is an attractive strategy for extending the plasma residence time of protein therapeutics. However, a protein fused with such a bacterial module could induce unfavorable immune reactions. To address this, we designed an alternative binding protein by imparting albumin-binding affinity to a human protein using molecular surface grafting. The result was a series of human-derived 6 helix-bundle proteins, one of which specifically binds to human serum albumin (HSA) with adequate affinity (KD = 100 nM). The proteins were designed by transferring key binding residues of a bacterial albumin-binding module, Finegoldia magna protein G-related albumin-binding domain (GA) module, onto the human protein scaffold. Despite 13-15 mutations, the designed proteins maintain the original secondary structure by virtue of careful grafting based on structural informatics. Competitive binding assays and thermodynamic analyses of the best binders show that the binding mode resembles that of the GA module, suggesting that the contacting surface of the GA module is mimicked well on the designed protein. These results indicate that the designed protein may act as an alternative low-risk binding module to HSA. Furthermore, molecular surface grafting in combination with structural informatics is an effective approach for avoiding deleterious mutations on a target protein and for imparting the binding function of one protein onto another.

Characterization of flavonoid-protein interactions using fluorescence spectroscopy: Binding of pelargonidin to dairy proteins.

PubMed

Arroyo-Maya, Izlia J; Campos-Terán, José; Hernández-Arana, Andrés; McClements, David Julian

2016-12-15

In this study, the interaction between the flavonoid pelargonidin and dairy proteins: β-lactoglobulin (β-LG), whey protein (WPI), and caseinate (CAS) was investigated. Fluorescence experiments demonstrated that pelargonidin quenched milk proteins fluorescence strongly. However, the protein secondary structure was not significantly affected by pelargonidin, as judged from far-UV circular dichroism. Analysis of fluorescence data indicated that pelargonidin-induced quenching does not arise from a dynamical mechanism, but instead is due to protein-ligand binding. Therefore, quenching data were analyzed using the model of independent binding sites. Both β-LG and CAS, but not WPI, showed hyperbolic binding isotherms indicating that these proteins firmly bound pelargonidin at both pH 7.0 and 3.0 (binding constants ca. 1.0×10(5) at 25.0°C). To investigate the underlying thermodynamics, binding constants were determined at 25.0, 35.0, and 45.0°C. These results pointed to binding processes that depend on the structural conformation of the milk proteins. Copyright © 2016 Elsevier Ltd. All rights reserved.

The Acetylase/Deacetylase Couple CREB-binding Protein/Sirtuin 1 Controls Hypoxia-inducible Factor 2 Signaling*

PubMed Central

Chen, Rui; Xu, Min; Hogg, Richard T.; Li, Jiwen; Little, Bertis; Gerard, Robert D.; Garcia, Joseph A.

2012-01-01

Hypoxia-inducible factors (HIFs) are oxygen-sensitive transcription factors. HIF-1α plays a prominent role in hypoxic gene induction. HIF-2α target genes are more restricted but include erythropoietin (Epo), one of the most highly hypoxia-inducible genes in mammals. We previously reported that HIF-2α is acetylated during hypoxia but is rapidly deacetylated by the stress-responsive deacetylase Sirtuin 1. We now demonstrate that the lysine acetyltransferases cAMP-response element-binding protein-binding protein (CBP) and p300 are required for efficient Epo induction during hypoxia. However, despite close structural similarity, the roles of CBP and p300 differ in HIF signaling. CBP acetylates HIF-2α, is a major coactivator for HIF-2-mediated Epo induction, and is required for Sirt1 augmentation of HIF-2 signaling during hypoxia in Hep3B cells. In comparison, p300 is a major contributor for HIF-1 signaling as indicated by induction of Pgk1. Whereas CBP can bind with HIF-2α independent of the HIF-2α C-terminal activation domain via enzyme/substrate interactions, p300 only complexes with HIF-2α through the C-terminal activation domain. Maximal CBP/HIF-2 signaling requires intact CBP acetyltransferase activity in both Hep3B cells as well as in mice. PMID:22807441

Acetyllysine-binding and function of bromodomain-containing proteins in chromatin.

PubMed

Dyson, M H; Rose, S; Mahadevan, L C

2001-08-01

Acetylated histones are generally associated with active chromatin. The bromodomain has recently been identified as a protein module capable of binding to acetylated lysine residues, and hence is able to mediate the recruitment of factors to acetylated chromatin. Functional studies of bromodomain-containing proteins indicate how this domain contributes to the activity of a number of nuclear factors including histone acetyltransferases and chromatin remodelling complexes. Here, we review the characteristics of acetyllysine-binding by bromodomains, discuss associated domains found in these proteins, and address the function of the bromodomain in the context of chromatin. Finally, the modulation of bromodomain binding by neighbouring post-translational modifications within histone tails might provide a mechanism through which combinations of covalent marks could exert control on chromatin function.

Time- and dose-related interactions between glucocorticoid and cyclic adenosine 3',5'-monophosphate on CCAAT/enhancer-binding protein-dependent insulin-like growth factor I expression by osteoblasts

NASA Technical Reports Server (NTRS)

McCarthy, T. L.; Ji, C.; Chen, Y.; Kim, K.; Centrella, M.

2000-01-01

Glucocorticoid has complex effects on osteoblasts. Several of these changes appear to be related to steroid concentration, duration of exposure, or specific effects on growth factor expression or activity within bone. One important bone growth factor, insulin-like growth factor I (IGF-I), is induced in osteoblasts by hormones such as PGE2 that increase intracellular cAMP levels. In this way, PGE2 activates transcription factor CCAAT/enhancer-binding protein-delta (C/EBPdelta) and enhances its binding to a specific control element found in exon 1 in the IGF-I gene. Our current studies show that preexposure to glucocorticoid enhanced C/EBPdelta and C/EBPbeta expression by osteoblasts and thereby potentiated IGF-I gene promoter activation in response to PGE2. Importantly, this directly contrasts with inhibitory effects on IGF-I expression that result from sustained or pharmacologically high levels of glucocorticoid exposure. Consistent with the stimulatory effect of IGF-I on bone protein synthesis, pretreatment with glucocorticoid sensitized osteoblasts to PGE2, and in this context significantly enhanced new collagen and noncollagen protein synthesis. Therefore, pharmacological levels of glucocorticoid may reduce IGF-I expression by osteoblasts and cause osteopenic disease, whereas physiological transient increases in glucocorticoid may permit or amplify the effectiveness of hormones that regulate skeletal tissue integrity. These events appear to converge on the important role of C/EBPdelta and C/EBPbeta on IGF-I expression by osteoblasts.

Phosphorylation of ETS Transcription Factor ER81 in a Complex with Its Coactivators CREB-Binding Protein and p300

PubMed Central

Papoutsopoulou, Stamatia; Janknecht, Ralf

2000-01-01

The ETS protein ER81 is a DNA-binding factor capable of enhancing gene transcription and is implicated in cellular transformation, but presently the mechanisms of its actions are unclear. In this report, ER81 is shown to coimmunoprecipitate with the transcriptional coactivator CREB-binding protein (CBP) and the related p300 protein (together referred to as CBP/p300). Moreover, confocal laser microscopic studies demonstrated that ER81 and p300 colocalized to nuclear speckles. In vitro and in vivo interaction studies revealed that ER81 amino acids 249 to 429, which encompass the ETS DNA-binding domain, are responsible for binding to CBP/p300. However, mutation of a putative protein-protein interaction motif, LXXLL, in the ETS domain of ER81 did not affect interaction with CBP/p300, whereas DNA binding of ER81 was abolished. Furthermore, two regions within CBP, amino acids 451 to 721 and 1891 to 2175, are capable of binding to ER81. Consistent with the physical interaction between ER81 and the coactivators CBP and p300, ER81 transcriptional activity was potentiated by CBP/p300 overexpression. Moreover, an ER81-associated protein kinase activity was enhanced upon p300 overexpression. This protein kinase phosphorylates ER81 on serines 191 and 216, and mutation of these phosphorylation sites increased ER81 transcriptional activity in Mv1Lu cells but not in HeLa cells. Altogether, our data elucidate the mechanism of how ER81 regulates gene transcription, through interaction with the coactivators CBP and p300 and an associated kinase that may cell type specifically modulate the ability of ER81 to activate gene transcription. PMID:10982847

Material-induced Senescence (MIS): Fluidity Induces Senescent Type Cell Death of Lung Cancer Cells via Insulin-Like Growth Factor Binding Protein 5.

PubMed

Mano, Sharmy Saimon; Uto, Koichiro; Ebara, Mitsuhiro

2017-01-01

Objective: We propose here material-induced senescence (MIS) as a new therapeutic concept that limits cancer progression by stable cell cycle arrest. This study examined for the first time the effect of material fluidity on cellular senescence in lung carcinoma using poly(ε-caprolactone- co -D, L-lactide) (P(CL- co -DLLA)) with tunable elasticity and fluidity. Methods: The fluidity was varied by chemically crosslinking the polymer networks: the crosslinked P(CL- co -DLLA) shows solid-like properties with a stiffness of 260 kPa, while the non-crosslinked polymer exists in a quasi-liquid state with loss and storage moduli of 33 kPa and 11 kPa, respectively. Results: We found that cancer cells growing on the non-crosslinked, fluidic substrate undergo a non-apoptotic form of cell death and the cell cycle was accumulated in a G0/G1 phase. Next, we investigated the expression of biomarkers that are associated with cancer pathways. The cancer cells on the fluidic substrate expressed several biomarkers associated with senescence such as insulin-like growth factor binding protein 5 (IGFBP5). This result indicates that when cancer cells sense fluidity in their surroundings, the cells express IGFBP5, which in turn triggers the expression of tumor suppressor protein 53 and initiates cell cycle arrest at the G1 phase followed by cellular senescence. Furthermore, the cancer cells on the fluidic substrate maintained their epithelial phenotype, suggesting that the cancer cells do not undergo epithelial to mesenchymal transition. Conclusion: By considering these results as the fundamental information for MIS, our system could be applied to induce senescence in treatment-resistant cancers such as metastatic cancer or cancer stem cells.

C/EBPβ (CCAAT/enhancer-binding protein β) mediates progesterone production through transcriptional regulation in co-operation with SF-1 (steroidogenic factor-1).

PubMed

Mizutani, Tetsuya; Ju, Yunfeng; Imamichi, Yoshitaka; Osaki, Tsukasa; Yazawa, Takashi; Kawabe, Shinya; Ishikane, Shin; Matsumura, Takehiro; Kanno, Masafumi; Kamiki, Yasue; Kimura, Kohei; Minamino, Naoto; Miyamoto, Kaoru

2014-06-15

The transcription factor SF-1 (steroidogenic factor-1) is a master regulator of steroidogenesis. Previously, we have found that SF-1 induces the differentiation of mesenchymal stem cells into steroidogenic cells. To elucidate the molecular mechanisms of SF-1-mediated functions, we attempted to identify protein components of the SF-1 nuclear protein complex in differentiated cells. SF-1 immunoaffinity chromatography followed by MS/MS analysis was performed, and 24 proteins were identified. Among these proteins, we focused on C/EBPβ (CCAAT/enhancer-binding protein β), which is an essential transcription factor for ovulation and luteinization, as the transcriptional mechanisms of C/EBPβ working together with SF-1 are poorly understood. C/EBPβ knockdown attenuated cAMP-induced progesterone production in granulosa tumour-derived KGN cells by altering STAR (steroidogenic acute regulatory protein), CYP11A1 (cytochrome P450, family 11, subfamily A, polypeptide 1) and HSD3B2 (hydroxy-δ-5-steroid dehydrogenase, 3β- and steroid δ-isomerase 2) expression. EMSA and ChIP assays revealed novel C/EBPβ-binding sites in the upstream regions of the HSD3B2 and CYP11A1 genes. These interactions were enhanced by cAMP stimulation. Luciferase assays showed that C/EBPβ-responsive regions were found in each promoter and C/EBPβ is involved in the cAMP-induced transcriptional activity of these genes together with SF-1. These results indicate that C/EBPβ is an important mediator of progesterone production by working together with SF-1, especially under tropic hormone-stimulated conditions.

Hyaluronate-binding proteins of murine brain.

PubMed

Marks, M S; Chi-Rosso, G; Toole, B P

1990-01-01

The distribution of hyaluronate-binding activity was determined in the soluble and membrane fractions derived from adult mouse brain by sonication in low-ionic-strength buffer. Approximately 60% of the total activity was recovered in the soluble fraction and 33% in membrane fractions. In both cases, the hyaluronate-binding activities were found to be of high affinity (KD = 10(-9) M), specific for hyaluronate, and glycoprotein in nature. Most of the hyaluronate-binding activity from the soluble fraction chromatographed in the void volume of Sepharose CL-4B and CL-6B. Approximately 50% of this activity was highly negatively charged, eluting from diethylaminoethyl (DEAE)-cellulose in 0.5 M NaCl, and contained chondroitin sulfate chains. This latter material also reacted with antibodies raised against cartilage link protein and the core protein of cartilage proteoglycan. Thus, the binding and physical characteristics of this hyaluronate-binding activity are consistent with those of a chondroitin sulfate proteoglycan aggregate similar to that found in cartilage. A 500-fold purification of this proteoglycan-like, hyaluronate-binding material was achieved by wheat germ agglutinin affinity chromatography, molecular sieve chromatography on Sepharose CL-6B, and ion exchange chromatography on DEAE-cellulose. Another class of hyaluronate-binding material (25-50% of that recovered) eluted from DEAE with 0.24 M NaCl; this material had the properties of a complex glycoprotein, did not contain chondroitin sulfate, and did not react with the antibodies against cartilage link protein and proteoglycan. Thus, adult mouse brain contains at least three different forms of hyaluronate-binding macromolecules. Two of these have properties similar to the link protein and proteoglycan of cartilage proteoglycan aggregates; the third is distinguishable from these entities.

Selective activators of protein phosphatase 5 target the auto-inhibitory mechanism.

PubMed

Haslbeck, Veronika; Drazic, Adrian; Eckl, Julia M; Alte, Ferdinand; Helmuth, Martin; Popowicz, Grzegorz; Schmidt, Werner; Braun, Frank; Weiwad, Matthias; Fischer, Gunter; Gemmecker, Gerd; Sattler, Michael; Striggow, Frank; Groll, Michael; Richter, Klaus

2015-04-20

Protein phosphatase 5 (PP5) is an evolutionary conserved serine/threonine phosphatase. Its dephosphorylation activity modulates a diverse set of cellular factors including protein kinases and the microtubule-associated tau protein involved in neurodegenerative disorders. It is auto-regulated by its heat-shock protein (Hsp90)-interacting tetratricopeptide repeat (TPR) domain and its C-terminal α-helix. In the present study, we report the identification of five specific PP5 activators [PP5 small-molecule activators (P5SAs)] that enhance the phosphatase activity up to 8-fold. The compounds are allosteric modulators accelerating efficiently the turnover rate of PP5, but do barely affect substrate binding or the interaction between PP5 and the chaperone Hsp90. Enzymatic studies imply that the compounds bind to the phosphatase domain of PP5. For the most promising compound crystallographic comparisons of the apo PP5 and the PP5-P5SA-2 complex indicate a relaxation of the auto-inhibited state of PP5. Residual electron density and mutation analyses in PP5 suggest activator binding to a pocket in the phosphatase/TPR domain interface, which may exert regulatory functions. These compounds thus may expose regulatory mechanisms in the PP5 enzyme and serve to develop optimized activators based on these scaffolds. © 2015 Authors.

Computational design of a pH-sensitive IgG binding protein.

PubMed

Strauch, Eva-Maria; Fleishman, Sarel J; Baker, David

2014-01-14

Computational design provides the opportunity to program protein-protein interactions for desired applications. We used de novo protein interface design to generate a pH-dependent Fc domain binding protein that buries immunoglobulin G (IgG) His-433. Using next-generation sequencing of naïve and selected pools of a library of design variants, we generated a molecular footprint of the designed binding surface, confirming the binding mode and guiding further optimization of the balance between affinity and pH sensitivity. In biolayer interferometry experiments, the optimized design binds IgG with a Kd of ∼ 4 nM at pH 8.2, and approximately 500-fold more weakly at pH 5.5. The protein is extremely stable, heat-resistant and highly expressed in bacteria, and allows pH-based control of binding for IgG affinity purification and diagnostic devices.

In Situ Protein Binding Assay Using Fc-Fusion Proteins.

PubMed

Padmanabhan, Nirmala; Siddiqui, Tabrez J

2017-01-01

This protocol describes an in situ protein-protein interaction assay between tagged recombinant proteins and cell-surface expressed synaptic proteins. The assay is arguably more sensitive than other traditional protein binding assays such as co-immunoprecipitation and pull-downs and provides a visual readout for binding. This assay has been widely used to determine the dissociation constant of binding of trans-synaptic adhesion proteins. The step-wise description in the protocol should facilitate the adoption of this method in other laboratories.

A calmodulin binding protein from Arabidopsis is induced by ethylene and contains a DNA-binding motif

NASA Technical Reports Server (NTRS)

Reddy, A. S.; Reddy, V. S.; Golovkin, M.

2000-01-01

Calmodulin (CaM), a key calcium sensor in all eukaryotes, regulates diverse cellular processes by interacting with other proteins. To isolate CaM binding proteins involved in ethylene signal transduction, we screened an expression library prepared from ethylene-treated Arabidopsis seedlings with 35S-labeled CaM. A cDNA clone, EICBP (Ethylene-Induced CaM Binding Protein), encoding a protein that interacts with activated CaM was isolated in this screening. The CaM binding domain in EICBP was mapped to the C-terminus of the protein. These results indicate that calcium, through CaM, could regulate the activity of EICBP. The EICBP is expressed in different tissues and its expression in seedlings is induced by ethylene. The EICBP contains, in addition to a CaM binding domain, several features that are typical of transcription factors. These include a DNA-binding domain at the N terminus, an acidic region at the C terminus, and nuclear localization signals. In database searches a partial cDNA (CG-1) encoding a DNA-binding motif from parsley and an ethylene up-regulated partial cDNA from tomato (ER66) showed significant similarity to EICBP. In addition, five hypothetical proteins in the Arabidopsis genome also showed a very high sequence similarity with EICBP, indicating that there are several EICBP-related proteins in Arabidopsis. The structural features of EICBP are conserved in all EICBP-related proteins in Arabidopsis, suggesting that they may constitute a new family of DNA binding proteins and are likely to be involved in modulating gene expression in the presence of ethylene.

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

PubMed

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.

Mechanistic events underlying odorant binding protein chemoreception.

PubMed

Golebiowski, Jérôme; Antonczak, Serge; Fiorucci, Sébastien; Cabrol-Bass, Daniel

2007-05-01

Odorant binding proteins (OBP's) are small hydrophilic proteins, belonging to the lipocalin family dedicated to bind and transport small hydrophobic ligands. Despite many works, the mechanism of ligand binding, together with the functional role of these proteins remains a topic of debate and little is known at the atomic level. The present work reports a computational study of odorants capture and release by an OBP, using both constrained and unconstrained simulations, giving a glimpse on the molecular mechanism of chemoreception. The residues at the origin of the regulation of the protein door opening are identified and a tyrosine amino-acid together with other nearby residues appear to play a crucial role in allowing this event to occur. The simulations reveal that this tyrosine and the protein's L5 loop are implicated in the ligand contact with the protein and act as an anchoring point for the ligand. The protein structural features required for the ligand entry are highly conserved among many transport proteins, suggesting that this mechanism could somewhat be extended to some members of the larger family of lipocalin. (c) 2007 Wiley-Liss, Inc.

DNA-repair protein hHR23a alters its protein structure upon binding proteasomal subunit S5a

PubMed Central

Walters, Kylie J.; Lech, Patrycja J.; Goh, Amanda M.; Wang, Qinghua; Howley, Peter M.

2003-01-01

The Rad23 family of proteins, including the human homologs hHR23a and hHR23b, stimulates nucleotide excision repair and has been shown to provide a novel link between proteasome-mediated protein degradation and DNA repair. In this work, we illustrate how the proteasomal subunit S5a regulates hHR23a protein structure. By using NMR spectroscopy, we have elucidated the structure and dynamic properties of the 40-kDa hHR23a protein and show it to contain four structured domains connected by flexible linker regions. In addition, we reveal that these domains interact in an intramolecular fashion, and by using residual dipolar coupling data in combination with chemical shift perturbation analysis, we present the hHR23a structure. By itself, hHR23a adopts a closed conformation defined by the interaction of an N-terminal ubiquitin-like domain with two ubiquitin-associated domains. Interestingly, binding of the proteasomal subunit S5a disrupts the hHR23a interdomain interactions and thereby causes it to adopt an opened conformation. PMID:14557549

The structure of myristoylated Mason-Pfizer monkey virus matrix protein and the role of phosphatidylinositol-(4,5)-bisphosphate in its membrane binding.

PubMed

Prchal, Jan; Srb, Pavel; Hunter, Eric; Ruml, Tomáš; Hrabal, Richard

2012-10-26

We determined the solution structure of myristoylated Mason-Pfizer monkey virus matrix protein by NMR spectroscopy. The myristoyl group is buried inside the protein and causes a slight reorientation of the helices. This reorientation leads to the creation of a binding site for phosphatidylinositols. The interaction between the matrix protein and phosphatidylinositols carrying C(8) fatty acid chains was monitored by observation of concentration-dependent chemical shift changes of the affected amino acid residues, a saturation transfer difference experiment and changes in (31)P chemical shifts. No differences in the binding mode or affinity were observed with differently phosphorylated phosphatidylinositols. The structure of the matrix protein-phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P(2)] complex was then calculated with HADDOCK software based on the intermolecular nuclear Overhauser enhancement contacts between the ligand and the matrix protein obtained from a (13)C-filtered/(13)C-edited nuclear Overhauser enhancement spectroscopy experiment. PI(4,5)P(2) binding was not strong enough for triggering of the myristoyl-switch. The structural changes of the myristoylated matrix protein were also found to result in a drop in the oligomerization capacity of the protein. Copyright © 2012. Published by Elsevier Ltd.

RNA binding properties of the US11 protein from four primate simplexviruses.

PubMed

Tohme, Sarah; Cukier, Cyprian D; Severini, Alberto

2011-11-03

The protein encoded by the Us11 gene of herpes simplex viruses is a dsRNA binding protein which inhibits protein kinase R activity, thereby preventing the interferon-induced shut down of protein synthesis following viral infection. Us11 protein is not essential for infectivity in vitro and in mice in herpes simplex virus type 1 (HSV1), however this virus has a second, and apparently more important, inhibitor of PKR activity, the γ134.5 protein. Recently sequenced simian simplexviruses SA8, HVP2 and B virus do not have an ORF corresponding to the γ134.5 protein, yet they have similar, or greater, infectivity as HSV1 and HSV2. We have expressed the US11 proteins of the simplexviruses HSV1, HSV2, HVP2 and B virus and measured their abilities to bind dsRNA, in order to investigate possible differences that could complement the absence of the γ134.5 protein. We employed a filter binding technique that allows binding of the Us11 protein under condition of excess dsRNA substrate and therefore a measurement of the true Kd value of Us11-dsRNA binding. The results show a Kd of binding in the range of 0.89 nM to 1.82 nM, with no significant difference among the four Us11 proteins.

RNA binding properties of the US11 protein from four primate simplexviruses

PubMed Central

2011-01-01

Background The protein encoded by the Us11 gene of herpes simplex viruses is a dsRNA binding protein which inhibits protein kinase R activity, thereby preventing the interferon-induced shut down of protein synthesis following viral infection. Us11 protein is not essential for infectivity in vitro and in mice in herpes simplex virus type 1 (HSV1), however this virus has a second, and apparently more important, inhibitor of PKR activity, the γ134.5 protein. Recently sequenced simian simplexviruses SA8, HVP2 and B virus do not have an ORF corresponding to the γ134.5 protein, yet they have similar, or greater, infectivity as HSV1 and HSV2. Methods We have expressed the US11 proteins of the simplexviruses HSV1, HSV2, HVP2 and B virus and measured their abilities to bind dsRNA, in order to investigate possible differences that could complement the absence of the γ134.5 protein. We employed a filter binding technique that allows binding of the Us11 protein under condition of excess dsRNA substrate and therefore a measurement of the true Kd value of Us11-dsRNA binding. Results and Conclusions The results show a Kd of binding in the range of 0.89 nM to 1.82 nM, with no significant difference among the four Us11 proteins. PMID:22054255

Electrostatic Forces as Dominant Interactions Between Proteins and Polyanions: an ESI MS Study of Fibroblast Growth Factor Binding to Heparin Oligomers

NASA Astrophysics Data System (ADS)

Minsky, Burcu Baykal; Dubin, Paul L.; Kaltashov, Igor A.

2017-04-01

The interactions between fibroblast growth factors (FGFs) and their receptors (FGFRs) are facilitated by heparan sulfate (HS) and heparin (Hp), highly sulfated biological polyelectrolytes. The molecular basis of FGF interactions with these polyelectrolytes is highly complex due to the structural heterogeneity of HS/Hp, and many details still remain elusive, especially the significance of charge density and minimal chain length of HS/Hp in growth factor recognition and multimerization. In this work, we use electrospray ionization mass spectrometry (ESI MS) to investigate the association of relatively homogeneous oligoheparins (octamer, dp8, and decamer, dp10) with acidic fibroblast growth factor (FGF-1). This growth factor forms 1:1, 2:1, and 3:1 protein/heparinoid complexes with both dp8 and dp10, and the fraction of bound protein is highly dependent on protein/heparinoid molar ratio. Multimeric complexes are preferentially formed on the highly sulfated Hp oligomers. Although a variety of oligomers appear to be binding-competent, there is a strong correlation between the affinity and the overall level of sulfation (the highest charge density polyanions binding FGF most strongly via multivalent interactions). These results show that the interactions between FGF-1 and Hp oligomers are primarily directed by electrostatics, and also demonstrate the power of ESI MS as a tool to study multiple binding equilibria between proteins and structurally heterogeneous polyanions.

Structural Basis for Ligand Regulation of the Fatty Acid-binding Protein 5, Peroxisome Proliferator-activated Receptor β/δ (FABP5-PPARβ/δ) Signaling Pathway*

PubMed Central

Armstrong, Eric H.; Goswami, Devrishi; Griffin, Patrick R.; Noy, Noa; Ortlund, Eric A.

2014-01-01

Fatty acid-binding proteins (FABPs) are a widely expressed group of calycins that play a well established role in solubilizing cellular fatty acids. Recent studies, however, have recast FABPs as active participants in vital lipid-signaling pathways. FABP5, like its family members, displays a promiscuous ligand binding profile, capable of interacting with numerous long chain fatty acids of varying degrees of saturation. Certain “activating” fatty acids induce the protein's cytoplasmic to nuclear translocation, stimulating PPARβ/δ transactivation; however, the rules that govern this process remain unknown. Using a range of structural and biochemical techniques, we show that both linoleic and arachidonic acid elicit FABP5's translocation by permitting allosteric communication between the ligand-sensing β2 loop and a tertiary nuclear localization signal within the α-helical cap of the protein. Furthermore, we show that more saturated, nonactivating fatty acids inhibit nuclear localization signal formation by destabilizing this activation loop, thus implicating FABP5 specifically in cis-bonded, polyunsaturated fatty acid signaling. PMID:24692551

Human Lineage-Specific Transcriptional Regulation through GA-Binding Protein Transcription Factor Alpha (GABPa)

PubMed Central

Perdomo-Sabogal, Alvaro; Nowick, Katja; Piccini, Ilaria; Sudbrak, Ralf; Lehrach, Hans; Yaspo, Marie-Laure; Warnatz, Hans-Jörg; Querfurth, Robert

2016-01-01

A substantial fraction of phenotypic differences between closely related species are likely caused by differences in gene regulation. While this has already been postulated over 30 years ago, only few examples of evolutionary changes in gene regulation have been verified. Here, we identified and investigated binding sites of the transcription factor GA-binding protein alpha (GABPa) aiming to discover cis-regulatory adaptations on the human lineage. By performing chromatin immunoprecipitation-sequencing experiments in a human cell line, we found 11,619 putative GABPa binding sites. Through sequence comparisons of the human GABPa binding regions with orthologous sequences from 34 mammals, we identified substitutions that have resulted in 224 putative human-specific GABPa binding sites. To experimentally assess the transcriptional impact of those substitutions, we selected four promoters for promoter-reporter gene assays using human and African green monkey cells. We compared the activities of wild-type promoters to mutated forms, where we have introduced one or more substitutions to mimic the ancestral state devoid of the GABPa consensus binding sequence. Similarly, we introduced the human-specific substitutions into chimpanzee and macaque promoter backgrounds. Our results demonstrate that the identified substitutions are functional, both in human and nonhuman promoters. In addition, we performed GABPa knock-down experiments and found 1,215 genes as strong candidates for primary targets. Further analyses of our data sets link GABPa to cognitive disorders, diabetes, KRAB zinc finger (KRAB-ZNF), and human-specific genes. Thus, we propose that differences in GABPa binding sites played important roles in the evolution of human-specific phenotypes. PMID:26814189

Binding Rate Constants Reveal Distinct Features of Disordered Protein Domains.

PubMed

Dogan, Jakob; Jonasson, Josefin; Andersson, Eva; Jemth, Per

2015-08-04

Intrinsically disordered proteins (IDPs) are abundant in the proteome and involved in key cellular functions. However, experimental data about the binding kinetics of IDPs as a function of different environmental conditions are scarce. We have performed an extensive characterization of the ionic strength dependence of the interaction between the molten globular nuclear co-activator binding domain (NCBD) of CREB binding protein and five different protein ligands, including the intrinsically disordered activation domain of p160 transcriptional co-activators (SRC1, TIF2, ACTR), the p53 transactivation domain, and the folded pointed domain (PNT) of transcription factor ETS-2. Direct comparisons of the binding rate constants under identical conditions show that the association rate constant, kon, for interactions between NCBD and disordered protein domains is high at low salt concentrations (90-350 × 10(6) M(-1) s(-1) at 4 °C) but is reduced significantly (10-30-fold) with an increasing ionic strength and reaches a plateau around physiological ionic strength. In contrast, the kon for the interaction between NCBD and the folded PNT domain is only 7 × 10(6) M(-1) s(-1) (4 °C and low salt) and displays weak ionic strength dependence, which could reflect a distinctly different association that relies less on electrostatic interactions. Furthermore, the basal rate constant (in the absence of electrostatic interactions) is high for the NCBD interactions, exceeding those typically observed for folded proteins. One likely interpretation is that disordered proteins have a large number of possible collisions leading to a productive on-pathway encounter complex, while folded proteins are more restricted in terms of orientation. Our results highlight the importance of electrostatic interactions in binding involving IDPs and emphasize the significance of including ionic strength as a factor in studies that compare the binding properties of IDPs to those of ordered proteins.

Involvement of the Eukaryote-Like Kinase-Phosphatase System and a Protein That Interacts with Penicillin-Binding Protein 5 in Emergence of Cephalosporin Resistance in Cephalosporin-Sensitive Class A Penicillin-Binding Protein Mutants in Enterococcus faecium.

PubMed

Desbonnet, Charlene; Tait-Kamradt, Amelia; Garcia-Solache, Monica; Dunman, Paul; Coleman, Jeffrey; Arthur, Michel; Rice, Louis B

2016-04-05

The intrinsic resistance of Enterococcus faecium to ceftriaxone and cefepime (here referred to as "cephalosporins") is reliant on the presence of class A penicillin-binding proteins (Pbps) PbpF and PonA. Mutants lacking these Pbps exhibit cephalosporin susceptibility that is reversible by exposure to penicillin and by selection on cephalosporin-containing medium. We selected two cephalosporin-resistant mutants (Cro1 and Cro2) of class A Pbp-deficient E. faecium CV598. Genome analysis revealed changes in the serine-threonine kinase Stk in Cro1 and a truncation in the associated phosphatase StpA in Cro2 whose respective involvements in resistance were confirmed in separate complementation experiments. In an additional effort to identify proteins linked to cephalosporin resistance, we performed tandem affinity purification using Pbp5 as bait in penicillin-exposed E. faecium; these experiments yielded a protein designated Pbp5-associated protein (P5AP). Transcription of the P5AP gene was increased after exposure to penicillin in wild-type strains and in Cro2 and suppressed in Cro2 complemented with the wild-type stpA Transformation of class A Pbp-deficient strains with the plasmid-carried P5AP gene conferred cephalosporin resistance. These data suggest that Pbp5-associated cephalosporin resistance in E. faecium devoid of typical class A Pbps is related to the presence of P5AP, whose expression is influenced by the activity of the serine-threonine phosphatase/kinase system. β-Lactam antibiotics remain our most effective therapies against susceptible Gram-positive bacteria. The intrinsic resistance of Enterococcus faecium to β-lactams, particularly to cephalosporins, therefore represents a major limitation of therapy. Although the primary mechanism of resistance to β-lactams in E. faecium is the presence of low-affinity monofunctional transpeptidase (class B) penicillin-binding protein Pbp5, the interaction of Pbp5 with other proteins is fundamental to maintain a

Spatial Analysis and Quantification of the Thermodynamic Driving Forces in Protein-Ligand Binding: Binding Site Variability

PubMed Central

Raman, E. Prabhu; MacKerell, Alexander D.

2015-01-01

The thermodynamic driving forces behind small molecule-protein binding are still not well understood, including the variability of those forces associated with different types of ligands in different binding pockets. To better understand these phenomena we calculate spatially resolved thermodynamic contributions of the different molecular degrees of freedom for the binding of propane and methanol to multiple pockets on the proteins Factor Xa and p38 MAP kinase. Binding thermodynamics are computed using a statistical thermodynamics based end-point method applied on a canonical ensemble comprising the protein-ligand complexes and the corresponding free states in an explicit solvent environment. Energetic and entropic contributions of water and ligand degrees of freedom computed from the configurational ensemble provides an unprecedented level of detail into the mechanisms of binding. Direct protein-ligand interaction energies play a significant role in both non-polar and polar binding, which is comparable to water reorganization energy. Loss of interactions with water upon binding strongly compensates these contributions leading to relatively small binding enthalpies. For both solutes, the entropy of water reorganization is found to favor binding in agreement with the classical view of the “hydrophobic effect”. Depending on the specifics of the binding pocket, both energy-entropy compensation and reinforcement mechanisms are observed. Notable is the ability to visualize the spatial distribution of the thermodynamic contributions to binding at atomic resolution showing significant differences in the thermodynamic contributions of water to the binding of propane versus methanol. PMID:25625202

Fatty acid-binding protein 5 limits the anti-inflammatory response in murine macrophages.

PubMed

Moore, Sherri M; Holt, Vivian V; Malpass, Lillie R; Hines, Ian N; Wheeler, Michael D

2015-10-01

The beginning stages of liver damage induced by various etiologies (i.e. high fat diet, alcohol consumption, toxin exposure) are characterized by abnormal accumulation of lipid in liver. Alterations in intracellular lipid transport, storage, and metabolism accompanied by cellular insult within the liver play an important role in the pathogenesis of liver disease, often involving a sustained inflammatory response. The intracellular lipid transporter, fatty acid binding protein 5 (FABP5), is highly expressed in macrophages and may play an important role in the hepatic inflammatory response after endotoxin exposure in mice. This study tested the hypothesis that FABP5 regulates macrophage response to LPS in male C57bl/6 (wild type) and FABP5 knockout mice, both in vitro and in vivo. Treatment with LPS revealed that loss of FABP5 enhances the number of hepatic F4/80(+) macrophages in the liver despite limited liver injury. Conversely, FABP5 knock out mice display higher mRNA levels of anti-inflammatory cytokines IL-10, arginase, YM-1, and Fizz-1 in liver compared to wild type mice. Bone marrow derived macrophages stimulated with inflammatory (LPS and IFN-γ) or anti-inflammatory (IL-4) mediators also showed significantly higher expression of anti-inflammatory/regulatory factors. These findings reveal a regulatory role of FABP5 in the acute inflammatory response to LPS-induced liver injury, which is consistent with the principle finding that FABP5 is a regulator of macrophage phenotype. Specifically, these findings demonstrate that loss of FABP5 promotes a more anti-inflammatory response. Copyright © 2015 Elsevier Ltd. All rights reserved.

Trans‐acting translational regulatory RNA binding proteins

PubMed Central

Harvey, Robert F.; Smith, Tom S.; Mulroney, Thomas; Queiroz, Rayner M. L.; Pizzinga, Mariavittoria; Dezi, Veronica; Villenueva, Eneko; Ramakrishna, Manasa

2018-01-01

The canonical molecular machinery required for global mRNA translation and its control has been well defined, with distinct sets of proteins involved in the processes of translation initiation, elongation and termination. Additionally, noncanonical, trans‐acting regulatory RNA‐binding proteins (RBPs) are necessary to provide mRNA‐specific translation, and these interact with 5′ and 3′ untranslated regions and coding regions of mRNA to regulate ribosome recruitment and transit. Recently it has also been demonstrated that trans‐acting ribosomal proteins direct the translation of specific mRNAs. Importantly, it has been shown that subsets of RBPs often work in concert, forming distinct regulatory complexes upon different cellular perturbation, creating an RBP combinatorial code, which through the translation of specific subsets of mRNAs, dictate cell fate. With the development of new methodologies, a plethora of novel RNA binding proteins have recently been identified, although the function of many of these proteins within mRNA translation is unknown. In this review we will discuss these methodologies and their shortcomings when applied to the study of translation, which need to be addressed to enable a better understanding of trans‐acting translational regulatory proteins. Moreover, we discuss the protein domains that are responsible for RNA binding as well as the RNA motifs to which they bind, and the role of trans‐acting ribosomal proteins in directing the translation of specific mRNAs. This article is categorized under: 1RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes2Translation > Translation Regulation3Translation > Translation Mechanisms PMID:29341429

Mutant forms of Escherichia coli protein L25 unable to bind to 5S rRNA are incorporated efficiently into the ribosome in vivo.

PubMed

Anikaev, A Y; Korepanov, A P; Korobeinikova, A V; Kljashtorny, V G; Piendl, W; Nikonov, S V; Garber, M B; Gongadze, G M

2014-08-01

5S rRNA-binding ribosomal proteins of the L25 family are an evolutional acquisition of bacteria. Earlier we showed that (i) single replacements in the RNA-binding module of the protein of this family result in destabilization or complete impossibility to form a complex with 5S rRNA in vitro; (ii) ΔL25 ribosomes of Escherichia coli are less efficient in protein synthesis in vivo than the control ribosomes. In the present work, the efficiency of incorporation of the E. coli protein L25 with mutations in the 5S rRNA-binding region into the ribosome in vivo was studied. It was found that the mutations in L25 that abolish its ability to form the complex with free 5S rRNA do not prevent its correct and efficient incorporation into the ribosome. This is supported by the fact that even the presence of a very weakly retained mutant form of the protein in the ribosome has a positive effect on the activity of the translational machinery in vivo. All this suggests the existence of an alternative incorporation pathway for this protein into the ribosome, excluding the preliminary formation of the complex with 5S rRNA. At the same time, the stable L25-5S rRNA contact is important for the retention of the protein within the ribosome, and the conservative amino acid residues of the RNA-binding module play a key role in this.

Deficiency of a Retinal Dystrophy Protein, Acyl-CoA Binding Domain-containing 5 (ACBD5), Impairs Peroxisomal β-Oxidation of Very-long-chain Fatty Acids*

PubMed Central

Yagita, Yuichi; Shinohara, Kyoko; Abe, Yuichi; Nakagawa, Keiko; Al-Owain, Mohammed; Alkuraya, Fowzan S.; Fujiki, Yukio

2017-01-01

Acyl-CoA binding domain-containing 5 (ACBD5) is a peroxisomal protein that carries an acyl-CoA binding domain (ACBD) at its N-terminal region. The recent identification of a mutation in the ACBD5 gene in patients with a syndromic form of retinal dystrophy highlights the physiological importance of ACBD5 in humans. However, the underlying pathogenic mechanisms and the precise function of ACBD5 remain unclear. We herein report that ACBD5 is a peroxisomal tail-anchored membrane protein exposing its ACBD to the cytosol. Using patient-derived fibroblasts and ACBD5 knock-out HeLa cells generated via genome editing, we demonstrate that ACBD5 deficiency causes a moderate but significant defect in peroxisomal β-oxidation of very-long-chain fatty acids (VLCFAs) and elevates the level of cellular phospholipids containing VLCFAs without affecting peroxisome biogenesis, including the import of membrane and matrix proteins. Both the N-terminal ACBD and peroxisomal localization of ACBD5 are prerequisite for efficient VLCFA β-oxidation in peroxisomes. Furthermore, ACBD5 preferentially binds very-long-chain fatty acyl-CoAs (VLC-CoAs). Together, these results suggest a direct role of ACBD5 in peroxisomal VLCFA β-oxidation. Based on our findings, we propose that ACBD5 captures VLC-CoAs on the cytosolic side of the peroxisomal membrane so that the transport of VLC-CoAs into peroxisomes and subsequent β-oxidation thereof can proceed efficiently. Our study reclassifies ACBD5-related phenotype as a novel peroxisomal disorder. PMID:27899449

Acanthamoeba castellanii contains a ribosomal RNA enhancer binding protein which stimulates TIF-IB binding and transcription under stringent conditions.

PubMed

Yang, Q; Radebaugh, C A; Kubaska, W; Geiss, G K; Paule, M R

1995-11-11

The intergenic spacer (IGS) of Acanthamoeba castellanii rRNA genes contains repeated elements which are weak enhancers for transcription by RNA polymerase I. A protein, EBF, was identified and partially purified which binds to the enhancers and to several other sequences within the IGS, but not to other DNA fragments, including the rRNA core promoter. No consensus binding sequence could be discerned in these fragments and bound factor is in rapid equilibrium with unbound. EBF has functional characteristics similar to vertebrate upstream binding factors (UBF). Not only does it bind to the enhancer and other IGS elements, but it also stimulates binding of TIF-IB, the fundamental transcription initiation factor, to the core promoter and stimulates transcription from the promoter. Attempts to identify polypeptides with epitopes similar to rat or Xenopus laevis UBF suggest that structurally the protein from A.castellanii is not closely related to vertebrate UBF.

A Crayfish Insulin-like-binding Protein

PubMed Central

Rosen, Ohad; Weil, Simy; Manor, Rivka; Roth, Ziv; Khalaila, Isam; Sagi, Amir

2013-01-01

Across the animal kingdom, the involvement of insulin-like peptide (ILP) signaling in sex-related differentiation processes is attracting increasing attention. Recently, a gender-specific ILP was identified as the androgenic sex hormone in Crustacea. However, moieties modulating the actions of this androgenic insulin-like growth factor were yet to be revealed. Through molecular screening of an androgenic gland (AG) cDNA library prepared from the crayfish Cherax quadricarinatus, we have identified a novel insulin-like growth factor-binding protein (IGFBP) termed Cq-IGFBP. Based on bioinformatics analyses, the deduced Cq-IGFBP was shown to share high sequence homology with IGFBP family members from both invertebrates and vertebrates. The protein also includes a sequence determinant proven crucial for ligand binding, which according to three-dimensional modeling is assigned to the exposed outer surface of the protein. Recombinant Cq-IGFBP (rCq-IGFBP) protein was produced and, using a “pulldown” methodology, was shown to specifically interact with the insulin-like AG hormone of the crayfish (Cq-IAG). Particularly, using both mass spectral analysis and an immunological tool, rCq-IGFBP was shown to bind the Cq-IAG prohormone. Furthermore, a peptide corresponding to residues 23–38 of the Cq-IAG A-chain was found sufficient for in vitro recognition by rCq-IGFBP. Cq-IGFBP is the first IGFBP family member shown to specifically interact with a gender-specific ILP. Unlike their ILP ligands, IGFBPs are highly conserved across evolution, from ancient arthropods, like crustaceans, to humans. Such conservation places ILP signaling at the center of sex-related phenomena in early animal development. PMID:23775079

A Plasmodium falciparum PHIST protein binds the virulence factor PfEMP1 and comigrates to knobs on the host cell surface

PubMed Central

Oberli, Alexander; Slater, Leanne M.; Cutts, Erin; Brand, Françoise; Mundwiler-Pachlatko, Esther; Rusch, Sebastian; Masik, Martin F. G.; Erat, Michèle C.; Beck, Hans-Peter; Vakonakis, Ioannis

2014-01-01

Uniquely among malaria parasites, Plasmodium falciparum-infected erythrocytes (iRBCs) develop membrane protrusions, known as knobs, where the parasite adhesion receptor P. falciparum erythrocyte membrane protein 1 (PfEMP1) clusters. Knob formation and the associated iRBC adherence to host endothelium are directly linked to the severity of malaria and are functional manifestations of protein export from the parasite to the iRBC. A family of exported proteins featuring Plasmodium helical interspersed subtelomeric (PHIST) domains has attracted attention, with members being implicated in host-parasite protein interactions and differentially regulated in severe disease and among parasite isolates. Here, we show that PHIST member PFE1605w binds the PfEMP1 intracellular segment directly with Kd = 5 ± 0.6 μM, comigrates with PfEMP1 during export, and locates in knobs. PHIST variants that do not locate in knobs (MAL8P1.4) or bind PfEMP1 30 times more weakly (PFI1780w) used as controls did not display the same pattern. We resolved the first crystallographic structure of a PHIST protein and derived a partial model of the PHIST-PfEMP1 interaction from nuclear magnetic resonance. We propose that PFE1605w reinforces the PfEMP1-cytoskeletal connection in knobs and discuss the possible role of PHIST proteins as interaction hubs in the parasite exportome.—Oberli, A., Slater, L. M., Cutts, E., Brand, F., Mundwiler-Pachlatko, E., Rusch, S., Masik, M. F. G., Erat, M. C., Beck, H.-P., Vakonakis, I. A Plasmodium falciparum PHIST protein binds the virulence factor PfEMP1 and comigrates to knobs on the host cell surface. PMID:24983468

Identification and characterization of novel IGFBP5 interacting protein: evidence IGFBP5-IP is a potential regulator of osteoblast cell proliferation

PubMed Central

Amaar, Yousef G.; Tapia, Blanca; Chen, Shin-Tai; Baylink, David J.; Mohan, Subburaman

2010-01-01

Insulin-like growth factor binding protein-5 (IGFBP5) is a multifunctional protein, which acts not only as a traditional binding protein, but also functions as a growth factor independent of IGFs to stimulate bone formation. It has been predicted that the intrinsic growth factor action of IGFBP5 involves binding of IGFBP5 to a putative receptor to induce downstream signaling pathways and/or nuclear translocation of IGFBP5 to influence transcription of genes involved in osteoblast cell proliferation/differentiation. Our study indentified proteins that bound to IGFBP5 using IGFBP5 as bait in a yeast two-hybrid screen of the U2 human osteosarcoma cell cDNA library. One of the clones that interacted strongly with the bait under high-stringency conditions corresponded to a novel IGFBP5 interacting protein (IGFBP5-IP) encoded by a gene that resides in mouse chromosome 10. The interaction between IGFBP5-IP and IGFBP5 is confirmed by in vitro coimmunoprecipitation studies that used pFlag and IGFBP5 polyclonal antibody, and cell lysates overexpressing both IGFBP5-IP and IGFBP5. Northern blot and RT-PCR analysis showed that the IGFBP-IP is expressed in both untransformed normal human osteoblasts and in osteosarcoma cell lines, which are known to produce IGFBP5. To determine the roles of IGFBP5-IP, we evaluated the effect of blocking the expression of IGFBP5-IP on osteoblast proliferation. We found that using a IGFBP5-IP-specific small interfering-hairpin plasmid resulted in a decrease in both basal and IGFBP5-induced osteoblast cell proliferation. On the basis of these findings, we predict that IGFBP5-IP may act as intracellular mediator of growth promoting actions of IGFBP5 and perhaps other osteoregulatory agents in bone cells. PMID:16269403

Calcium-binding proteins and development

NASA Technical Reports Server (NTRS)

Beckingham, K.; Lu, A. Q.; Andruss, B. F.; McIntire, L. V. (Principal Investigator)

1998-01-01

The known roles for calcium-binding proteins in developmental signaling pathways are reviewed. Current information on the calcium-binding characteristics of three classes of cell-surface developmental signaling proteins (EGF-domain proteins, cadherins and integrins) is presented together with an overview of the intracellular pathways downstream of these surface receptors. The developmental roles delineated to date for the universal intracellular calcium sensor, calmodulin, and its targets, and for calcium-binding regulators of the cytoskeleton are also reviewed.

Localized frustration and binding-induced conformational change in recognition of 5S RNA by TFIIIA zinc finger.

PubMed

Tan, Cheng; Li, Wenfei; Wang, Wei

2013-12-19

Protein TFIIIA is composed of nine tandemly arranged Cys2His2 zinc fingers. It can bind either to the 5S RNA gene as a transcription factor or to the 5S RNA transcript as a chaperone. Although structural and biochemical data provided valuable information on the recognition between the TFIIIIA and the 5S DNA/RNA, the involved conformational motions and energetic factors contributing to the binding affinity and specificity remain unclear. In this work, we conducted MD simulations and MM/GBSA calculations to investigate the binding-induced conformational changes in the recognition of the 5S RNA by the central three zinc fingers of TFIIIA and the energetic factors that influence the binding affinity and specificity at an atomistic level. Our results revealed drastic interdomain conformational changes between these three zinc fingers, involving the exposure/burial of several crucial DNA/RNA binding residues, which can be related to the competition between DNA and RNA for the binding of TFIIIA. We also showed that the specific recognition between finger 4/finger 6 and the 5S RNA introduces frustrations to the nonspecific interactions between finger 5 and the 5S RNA, which may be important to achieve optimal binding affinity and specificity.

Engineered proteins as specific binding reagents.

PubMed

Binz, H Kaspar; Plückthun, Andreas

2005-08-01

Over the past 30 years, monoclonal antibodies have become the standard binding proteins and currently find applications in research, diagnostics and therapy. Yet, monoclonal antibodies now face strong competition from synthetic antibody libraries in combination with powerful library selection technologies. More recently, an increased understanding of other natural binding proteins together with advances in protein engineering, selection and evolution technologies has also triggered the exploration of numerous other protein architectures for the generation of designed binding molecules. Valuable protein-binding scaffolds have been obtained and represent promising alternatives to antibodies for biotechnological and, potentially, clinical applications.

Protein Binding Capacity of Different Forages Tannin

NASA Astrophysics Data System (ADS)

Yusiati, L. M.; Kurniawati, A.; Hanim, C.; Anas, M. A.

2018-02-01

Eight forages of tannin sources(Leucaena leucocephala, Arachis hypogaea, Mimosa pudica, Morus alba L, Swietenia mahagoni, Manihot esculenta, Gliricidia sepium, and Bauhinia purpurea)were evaluated their tannin content and protein binding capacity. The protein binding capacity of tannin were determined using precipitation of bovine serum albumin (BSA). Swietenia mahagonihas higest total tannin level and condensed tannin (CT) compared with other forages (P<0.01). The Leucaena leucocephala has highest hydrolysable tannin (HT) level (P<0.01). The total and condensed tannin content of Swietenia mahagoni were 11.928±0.04 mg/100 mg and 9.241±0.02mg/100mg dry matter (DM) of leaves. The hydrolysable tannin content of Leucaena leucocephala was 5.338±0.03 mg/100 mg DM of leaves. Binding capacity was highest in Swietenia mahagoni and Leucaena leucocephala compared to the other forages (P<0.01). The optimum binding of BSA to tannin in Leucaena leucocephala and Swietenia mahagoniwere1.181±0.44 and 1.217±0.60mg/mg dry matter of leaves. The present study reports that Swietenia mahagoni has highest of tannin content and Leucaena leucocephala and Swietenia mahagoni capacity of protein binding.

Structure, Function, and Evolution of Biogenic Amine-binding Proteins in Soft Ticks

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mans, Ben J.; Ribeiro, Jose M.C.; Andersen, John F.

2008-08-19

Two highly abundant lipocalins, monomine and monotonin, have been isolated from the salivary gland of the soft tick Argas monolakensis and shown to bind histamine and 5-hydroxytryptamine (5-HT), respectively. The crystal structures of monomine and a paralog of monotonin were determined in the presence of ligands to compare the determinants of ligand binding. Both the structures and binding measurements indicate that the proteins have a single binding site rather than the two sites previously described for the female-specific histamine-binding protein (FS-HBP), the histamine-binding lipocalin of the tick Rhipicephalus appendiculatus. The binding sites of monomine and monotonin are similar to themore » lower, low affinity site of FS-HBP. The interaction of the protein with the aliphatic amine group of the ligand is very similar for the all of the proteins, whereas specificity is determined by interactions with the aromatic portion of the ligand. Interestingly, protein interaction with the imidazole ring of histamine differs significantly between the low affinity binding site of FS-HBP and monomine, suggesting that histamine binding has evolved independently in the two lineages. From the conserved features of these proteins, a tick lipocalin biogenic amine-binding motif could be derived that was used to predict biogenic amine-binding function in other tick lipocalins. Heterologous expression of genes from salivary gland libraries led to the discovery of biogenic amine-binding proteins in soft (Ornithodoros) and hard (Ixodes) tick genera. The data generated were used to reconstruct the most probable evolutionary pathway for the evolution of biogenic amine-binding in tick lipocalins.« less

High-mobility group (HMG) protein HMG-1 and TATA-binding protein-associated factor TAF(II)30 affect estrogen receptor-mediated transcriptional activation.

PubMed

Verrier, C S; Roodi, N; Yee, C J; Bailey, L R; Jensen, R A; Bustin, M; Parl, F F

1997-07-01

The estrogen receptor (ER) belongs to a family of ligand-inducible nuclear receptors that exert their effects by binding to cis-acting DNA elements in the regulatory region of target genes. The detailed mechanisms by which ER interacts with the estrogen response element (ERE) and affects transcription still remain to be elucidated. To study the ER-ERE interaction and transcription initiation, we employed purified recombinant ER expressed in both the baculovirus-Sf9 and his-tagged bacterial systems. The effect of high-mobility group (HMG) protein HMG-1 and purified recombinant TATA-binding protein-associated factor TAF(II)30 on ER-ERE binding and transcription initiation were assessed by electrophoretic mobility shift assay and in vitro transcription from an ERE-containing template (pERE2LovTATA), respectively. We find that purified, recombinant ER fails to bind to ERE in spite of high ligand-binding activity and electrophoretic and immunological properties identical to ER in MCF-7 breast cancer cells. HMG-1 interacts with ER and promotes ER-ERE binding in a concentration- and time-dependent manner. The effectiveness of HMG-1 to stimulate ER-ERE binding in the electrophoretic mobility shift assay depends on the sequence flanking the ERE consensus as well as the position of the latter in the oligonucleotide. We find that TAF(II)30 has no effect on ER-ERE binding either alone or in combination with ER and HMG-1. Although HMG-1 promotes ER-ERE binding, it fails to stimulate transcription initiation either in the presence or absence of hormone. In contrast, TAF(II)30, while not affecting ER-ERE binding, stimulates transcription initiation 20-fold in the presence of HMG-1. These results indicate that HMG-1 and TAF(II)30 act in sequence, the former acting to promote ER-ERE binding followed by the latter to stimulate transcription initiation.

Erythropoietin binding protein from mammalian serum

DOEpatents

Clemons, Gisela K.

1997-01-01

Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described.

Production of insulin-like growth factor binding proteins by small-cell lung cancer cell lines

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jaques, G.; Kiefer, P.; Rotsch, M.

1989-10-01

Conditioned serum-free media (CM) from small-cell lung cancer (SCLC) cell lines were examined for the presence of insulin-like growth-factor-binding proteins (IGF-BP). 6/9 SCLC cell lines secreted binding proteins with high affinity for IGFs. When ({sup 125}I)IGF-1 or ({sup 125}I)IGF-II was incubated with the CMs, complexes of tracer with proteins could be demonstrated by gel filtration, by precipitation with polyethylenglycol, and after adsorption of unbound tracer with activated charcoal. Analysis of binding data according to the method of Scatchard resulted in linear plots for IGF-I and IGF-II. Cross-linking of ({sup 125}I)IGF-I or ({sup 125}I)IGF-II to the CMs followed by sodium dodecylmore » sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions revealed the presence of IGF-BPs with molecular masses in the range of 24-32 kDa. Northern blot hybridization with an IGF-BP cDNA probe encoding a low-molecular-weight IGF-BP from a human placenta cDNA library and Western blot analysis with a corresponding polyclonal antibody showed no expression of this gene. These data demonstrate that SCLC cell lines release IGF-BPs in culture supernatants, which differ from IGF-BPs detected in liver and placenta. These IGF-BPs might be important mediators in the autocrine/paracrine growth regulation of IGFs in SCLC.« less

Identification of neuronal target genes for CCAAT/Enhancer Binding Proteins

PubMed Central

Kfoury, N.; Kapatos, G.

2009-01-01

CCAAT/Enhancer Binding Proteins (C/EBPs) play pivotal roles in development and plasticity of the nervous system. Identification of the physiological targets of C/EBPs (C/EBP target genes) should therefore provide insight into the underlying biology of these processes. We used unbiased genome-wide mapping to identify 115 C/EBPβ target genes in PC12 cells that include transcription factors, neurotransmitter receptors, ion channels, protein kinases and synaptic vesicle proteins. C/EBPβ binding sites were located primarily within introns, suggesting novel regulatory functions, and were associated with binding sites for other developmentally important transcription factors. Experiments using dominant negatives showed C/EBPβ to repress transcription of a subset of target genes. Target genes in rat brain were subsequently found to preferentially bind C/EBPα, β and δ. Analysis of the hippocampal transcriptome of C/EBPβ knockout mice revealed dysregulation of a high percentage of transcripts identified as C/EBP target genes. These results support the hypothesis that C/EBPs play non-redundant roles in the brain. PMID:19103292

A DNA-binding protein from Candida albicans that binds to the RPG box of Saccharomyces cerevisiae and the telomeric repeat sequence of C. albicans.

PubMed

Ishii, N; Yamamoto, M; Lahm, H W; Iizumi, S; Yoshihara, F; Nakayama, H; Arisawa, M; Aoki, Y

1997-02-01

Electromobility shift assays with a DNA probe containing the Saccharomyces cerevisiae ENO1 RPG box identified a specific DNA-binding protein in total protein extracts of Candida albicans. The protein, named Rbf1p (RPG-box-binding protein 1), bound to other S. cerevisiae RPG boxes, although the nucleotide recognition profile was not completely the same as that of S. cerevisiae Rap 1p (repressor-activator protein 1), an RPG-box-binding protein. The repetitive sequence of the C. albicans chromosomal telomere also competed with RPG-box binding to Rbf1p. For further analysis, we purified Rbf1p 57,600-fold from C. albicans total protein extracts, raised mAbs against the purified protein and immunologically cloned the gene, whose ORF specified a protein of 527 aa. The bacterially expressed protein showed RPG-box-binding activity with the same profile as that of the purified one. The Rbf1p, containing two glutamine-rich regions that are found in many transcription factors, showed transcriptional activation capability in S. cerevisiae and was predominantly observed in nuclei. These results suggest that Rbf1p is a transcription factor with telomere-binding activity in C. albicans.

Porcine deltacoronavirus accessory protein NS6 antagonizes IFN-β production by interfering with the binding of RIG-I/MDA5 to double-stranded RNA.

PubMed

Fang, Puxian; Fang, Liurong; Ren, Jie; Hong, Yingying; Liu, Xiaorong; Zhao, Yunyang; Wang, Dang; Peng, Guiqing; Xiao, Shaobo

2018-05-16

Porcine deltacoronavirus (PDCoV) has recently emerged as an enteric pathogen that can cause serious vomiting and diarrhea in suckling piglets. The first outbreak of PDCoV occurred in the United States in 2014 and was followed by reports of PDCoV in South Korea, China, Thailand, Lao people's Democratic Republic, and Vietnam, leading to economic losses for pig farms and posing considerable threat to the swine industry worldwide. Our previous studies have shown that PDCoV encodes three accessory proteins, NS6, NS7, and NS7a, but the functions of these proteins in viral replication, pathogenesis, and immune regulation remain unclear. Here, we found that ectopic expression of accessory protein NS6 significantly inhibits Sendai virus-induced interferon-β (IFN-β) production, as well as the activation of transcription factors IRF3 and NF-κB. Interestingly, NS6 does not impede the IFN-β promoter activation mediated via key molecules in the RIG-I-like receptor (RLR) signaling pathway, specifically RIG-I, MDA5, and their downstream molecules MAVS, TBK1, IKKϵ, and IRF3. Further analyses revealed that NS6 is not a RNA-binding protein; however, it interacts with RIG-I/MDA5. This interaction attenuates the binding of double-stranded RNA by RIG-I/MDA5, resulting in the reduction of RLR-mediated IFN-β production. Taken together, our results demonstrate that ectopic expression of NS6 antagonizes IFN-β production by interfering with the binding of RIG-I/MDA5 to double-stranded RNA, revealing a new strategy employed by PDCoV accessory proteins to counteract the host innate antiviral immune response. IMPORTANCE Coronavirus accessory proteins are species-specific, and they perform multiple functions in viral pathogenicity and immunity, such as acting as interferon (IFN) antagonists and cell death inducers. Our previous studies have shown that porcine deltacoronavirus (PDCoV) encodes three accessory proteins. Here, we demonstrated for the first time that PDCoV accessory protein NS

SONAR Discovers RNA-Binding Proteins from Analysis of Large-Scale Protein-Protein Interactomes.

PubMed

Brannan, Kristopher W; Jin, Wenhao; Huelga, Stephanie C; Banks, Charles A S; Gilmore, Joshua M; Florens, Laurence; Washburn, Michael P; Van Nostrand, Eric L; Pratt, Gabriel A; Schwinn, Marie K; Daniels, Danette L; Yeo, Gene W

2016-10-20

RNA metabolism is controlled by an expanding, yet incomplete, catalog of RNA-binding proteins (RBPs), many of which lack characterized RNA binding domains. Approaches to expand the RBP repertoire to discover non-canonical RBPs are currently needed. Here, HaloTag fusion pull down of 12 nuclear and cytoplasmic RBPs followed by quantitative mass spectrometry (MS) demonstrates that proteins interacting with multiple RBPs in an RNA-dependent manner are enriched for RBPs. This motivated SONAR, a computational approach that predicts RNA binding activity by analyzing large-scale affinity precipitation-MS protein-protein interactomes. Without relying on sequence or structure information, SONAR identifies 1,923 human, 489 fly, and 745 yeast RBPs, including over 100 human candidate RBPs that contain zinc finger domains. Enhanced CLIP confirms RNA binding activity and identifies transcriptome-wide RNA binding sites for SONAR-predicted RBPs, revealing unexpected RNA binding activity for disease-relevant proteins and DNA binding proteins. Copyright © 2016 Elsevier Inc. All rights reserved.

Erythropoietin binding protein from mammalian serum

DOEpatents

Clemons, G.K.

1997-04-29

Purified mammalian erythropoietin binding-protein is disclosed, and its isolation, identification, characterization, purification, and immunoassay are described. The erythropoietin binding protein can be used for regulation of erythropoiesis by regulating levels and half-life of erythropoietin. A diagnostic kit for determination of level of erythropoietin binding protein is also described. 11 figs.

Accurate and sensitive quantification of protein-DNA binding affinity.

PubMed

Rastogi, Chaitanya; Rube, H Tomas; Kribelbauer, Judith F; Crocker, Justin; Loker, Ryan E; Martini, Gabriella D; Laptenko, Oleg; Freed-Pastor, William A; Prives, Carol; Stern, David L; Mann, Richard S; Bussemaker, Harmen J

2018-04-17

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. Copyright © 2018 the Author(s). Published by PNAS.

Accurate and sensitive quantification of protein-DNA binding affinity

PubMed Central

Rastogi, Chaitanya; Rube, H. Tomas; Kribelbauer, Judith F.; Crocker, Justin; Loker, Ryan E.; Martini, Gabriella D.; Laptenko, Oleg; Freed-Pastor, William A.; Prives, Carol; Stern, David L.; Mann, Richard S.; Bussemaker, Harmen J.

2018-01-01

Transcription factors (TFs) control gene expression by binding to genomic DNA in a sequence-specific manner. Mutations in TF binding sites are increasingly found to be associated with human disease, yet we currently lack robust methods to predict these sites. Here, we developed a versatile maximum likelihood framework named No Read Left Behind (NRLB) that infers a biophysical model of protein-DNA recognition across the full affinity range from a library of in vitro selected DNA binding sites. NRLB predicts human Max homodimer binding in near-perfect agreement with existing low-throughput measurements. It can capture the specificity of the p53 tetramer and distinguish multiple binding modes within a single sample. Additionally, we confirm that newly identified low-affinity enhancer binding sites are functional in vivo, and that their contribution to gene expression matches their predicted affinity. Our results establish a powerful paradigm for identifying protein binding sites and interpreting gene regulatory sequences in eukaryotic genomes. PMID:29610332

Acanthamoeba castellanii contains a ribosomal RNA enhancer binding protein which stimulates TIF-IB binding and transcription under stringent conditions.

PubMed Central

Yang, Q; Radebaugh, C A; Kubaska, W; Geiss, G K; Paule, M R

1995-01-01

The intergenic spacer (IGS) of Acanthamoeba castellanii rRNA genes contains repeated elements which are weak enhancers for transcription by RNA polymerase I. A protein, EBF, was identified and partially purified which binds to the enhancers and to several other sequences within the IGS, but not to other DNA fragments, including the rRNA core promoter. No consensus binding sequence could be discerned in these fragments and bound factor is in rapid equilibrium with unbound. EBF has functional characteristics similar to vertebrate upstream binding factors (UBF). Not only does it bind to the enhancer and other IGS elements, but it also stimulates binding of TIF-IB, the fundamental transcription initiation factor, to the core promoter and stimulates transcription from the promoter. Attempts to identify polypeptides with epitopes similar to rat or Xenopus laevis UBF suggest that structurally the protein from A.castellanii is not closely related to vertebrate UBF. Images PMID:7501455

NF-Y, a CCAAT box-binding protein, is one of the trans-acting factors necessary for the response of the murine ERp72 gene to protein traffic.

PubMed

Marcus, N; Green, M

1997-09-01

The accumulation of incompletely assembled immunoglobulin mu heavy chain in transfected COS cells stimulates the cellular response to protein traffic that results in the increased transcription and elevated synthesis of several ER chaperones, including ERP72, a member of the protein disulfide isomerase family of molecular chaperones. The ERp72 promoter contains an 82 bp ER protein traffic response element (ERPTRE) that is sufficient to mediate this response. Previously, it had been shown that the alteration of a putative AP-2 site and a CCAAT and inverted CCAAT site within the ERPTRE significantly decreased the response of ERp72 promoter to mu chain accumulation. We have extended these findings by demonstrating a role for NF-Y and a potentially novel DNA-binding protein in the regulation of transcription from the ERp72 promoter. The fact that NF-Y binding to the ERPTRE is observed in extracts from both control cells and cells in which the response to protein traffic has been activated indicates that the binding of NF-Y, while necessary, is not sufficient to account for the response. Each of the two CCAAT sites in the ERPTRE can bind NF-Y independently, but both sites must be intact for full ERPTRE function. A second protein can bind to the ERPTRE independently of NF-Y and at a site overlapping or close to the 3' end of the reverse CCAAT site. It is possible that interactions between NF-Y, this protein and perhaps other factors are responsible for the regulation of the protein traffic response.

Trans-acting translational regulatory RNA binding proteins.

PubMed

Harvey, Robert F; Smith, Tom S; Mulroney, Thomas; Queiroz, Rayner M L; Pizzinga, Mariavittoria; Dezi, Veronica; Villenueva, Eneko; Ramakrishna, Manasa; Lilley, Kathryn S; Willis, Anne E

2018-05-01

The canonical molecular machinery required for global mRNA translation and its control has been well defined, with distinct sets of proteins involved in the processes of translation initiation, elongation and termination. Additionally, noncanonical, trans-acting regulatory RNA-binding proteins (RBPs) are necessary to provide mRNA-specific translation, and these interact with 5' and 3' untranslated regions and coding regions of mRNA to regulate ribosome recruitment and transit. Recently it has also been demonstrated that trans-acting ribosomal proteins direct the translation of specific mRNAs. Importantly, it has been shown that subsets of RBPs often work in concert, forming distinct regulatory complexes upon different cellular perturbation, creating an RBP combinatorial code, which through the translation of specific subsets of mRNAs, dictate cell fate. With the development of new methodologies, a plethora of novel RNA binding proteins have recently been identified, although the function of many of these proteins within mRNA translation is unknown. In this review we will discuss these methodologies and their shortcomings when applied to the study of translation, which need to be addressed to enable a better understanding of trans-acting translational regulatory proteins. Moreover, we discuss the protein domains that are responsible for RNA binding as well as the RNA motifs to which they bind, and the role of trans-acting ribosomal proteins in directing the translation of specific mRNAs. This article is categorized under: RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Translation > Translation Regulation Translation > Translation Mechanisms. © 2018 Medical Research Council and University of Cambridge. WIREs RNA published by Wiley Periodicals, Inc.

UO₂²⁺ uptake by proteins: understanding the binding features of the super uranyl binding protein and design of a protein with higher affinity.

PubMed

Odoh, Samuel O; Bondarevsky, Gary D; Karpus, Jason; Cui, Qiang; He, Chuan; Spezia, Riccardo; Gagliardi, Laura

2014-12-17

The capture of uranyl, UO2(2+), by a recently engineered protein (Zhou et al. Nat. Chem. 2014, 6, 236) with high selectivity and femtomolar sensitivity has been examined by a combination of density functional theory, molecular dynamics, and free-energy simulations. It was found that UO2(2+) is coordinated to five carboxylate oxygen atoms from four amino acid residues of the super uranyl binding protein (SUP). A network of hydrogen bonds between the amino acid residues coordinated to UO2(2+) and residues in its second coordination sphere also affects the protein's uranyl binding affinity. Free-energy simulations show how UO2(2+) capture is governed by the nature of the amino acid residues in the binding site, the integrity and strength of the second-sphere hydrogen bond network, and the number of water molecules in the first coordination sphere. Alteration of any of these three factors through mutations generally results in a reduction of the binding free energy of UO2(2+) to the aqueous protein as well as of the difference between the binding free energies of UO2(2+) and other ions (Ca(2+), Cu(2+), Mg(2+), and Zn(2+)), a proxy for the protein's selectivity over these ions. The results of our free-energy simulations confirmed the previously reported experimental results and allowed us to discover a mutant of SUP, specifically the GLU64ASP mutant, that not only binds UO2(2+) more strongly than SUP but that is also more selective for UO2(2+) over other ions. The predictions from the computations were confirmed experimentally.

The pure estrogen receptor antagonist ICI 182,780 promotes a novel interaction of estrogen receptor-alpha with the 3',5'-cyclic adenosine monophosphate response element-binding protein-binding protein/p300 coactivators.

PubMed

Jaber, Basem M; Gao, Tong; Huang, Luping; Karmakar, Sudipan; Smith, Carolyn L

2006-11-01

Estrogen receptor-alpha (ERalpha) is a member of the nuclear receptor superfamily of ligand-activated transcription factors. Abundant evidence demonstrates that ERalpha agonists promote, whereas antagonists inhibit, receptor binding to coactivators. In this report we demonstrate that binding of the ICI 182,780 (ICI) pure antiestrogen to ERalpha promotes its interaction with the cAMP response element-binding protein-binding protein (CBP)/p300 but not the p160 family of coactivators, demonstrating the specificity of this interaction. Amino acid mutations within the coactivator binding surface of the ERalpha ligand-binding domain revealed that CBP binds to this region of the ICI-liganded receptor. The carboxy-terminal cysteine-histidine rich domain 3 of CBP, rather than its amino-terminal nuclear interacting domain, shown previously to mediate agonist-dependent interactions of CBP with nuclear receptors, is required for binding to ICI-liganded ERalpha. Chromatin immunoprecipitation assays revealed that ICI but not the partial agonist/antagonist 4-hydroxytamoxifen is able to recruit CBP to the pS2 promoter, and this distinguishes ICI from this class of antiestrogens. Chromatin immunoprecipitation assays for pS2 and cytochrome P450 1B1 promoter regions revealed that ICI-dependent recruitment of CBP, but not receptor, to ERalpha targets is gene specific. ICI treatment did not recruit the steroid receptor coactivator 1 to the pS2 promoter, and it failed to induce the expression of this gene. Taken together, these data indicate that recruitment of the CBP coactivator/cointegrator without steroid receptor coactivator 1 to ERalpha is insufficient to promote transcription of ERalpha target genes.

Insight into the Role of Ca2+-Binding Protein 5 in Vesicle Exocytosis

PubMed Central

Sokal, Izabela

2011-01-01

Purpose. CaBP5 is a neuronal calmodulin-like Ca2+-binding protein that is expressed in the retina and in the cochlea. Although CaBP5 knockout mice displayed reduced sensitivity of retinal ganglion cell light responses, the function of CaBP5 in vivo is still unknown. To gain further insight into CaBP5 function, the authors screened for CaBP5-interacting partners. Methods. Potential retinal interacting partners for CaBP5 were identified using affinity chromatography followed by mass spectrometry and by yeast two-hybrid screening of a bovine retina cDNA library. Interacting partners were further analyzed using coimmunoprecipitation. Immunohistochemistry and subcellular fractionation were performed to determine their colocalization in the retina. The effect of CaBP5 on dopamine release and neurite outgrowth of PC12 cells was analyzed using ELISA and fluorescent labeling. Results. Using affinity chromatography, the authors identified Munc18–1 and myosin VI as interacting partners for CaBP5. Munc18–1 was also identified using the yeast two-hybrid system. Colocalization and coimmunoprecipitation of CaBP5 with these two proteins in retinal tissue further established their physiological interactions. Furthermore, CaBP5 expression in NGF-stimulated PC12 cells stimulates neurite outgrowth and dopamine exocytosis. Conclusions. This study shows that CaBP5 interacts with Munc18–1 and myosin VI, two proteins involved in the synaptic vesicle cycle. Together with the effect of CaBP5 in stimulating neurite outgrowth and vesicle exocytosis in PC12 cells, these results suggest that CaBP5 plays a role in neurotransmitter release. PMID:22039235

Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting

NASA Astrophysics Data System (ADS)

Zhu, Yuanjun; Li, Ruyi; Lin, Yuan; Shui, Mengyang; Liu, Xiaoyan; Chen, Huan; Wang, Yinye

2016-07-01

Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency.

Structural basis for ligand regulation of the fatty acid-binding protein 5, peroxisome proliferator-activated receptor β/δ (FABP5-PPARβ/δ) signaling pathway.

PubMed

Armstrong, Eric H; Goswami, Devrishi; Griffin, Patrick R; Noy, Noa; Ortlund, Eric A

2014-05-23

Fatty acid-binding proteins (FABPs) are a widely expressed group of calycins that play a well established role in solubilizing cellular fatty acids. Recent studies, however, have recast FABPs as active participants in vital lipid-signaling pathways. FABP5, like its family members, displays a promiscuous ligand binding profile, capable of interacting with numerous long chain fatty acids of varying degrees of saturation. Certain "activating" fatty acids induce the protein's cytoplasmic to nuclear translocation, stimulating PPARβ/δ transactivation; however, the rules that govern this process remain unknown. Using a range of structural and biochemical techniques, we show that both linoleic and arachidonic acid elicit FABP5's translocation by permitting allosteric communication between the ligand-sensing β2 loop and a tertiary nuclear localization signal within the α-helical cap of the protein. Furthermore, we show that more saturated, nonactivating fatty acids inhibit nuclear localization signal formation by destabilizing this activation loop, thus implicating FABP5 specifically in cis-bonded, polyunsaturated fatty acid signaling. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

[Glutamate-binding membrane proteins from human platelets].

PubMed

Gurevich, V S; Popov, Iu G; Gorodinskiĭ, A I; Dambinova, S A

1991-09-01

Solubilization of the total membrane fraction of human platelets in a 2% solution of sodium deoxycholate and subsequent affinity chromatography on glutamate agarose resulted in two protein fractions possessing a glutamate-binding activity. As can be evidenced from radioligand binding data, the first fraction contains two types of binding sites (Kd1 = 1 microM, Bmax 1 = 100 pmol/mg of protein; Kd2 = 9.3 microMm Bmax2 = 395 pmol/mg of protein). The second fraction has only one type of binding sites (Kd = 1 microM, Bmax = = 110 pmol/mg of protein). SDS-PAAG electrophoresis revealed the presence in the first fraction of proteins with Mr of 14, 24, 56 and 155 kDa, whereas the second fraction was found to contain 14, 46, 71 and 155 kDa proteins. Solid phase immunoenzymatic analysis using poly- and monoclonal specific antibodies against mammalian brain glutamate-binding proteins revealed a marked immunochemical similarity of the isolated protein fractions with human brain synaptic membrane glutamate-binding proteins.

Distant homologs of anti-apoptotic factor HAX1 encode parvalbumin-like calcium binding proteins.

PubMed

Kokoszyńska, Katarzyna; Rychlewski, Leszek; Wyrwicz, Lucjan S

2010-07-15

Apoptosis is a highly ordered and orchestrated multiphase process controlled by the numerous cellular and extra-cellular signals, which executes the programmed cell death via release of cytochrome c alterations in calcium signaling, caspase-dependent limited proteolysis and DNA fragmentation. Besides the general modifiers of apoptosis, several tissue-specific regulators of this process were identified including HAX1 (HS-1 associated protein X-1) - an anti-apoptotic factor active in myeloid cells. Although HAX1 was the subject of various experimental studies, the mechanisms of its action and a functional link connected with the regulation of apoptosis still remains highly speculative. Here we provide the data which suggests that HAX1 may act as a regulator or as a sensor of calcium. On the basis of iterative similarity searches, we identified a set of distant homologs of HAX1 in insects. The applied fold recognition protocol gives us strong evidence that the distant insects' homologs of HAX1 are novel parvalbumin-like calcium binding proteins. Although the whole three EF-hands fold is not preserved in vertebrate our analysis suggests that there is an existence of a potential single EF-hand calcium binding site in HAX1. The molecular mechanism of its action remains to be identified, but the risen hypothesis easily translates into previously reported lines of various data on the HAX1 biology as well as, provides us a direct link to the regulation of apoptosis. Moreover, we also report that other family of myeloid specific apoptosis regulators - myeloid leukemia factors (MLF1, MLF2) share the homologous C-terminal domain and taxonomic distribution with HAX1. Performed structural and active sites analyses gave new insights into mechanisms of HAX1 and MLF families in apoptosis process and suggested possible role of HAX1 in calcium-binding, still the analyses require further experimental verification.

Distant homologs of anti-apoptotic factor HAX1 encode parvalbumin-like calcium binding proteins

PubMed Central

2010-01-01

Background Apoptosis is a highly ordered and orchestrated multiphase process controlled by the numerous cellular and extra-cellular signals, which executes the programmed cell death via release of cytochrome c alterations in calcium signaling, caspase-dependent limited proteolysis and DNA fragmentation. Besides the general modifiers of apoptosis, several tissue-specific regulators of this process were identified including HAX1 (HS-1 associated protein X-1) - an anti-apoptotic factor active in myeloid cells. Although HAX1 was the subject of various experimental studies, the mechanisms of its action and a functional link connected with the regulation of apoptosis still remains highly speculative. Findings Here we provide the data which suggests that HAX1 may act as a regulator or as a sensor of calcium. On the basis of iterative similarity searches, we identified a set of distant homologs of HAX1 in insects. The applied fold recognition protocol gives us strong evidence that the distant insects' homologs of HAX1 are novel parvalbumin-like calcium binding proteins. Although the whole three EF-hands fold is not preserved in vertebrate our analysis suggests that there is an existence of a potential single EF-hand calcium binding site in HAX1. The molecular mechanism of its action remains to be identified, but the risen hypothesis easily translates into previously reported lines of various data on the HAX1 biology as well as, provides us a direct link to the regulation of apoptosis. Moreover, we also report that other family of myeloid specific apoptosis regulators - myeloid leukemia factors (MLF1, MLF2) share the homologous C-terminal domain and taxonomic distribution with HAX1. Conclusions Performed structural and active sites analyses gave new insights into mechanisms of HAX1 and MLF families in apoptosis process and suggested possible role of HAX1 in calcium-binding, still the analyses require further experimental verification. PMID:20633251

Protein Binding: Do We Ever Learn?▿

PubMed Central

Zeitlinger, Markus A.; Derendorf, Hartmut; Mouton, Johan W.; Cars, Otto; Craig, William A.; Andes, David; Theuretzbacher, Ursula

2011-01-01

Although the influence of protein binding (PB) on antibacterial activity has been reported for many antibiotics and over many years, there is currently no standardization for pharmacodynamic models that account for the impact of protein binding of antimicrobial agents in vitro. This might explain the somewhat contradictory results obtained from different studies. Simple in vitro models which compare the MIC obtained in protein-free standard medium versus a protein-rich medium are prone to methodological pitfalls and may lead to flawed conclusions. Within in vitro test systems, a range of test conditions, including source of protein, concentration of the tested antibiotic, temperature, pH, electrolytes, and supplements may influence the impact of protein binding. As new antibiotics with a high degree of protein binding are in clinical development, attention and action directed toward the optimization and standardization of testing the impact of protein binding on the activity of antibiotics in vitro become even more urgent. In addition, the quantitative relationship between the effects of protein binding in vitro and in vivo needs to be established, since the physiological conditions differ. General recommendations for testing the impact of protein binding in vitro are suggested. PMID:21537013

Carotenoid Antenna Binding and Function in Retinal Proteins

DTIC Science & Technology

2012-08-13

REPORT Carotenoid antenna binding and function in retinal proteins 14. ABSTRACT 16. SECURITY CLASSIFICATION OF: Xanthorhodopsin, a proton pump from the...eubacterium Salinibacter ruber, is a unique dual chromophore system that contains, in addition to retinal, the carotenoid salinixanthin as a light... carotenoid ring near the retinal ring. Substitution of the small glycine with bulky tryptophan in this site eliminates binding. The second factor is the 4

An ensemble model of competitive multi-factor binding of the genome

PubMed Central

Wasson, Todd; Hartemink, Alexander J.

2009-01-01

Hundreds of different factors adorn the eukaryotic genome, binding to it in large number. These DNA binding factors (DBFs) include nucleosomes, transcription factors (TFs), and other proteins and protein complexes, such as the origin recognition complex (ORC). DBFs compete with one another for binding along the genome, yet many current models of genome binding do not consider different types of DBFs together simultaneously. Additionally, binding is a stochastic process that results in a continuum of binding probabilities at any position along the genome, but many current models tend to consider positions as being either binding sites or not. Here, we present a model that allows a multitude of DBFs, each at different concentrations, to compete with one another for binding sites along the genome. The result is an “occupancy profile,” a probabilistic description of the DNA occupancy of each factor at each position. We implement our model efficiently as the software package COMPETE. We demonstrate genome-wide and at specific loci how modeling nucleosome binding alters TF binding, and vice versa, and illustrate how factor concentration influences binding occupancy. Binding cooperativity between nearby TFs arises implicitly via mutual competition with nucleosomes. Our method applies not only to TFs, but also recapitulates known occupancy profiles of a well-studied replication origin with and without ORC binding. Importantly, the sequence preferences our model takes as input are derived from in vitro experiments. This ensures that the calculated occupancy profiles are the result of the forces of competition represented explicitly in our model and the inherent sequence affinities of the constituent DBFs. PMID:19720867

The Scl1 protein of M6-type group A Streptococcus binds the human complement regulatory protein, factor H, and inhibits the alternative pathway of complement.

PubMed

Caswell, Clayton C; Han, Runlin; Hovis, Kelley M; Ciborowski, Pawel; Keene, Douglas R; Marconi, Richard T; Lukomski, Slawomir

2008-02-01

Non-specific activation of the complement system is regulated by the plasma glycoprotein factor H (FH). Bacteria can avoid complement-mediated opsonization and phagocytosis through acquiring FH to the cell surface. Here, we characterize an interaction between the streptococcal collagen-like protein Scl1.6 of M6-type group A Streptococcus (GAS) and FH. Using affinity chromatography with immobilized recombinant Scl1.6 protein, we co-eluted human plasma proteins with molecular weight of 155 kDa, 43 kDa and 38 kDa. Mass spectrometry identified the 155 kDa band as FH and two other bands as isoforms of the FH-related protein-1. The identities of all three bands were confirmed by Western immunoblotting with specific antibodies. Structure-function relation studies determined that the globular domain of the Scl1.6 variant specifically binds FH while fused to collagenous tails of various lengths. This binding is not restricted to Scl1.6 as the phylogenetically linked Scl1.55 variant also binds FH. Functional analyses demonstrated the cofactor activity of the rScl1.6-bound FH for factor I-mediated cleavage of C3b. Finally, purified FH bound to the Scl1.6 protein present in the cell wall material obtained from M6-type GAS. In conclusion, we have identified a functional interaction between Scl1 and plasma FH, which may contribute to GAS evasion of complement-mediated opsonization and phagocytosis.

The brain-specific double-stranded RNA-binding protein Staufen2: nucleolar accumulation and isoform-specific exportin-5-dependent export.

PubMed

Macchi, Paolo; Brownawell, Amy M; Grunewald, Barbara; DesGroseillers, Luc; Macara, Ian G; Kiebler, Michael A

2004-07-23

The mammalian double-stranded RNA-binding proteins Staufen (Stau1 and Stau2) are involved in RNA localization in polarized neurons. In contrast to the more ubiquitously expressed Stau1, Stau2 is mainly expressed in the nervous system. In Drosophila, the third double-stranded RNA-binding domain (RBD3) of Staufen is essential for RNA interaction. When conserved amino acids within the RBD3 of Stau2 were mutated to render Stau2 defective for RNA binding, the mutant Stau2 proteins accumulate predominantly in the nucleolus. This is in contrast to wild type Stau2 that mostly localizes in the cytosol. The nuclear import is dependent on a nuclear localization signal in close proximity to the RBD3. The nuclear export of Stau2 is not dependent on CRM1 but rather on Exportin-5. We show that Exportin-5 interacts with the RBD3 of wild type Stau2 in an RNA-dependent manner in vitro but not with mutant Stau2. When Exportin-5 is down-regulated by RNA interference, only the largest isoform of Stau2 (Stau2(62)) preferentially accumulates in the nucleolus. It is tempting to speculate that Stau2(62) binds RNA in the nucleus and assembles into ribonucleoparticles, which are then exported via the Exportin-5 pathway to their final destination.

The promiscuous protein binding ability of erythrosine B studied by metachromasy (metachromasia).

PubMed

Ganesan, Lakshmi; Buchwald, Peter

2013-04-01

The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein-protein interactions (PPIs) with a remarkably consistent median inhibitory concentration (IC50 ) in the 5- to 30-μM range. Because ErB exhibits metachromasy, that is, color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd ) and stoichiometry (nb ) using spectrophotometric methods. Binding was reversible, and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 μM for BSA and CD40L, respectively) were in good agreement with that expected from the PPI inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5-6 and 8-9 for BSA and CD40L, respectively), indicating the possibility of nonspecific binding of the flat and rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. Copyright © 2013 John Wiley & Sons, Ltd.

The Promiscuous Protein Binding Ability of Erythrosine B Studied by Metachromasy (Metachromasia)

PubMed Central

Ganesan, Lakshmi; Buchwald, Peter

2013-01-01

The present study aims to elucidate aspects of the protein binding ability of erythrosine B (ErB), a poly-iodinated xanthene dye and an FDA-approved food colorant (FD&C Red No. 3), which we have identified recently as a promiscuous inhibitor of protein–protein interactions (PPI) with a remarkably consistent median inhibitory concentration (IC50) in the 5–30 µM range. Because ErB exhibits metachromasy, i.e., color change upon binding to several proteins, we exploited this property to quantify its binding to proteins such as bovine serum albumin (BSA) and CD40L (CD154) and to determine the corresponding binding constants (Kd) and stoichiometry (nb) using spectrophotometric methods. Binding was reversible and the estimated affinities for both protein targets obtained here (Kd values of 14 and 20 µM for BSA and CD40L, respectively) were in good agreement with that expected from the protein–protein interaction (PPI) inhibitory activity of ErB. A stoichiometry greater than one was observed both for CD40L and BSA binding (nb of 5–6 and 8–9 for BSA and CD40L, respectively) indicating the possibility of nonspecific binding of the flat an rigid ErB molecule at multiple sites, which could explain the promiscuous PPI inhibitory activity if some of these overlap with the binding site of the protein partner and interfere with the binding. PMID:23456742

Definition of IgG- and albumin-binding regions of streptococcal protein G.

PubMed

Akerström, B; Nielsen, E; Björck, L

1987-10-05

Protein G, the immunoglobin G-binding surface protein of group C and G streptococci, also binds serum albumin. The albumin-binding site on protein G is distinct from the immunoglobulin G-binding site. By mild acid hydrolysis of the papain-liberated protein G fragment (35 kDa), a 28-kDa fragment was produced which retained full immunoglobulin G-binding activity (determined by Scatchard plotting) but had lost all albumin-binding capacity. A protein G (65 kDa), isolated after cloning and expression of the protein G gene in Escherichia coli, had comparable affinity to immunoglobulin G (5-10 X 10(10)M-1), but much higher affinity to albumin than the 35- and 28-kDa protein G fragments (31, 2.6, and 0 X 10(9)M-1, respectively). The amino-terminal amino acid sequences of the 65-, 35-, and 28-kDa fragments allowed us to exactly locate the three fragments in an overall sequence map of protein G, based on the partial gene sequences published by Guss et al. (Guss, B., Eliasson, M., Olsson, A., Uhlen, M., Frej, A.-K., Jörnvall, H., Flock, J.-I., and Lindberg, M. (1986) EMBO J. 5, 1567-1575) and Fahnestock et al. (Fahnestock, S. R., Alexander, P., Nagle, J., and Filpula, D. (1986) J. Bacteriol. 167, 870-880). In this map could then be deduced the location of three homologous albumin-binding regions and three homologous immunoglobulin G-binding regions.

Inhibition of PKR Activation by the Proline-Rich RNA Binding Domain of the Herpes Simplex Virus Type 1 Us11 Protein

PubMed Central

Poppers, Jeremy; Mulvey, Matthew; Khoo, David; Mohr, Ian

2000-01-01

Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The γ34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the γ34.5 gene specifies a regulatory subunit for protein phosphatase 1α, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. γ34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of γ34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation. PMID:11070019

Inhibition of PKR activation by the proline-rich RNA binding domain of the herpes simplex virus type 1 Us11 protein.

PubMed

Poppers, J; Mulvey, M; Khoo, D; Mohr, I

2000-12-01

Upon activation by double-stranded RNA in virus-infected cells, the cellular PKR kinase phosphorylates the translation initiation factor eukaryotic initiation factor 2 (eIF2) and thereby inhibits protein synthesis. The gamma 34.5 and Us11 gene products encoded by herpes simplex virus type 1 (HSV-1) are dedicated to preventing the accumulation of phosphorylated eIF2. While the gamma 34.5 gene specifies a regulatory subunit for protein phosphatase 1 alpha, the Us11 gene encodes an RNA binding protein that also prevents PKR activation. gamma 34.5 mutants fail to grow on a variety of human cells as phosphorylated eIF2 accumulates and protein synthesis ceases prior to the completion of the viral life cycle. We demonstrate that expression of a 68-amino-acid fragment of Us11 containing a novel proline-rich basic RNA binding domain allows for sustained protein synthesis and enhanced growth of gamma 34.5 mutants. Furthermore, this fragment is sufficient to inhibit activation of the cellular PKR kinase in a cell-free system, suggesting that the intrinsic activities of this small fragment, notably RNA binding and ribosome association, may be required to prevent PKR activation.

Differences in DNA Binding Specificity of Floral Homeotic Protein Complexes Predict Organ-Specific Target Genes.

PubMed

Smaczniak, Cezary; Muiño, Jose M; Chen, Dijun; Angenent, Gerco C; Kaufmann, Kerstin

2017-08-01

Floral organ identities in plants are specified by the combinatorial action of homeotic master regulatory transcription factors. However, how these factors achieve their regulatory specificities is still largely unclear. Genome-wide in vivo DNA binding data show that homeotic MADS domain proteins recognize partly distinct genomic regions, suggesting that DNA binding specificity contributes to functional differences of homeotic protein complexes. We used in vitro systematic evolution of ligands by exponential enrichment followed by high-throughput DNA sequencing (SELEX-seq) on several floral MADS domain protein homo- and heterodimers to measure their DNA binding specificities. We show that specification of reproductive organs is associated with distinct binding preferences of a complex formed by SEPALLATA3 and AGAMOUS. Binding specificity is further modulated by different binding site spacing preferences. Combination of SELEX-seq and genome-wide DNA binding data allows differentiation between targets in specification of reproductive versus perianth organs in the flower. We validate the importance of DNA binding specificity for organ-specific gene regulation by modulating promoter activity through targeted mutagenesis. Our study shows that intrafamily protein interactions affect DNA binding specificity of floral MADS domain proteins. Differential DNA binding of MADS domain protein complexes plays a role in the specificity of target gene regulation. © 2017 American Society of Plant Biologists. All rights reserved.

Small Molecule Ligands of Methyl-Lysine Binding Proteins

PubMed Central

Herold, J. Martin; Wigle, Tim J.; Norris, Jacqueline L.; Lam, Robert; Korboukh, Victoria K.; Gao, Cen; Ingerman, Lindsey A.; Kireev, Dmitri B.; Senisterra, Guillermo; Vedadi, Masoud; Tripathy, Ashutosh; Brown, Peter J.; Arrowsmith, Cheryl H.; Jin, Jian; Janzen, William P.; Frye, Stephen V.

2011-01-01

Proteins which bind methylated lysines (“readers” of the histone code) are important components in the epigenetic regulation of gene expression and can also modulate other proteins that contain methyl-lysine such as p53 and Rb. Recognition of methyl-lysine marks by MBT domains leads to compaction of chromatin and a repressed transcriptional state. Antagonists of MBT domains would serve as probes to interrogate the functional role of these proteins and initiate the chemical biology of methyl-lysine readers as a target class. Small molecule MBT antagonists were designed based on the structure of histone peptide-MBT complexes and their interaction with MBT domains determined using a chemiluminescent assay and ITC. The ligands discovered antagonize native histone peptide binding, exhibiting 5-fold stronger binding affinity to L3MBTL1 than its preferred histone peptide. The first co-crystal structure of a small molecule bound to L3MBTL1 was determined and provides new insights into binding requirements for further ligand design. PMID:21417280

Monoclonal antibodies to meningococcal factor H binding protein with overlapping epitopes and discordant functional activity.

PubMed

Giuntini, Serena; Beernink, Peter T; Reason, Donald C; Granoff, Dan M

2012-01-01

Meningococcal factor H binding protein (fHbp) is a promising vaccine candidate. Anti-fHbp antibodies can bind to meningococci and elicit complement-mediated bactericidal activity directly. The antibodies also can block binding of the human complement down-regulator, factor H (fH). Without bound fH, the organism would be expected to have increased susceptibility to bacteriolysis. Here we describe bactericidal activity of two anti-fHbp mAbs with overlapping epitopes in relation to their different effects on fH binding and bactericidal activity. Both mAbs recognized prevalent fHbp sequence variants in variant group 1. Using yeast display and site-specific mutagenesis, binding of one of the mAbs (JAR 1, IgG3) to fHbp was eliminated by a single amino acid substitution, R204A, and was decreased by K143A but not by R204H or D142A. The JAR 1 epitope overlapped that of previously described mAb (mAb502, IgG2a) whose binding to fHbp was eliminated by R204A or R204H substitutions, and was decreased by D142A but not by K143A. Although JAR 1 and mAb502 appeared to have overlapping epitopes, only JAR 1 inhibited binding of fH to fHbp and had human complement-mediated bactericidal activity. mAb502 enhanced fH binding and lacked human complement-mediated bactericidal activity. To control for confounding effects of different mouse IgG subclasses on complement activation, we created chimeric mAbs in which the mouse mAb502 or JAR 1 paratopes were paired with human IgG1 constant regions. While both chimeric mAbs showed similar binding to fHbp, only JAR 1, which inhibited fH binding, had human complement-mediated bactericidal activity. The lack of human complement-mediated bactericidal activity by anti-fHbp mAb502 appeared to result from an inability to inhibit binding of fH. These results underscore the importance of inhibition of fH binding for anti-fHbp mAb bactericidal activity.

Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms.

PubMed

Kahvejian, Avak; Svitkin, Yuri V; Sukarieh, Rami; M'Boutchou, Marie-Noël; Sonenberg, Nahum

2005-01-01

Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5'-end of the mRNA to promote the recruitment of the ribosome. Although the 3' poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for the involvement of PABP in key steps of the translation initiation pathway. Using a new technique to deplete PABP from mammalian cell extracts, we show that extracts lacking PABP exhibit dramatically reduced rates of translation, reduced efficiency of 48S and 80S ribosome initiation complex formation, and impaired interaction of eIF4E with the mRNA cap structure. Supplementing PABP-depleted extracts with wild-type PABP completely rectified these deficiencies, whereas a mutant of PABP, M161A, which is incapable of interacting with eIF4G, failed to restore translation. In addition, a stronger inhibition (approximately twofold) of 80S as compared to 48S ribosome complex formation (approximately 65% vs. approximately 35%, respectively) by PABP depletion suggests that PABP plays a direct role in 60S subunit joining. PABP can thus be considered a canonical translation initiation factor, integral to initiation complex formation at the 5'-end of mRNA.

Mammalian poly(A)-binding protein is a eukaryotic translation initiation factor, which acts via multiple mechanisms

PubMed Central

Kahvejian, Avak; Svitkin, Yuri V.; Sukarieh, Rami; M'Boutchou, Marie-Noël; Sonenberg, Nahum

2005-01-01

Translation initiation is a multistep process involving several canonical translation factors, which assemble at the 5′-end of the mRNA to promote the recruitment of the ribosome. Although the 3′ poly(A) tail of eukaryotic mRNAs and its major bound protein, the poly(A)-binding protein (PABP), have been studied extensively, their mechanism of action in translation is not well understood and is confounded by differences between in vivo and in vitro systems. Here, we provide direct evidence for the involvement of PABP in key steps of the translation initiation pathway. Using a new technique to deplete PABP from mammalian cell extracts, we show that extracts lacking PABP exhibit dramatically reduced rates of translation, reduced efficiency of 48S and 80S ribosome initiation complex formation, and impaired interaction of eIF4E with the mRNA cap structure. Supplementing PABP-depleted extracts with wild-type PABP completely rectified these deficiencies, whereas a mutant of PABP, M161A, which is incapable of interacting with eIF4G, failed to restore translation. In addition, a stronger inhibition (approximately twofold) of 80S as compared to 48S ribosome complex formation (∼65% vs. ∼35%, respectively) by PABP depletion suggests that PABP plays a direct role in 60S subunit joining. PABP can thus be considered a canonical translation initiation factor, integral to initiation complex formation at the 5′-end of mRNA. PMID:15630022

Phosphorylation of poly(rC) binding protein 1 (PCBP1) contributes to stabilization of mu opioid receptor (MOR) mRNA via interaction with AU-rich element RNA-binding protein 1 (AUF1) and poly A binding protein (PABP)

PubMed Central

Hwang, Cheol Kyu; Wagley, Yadav; Law, Ping-Yee; Wei, Li-Na; Loh, Horace H.

2016-01-01

Gene regulation at the post-transcriptional level is frequently based on cis- and trans-acting factors on target mRNAs. We found a C-rich element (CRE) in mu-opioid receptor (MOR) 3′-untranslated region (UTR) to which poly (rC) binding protein 1 (PCBP1) binds, resulting in MOR mRNA stabilization. RNA immunoprecipitation and RNA EMSA revealed the formation of PCBP1-RNA complexes at the element. Knockdown of PCBP1 decreased MOR mRNA half-life and protein expression. Stimulation by forskolin increased cytoplasmic localization of PCBP1 and PCBP1/MOR 3′-UTR interactions via increased serine phosphorylation that was blocked by protein kinase A (PKA) or (phosphatidyl inositol-3) PI3-kinase inhibitors. The forskolin treatment also enhanced serine- and tyrosine-phosphorylation of AU-rich element binding protein (AUF1), concurrent with its increased binding to the CRE, and led to an increased interaction of poly A binding protein (PABP) with the CRE and poly(A) sites. AUF1 phosphorylation also led to an increased interaction with PCBP1. These findings suggest that a single co-regulator, PCBP1, plays a crucial role in stabilizing MOR mRNA, and is induced by PKA signaling by conforming to AUF1 and PABP. PMID:27836661

Interactions of cullin3/KCTD5 complexes with both cytoplasmic and nuclear proteins: Evidence for a role in protein stabilization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rutz, Natalja; Heilbronn, Regine; Weger, Stefan, E-mail: stefan.weger@charite.de

2015-08-28

Based on its specific interaction with cullin3 mediated by an N-terminal BTB/POZ homologous domain, KCTD5 has been proposed to function as substrate adapter for cullin3 based ubiquitin E3 ligases. In the present study we tried to validate this hypothesis through identification and characterization of additional KCTD5 interaction partners. For the replication protein MCM7, the zinc finger protein ZNF711 and FAM193B, a yet poorly characterized cytoplasmic protein, we could demonstrate specific interaction with KCTD5 both in yeast two-hybrid and co-precipitation studies in mammalian cells. Whereas trimeric complexes of cullin3 and KCTD5 with the respective KCTD5 binding partner were formed, KCTD5/cullin3 inducedmore » polyubiquitylation and/or proteasome-dependent degradation of these binding partners could not be demonstrated. On the contrary, KCTD5 or Cullin3 overexpression increased ZNF711 protein stability. - Highlights: • KCTD5 nuclear translocation depends upon M phase and protein oligomerization. • Identification of MCM7, ZNF711 and FAM193 as KCTD5 interaction partners. • Formation of trimeric complexes of KCTD5/cullin3 with MCM7, ZNF711 and FAM193B. • KCTD5 is not involved in polyubiquitylation of MCM7 replication factor. • The KCTD5/cullin3 complex stabilizes ZNF711 transcription factor.« less

Identification of DNA-binding proteins that interact with the 5'-flanking region of the human D-amino acid oxidase gene by pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry.

PubMed

Tran, Diem Hong; Shishido, Yuji; Chung, Seong Pil; Trinh, Huong Thi Thanh; Yorita, Kazuko; Sakai, Takashi; Fukui, Kiyoshi

2015-12-10

D-Amino acid oxidase (DAO) is a flavoenzyme that metabolizes D-amino acids and is expected to be a promising therapeutic target of schizophrenia and glioblastoma. The study of DNA-binding proteins has yielded much information in the regulation of transcription and other biological processes. However, proteins interacting with DAO gene have not been elucidated. Our assessment of human DAO promoter activity using luciferase reporter system indicated the 5'-flanking region of this gene (-4289 bp from transcription initiation site) has a regulatory sequence for gene expression, which is regulated by multi-protein complexes interacting with this region. By using pull-down assay coupled with two-dimensional gel electrophoresis and mass spectrometry, we identified six proteins binding to the 5'-flanking region of the human DAO gene (zinc finger C2HC domain-containing protein 1A; histidine-tRNA ligase, cytoplasmic; molybdenum cofactor biosynthesis protein; 60S ribosomal protein L37; calponin-1; calmodulin binding protein and heterogeneous nuclear ribonucleoprotein A2/B1). These preliminary results will contribute to the advance in the understanding of the potential factors associated with the regulatory mechanism of DAO expression. Copyright © 2015 Elsevier B.V. All rights reserved.

Monoclonal antibodies to human vitamin D-binding protein.

PubMed Central

Pierce, E A; Dame, M C; Bouillon, R; Van Baelen, H; DeLuca, H F

1985-01-01

Monoclonal antibodies to vitamin D-binding protein isolated from human serum have been produced. The antibodies obtained have been shown to be specific for human vitamin D-binding protein by three independent assays. The antibodies recognize human vitamin D-binding protein specifically in an enzyme-linked immunosorbent assay. Human vitamin D-binding protein is detected specifically in both pure and crude samples by a radiometric immunosorbent assay (RISA) and by an immunoprecipitation assay. The anti-human vitamin D-binding protein antibodies cross-react with monkey and pig vitamin D-binding protein, but not with vitamin D-binding protein from rat, mouse, or chicken, as determined by the RISA and immunoprecipitation assays. Images PMID:3936035

Multivalent binding of formin-binding protein 21 (FBP21)-tandem-WW domains fosters protein recognition in the pre-spliceosome.

PubMed

Klippel, Stefan; Wieczorek, Marek; Schümann, Michael; Krause, Eberhard; Marg, Berenice; Seidel, Thorsten; Meyer, Tim; Knapp, Ernst-Walter; Freund, Christian

2011-11-04

The high abundance of repetitive but nonidentical proline-rich sequences in spliceosomal proteins raises the question of how these known interaction motifs recruit their interacting protein domains. Whereas complex formation of these adaptors with individual motifs has been studied in great detail, little is known about the binding mode of domains arranged in tandem repeats and long proline-rich sequences including multiple motifs. Here we studied the interaction of the two adjacent WW domains of spliceosomal protein FBP21 with several ligands of different lengths and composition to elucidate the hallmarks of multivalent binding for this class of recognition domains. First, we show that many of the proteins that define the cellular proteome interacting with FBP21-WW1-WW2 contain multiple proline-rich motifs. Among these is the newly identified binding partner SF3B4. Fluorescence resonance energy transfer (FRET) analysis reveals the tandem-WW domains of FBP21 to interact with splicing factor 3B4 (SF3B4) in nuclear speckles where splicing takes place. Isothermal titration calorimetry and NMR shows that the tandem arrangement of WW domains and the multivalency of the proline-rich ligands both contribute to affinity enhancement. However, ligand exchange remains fast compared with the NMR time scale. Surprisingly, a N-terminal spin label attached to a bivalent ligand induces NMR line broadening of signals corresponding to both WW domains of the FBP21-WW1-WW2 protein. This suggests that distinct orientations of the ligand contribute to a delocalized and semispecific binding mode that should facilitate search processes within the spliceosome.

Purification and characterization of a membrane-associated 3,3',5-triiodo-L-thyronine binding protein from a human carcinoma cell line.

PubMed Central

Cheng, S Y; Hasumura, S; Willingham, M C; Pastan, I

1986-01-01

A membrane-associated binding protein for 3,3',5-triiodo-L-thyronine (T3) was purified to apparent homogeneity from A431 human epidermoid carcinoma cells. A431 cells were specifically labeled with the N-bromoacetyl derivative of T3 labeled with 125I at the 3' position (BrAc[125I]T3) and were extracted with 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), a zwitterionic detergent. The solubilized BrAc[125I]T3-labeled protein was successively purified by chromatography on Sephadex G-200 and QAE-Sephadex followed by NaDod-SO4/PAGE. Approximately 0.2 mg of purified protein was obtained from 2.5 X 10(9) cells, which represents a 3000-fold purification. The membrane-associated T3 binding protein is an acidic protein with a pI of 5.1 and an apparent molecular mass of 55,000 daltons determined by NaDodSO4/PAGE. Polyclonal antibodies against the 55-kDa protein were prepared and used in indirect immunofluorescence to show that the 55-kDa protein was mainly found in the nuclear envelope and endoplasmic reticulum. Images PMID:3006034

Phosphorylation of Krüppel-like factor 3 (KLF3/BKLF) and C-terminal binding protein 2 (CtBP2) by homeodomain-interacting protein kinase 2 (HIPK2) modulates KLF3 DNA binding and activity.

PubMed

Dewi, Vitri; Kwok, Alister; Lee, Stella; Lee, Ming Min; Tan, Yee Mun; Nicholas, Hannah R; Isono, Kyo-ichi; Wienert, Beeke; Mak, Ka Sin; Knights, Alexander J; Quinlan, Kate G R; Cordwell, Stuart J; Funnell, Alister P W; Pearson, Richard C M; Crossley, Merlin

2015-03-27

Krüppel-like factor 3 (KLF3/BKLF), a member of the Krüppel-like factor (KLF) family of transcription factors, is a widely expressed transcriptional repressor with diverse biological roles. Although there is considerable understanding of the molecular mechanisms that allow KLF3 to silence the activity of its target genes, less is known about the signal transduction pathways and post-translational modifications that modulate KLF3 activity in response to physiological stimuli. We observed that KLF3 is modified in a range of different tissues and found that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) can both bind and phosphorylate KLF3. Mass spectrometry identified serine 249 as the primary phosphorylation site. Mutation of this site reduces the ability of KLF3 to bind DNA and repress transcription. Furthermore, we also determined that HIPK2 can phosphorylate the KLF3 co-repressor C-terminal binding protein 2 (CtBP2) at serine 428. Finally, we found that phosphorylation of KLF3 and CtBP2 by HIPK2 strengthens the interaction between these two factors and increases transcriptional repression by KLF3. Taken together, our results indicate that HIPK2 potentiates the activity of KLF3. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Cross-talk between cognate and noncognate RpoE sigma factors and Zn(2+)-binding anti-sigma factors regulates photooxidative stress response in Azospirillum brasilense.

PubMed

Gupta, Namrata; Gupta, Ankush; Kumar, Santosh; Mishra, Rajeev; Singh, Chhaya; Tripathi, Anil Kumar

2014-01-01

Azospirillum brasilense harbors two redox-sensitive Zinc-binding anti-sigma (ZAS) factors (ChrR1 and ChrR2), which negatively regulate the activity of their cognate extra-cytoplasmic function (ECF) σ factors (RpoE1 and RpoE2) by occluding their binding to the core enzyme. Both pairs of RpoE-ChrR control responses to photooxidative stress. The aim of this study was to investigate whether the two RpoE-ChrR pairs cross-talk while responding to the stress. In silico analysis showed a high sequence similarity between ChrR1 and ChrR2 proteins, but differences in redox sensitivity. Using in silico and in vitro methods of protein-protein interaction, we have shown that both ChrR1 and ChrR2 proteins physically bind to their noncognate RpoE proteins. Restoration of the phenotypes of chrR1::Tn5 and chrR2::Km mutants related to carotenoid biosynthesis and photooxidative stress tolerance by expressing chrR1 or chrR2 provided in vivo evidence for the cross-talk. In addition, up- or down-regulation of several identical proteins by expressing chrR1 or chrR2 in the chrR1::Tn5 mutant provided another in vivo evidence for the cross-talk. Although multiple redox-sensitive ZAS anti-σ factors occur in some Gram-positive bacteria, no cross-talk is reported among them. We report here, for the first time, that the two ZAS anti-σ factors of A. brasilense also interact with their noncognate σ factors and affect gene expression. The two redox-sensitive ZAS anti-σ factors in A. brasilense may interact with their cognate as well as noncognate ECF σ factors to play an important role in redox homeostasis by facilitating recovery from the oxidative stress.

A mammary cell-specific enhancer in mouse mammary tumor virus DNA is composed of multiple regulatory elements including binding sites for CTF/NFI and a novel transcription factor, mammary cell-activating factor.

PubMed Central

Mink, S; Härtig, E; Jennewein, P; Doppler, W; Cato, A C

1992-01-01

Mouse mammary tumor virus (MMTV) is a milk-transmitted retrovirus involved in the neoplastic transformation of mouse mammary gland cells. The expression of this virus is regulated by mammary cell type-specific factors, steroid hormones, and polypeptide growth factors. Sequences for mammary cell-specific expression are located in an enhancer element in the extreme 5' end of the long terminal repeat region of this virus. This enhancer, when cloned in front of the herpes simplex thymidine kinase promoter, endows the promoter with mammary cell-specific response. Using functional and DNA-protein-binding studies with constructs mutated in the MMTV long terminal repeat enhancer, we have identified two main regulatory elements necessary for the mammary cell-specific response. These elements consist of binding sites for a transcription factor in the family of CTF/NFI proteins and the transcription factor mammary cell-activating factor (MAF) that recognizes the sequence G Pu Pu G C/G A A G G/T. Combinations of CTF/NFI- and MAF-binding sites or multiple copies of either one of these binding sites but not solitary binding sites mediate mammary cell-specific expression. The functional activities of these two regulatory elements are enhanced by another factor that binds to the core sequence ACAAAG. Interdigitated binding sites for CTF/NFI, MAF, and/or the ACAAAG factor are also found in the 5' upstream regions of genes encoding whey milk proteins from different species. These findings suggest that mammary cell-specific regulation is achieved by a concerted action of factors binding to multiple regulatory sites. Images PMID:1328867

SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family.

PubMed

Antonets, Denis V; Nepomnyashchikh, Tatyana S; Shchelkunov, Sergei N

2010-10-27

Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein.

Factor H-related proteins.

PubMed

Józsi, Mihály; Meri, Seppo

2014-01-01

Factor H-related proteins (CFHRs) are plasma glycoproteins related in structure and antigenicity to each other and to the complement inhibitory protein factor H. Such proteins are found in most mammals but their number and domain composition vary. This chapter summarizes our current knowledge on the human factor H-related proteins. In contrast to factor H, they have no strong complement inhibitory activity, although for some of them regulatory or complement modulatory activity has been reported. A common feature of CFHRs is that they bind to the C3b component of complement. Novel links between CFHRs and various diseases (C3 glomerulopathies, atypical hemolytic uremic syndrome and age-related macular degeneration) have been revealed in recent years, but we are still far from understanding their biological function.

Serum lipopolysaccharide-binding protein prediction of severe bacterial infection in cirrhotic patients with ascites.

PubMed

Albillos, Agustín; de-la-Hera, Antonio; Alvarez-Mon, Melchor

2004-05-15

Serum lipopolysaccharide-binding protein is increased in a subset of non-infected ascitic cirrhotic patients, a finding previously related to bacterial passage from the gut to the circulation without overt infection. We prospectively analysed the risk factors associated with a first episode of severe bacterial infection in 84 ascitic cirrhotics, followed up for a median of 46 weeks. The cumulative probability of such infection in patients with raised and normal lipopolysaccharide-binding protein was 32.4% and 8.0% (p=0.004), respectively. Increased lipopolysaccharide-binding protein was the only factor independently associated with severe bacterial infection in a multivariate analysis (relative risk 4.49, 95% CI 1.42-14.1). Monitoring of serum lipopolysaccharide-binding protein could, therefore, help to target cirrhotic patients with ascites for antibiotic prophylaxis.

Odorant-binding proteins from a primitive termite.

PubMed

Ishida, Yuko; Chiang, Vicky P; Haverty, Michael I; Leal, Walter S

2002-09-01

Hitherto, odorant-binding proteins (OBPs) have been identified from insects belonging to more highly evolved insect orders (Lepidoptera, Coleoptera, Diptera, Hymenoptera, and Hemiptera), whereas only chemosensory proteins have been identified from more primitive species, such as orthopteran and phasmid species. Here, we report for the first time the isolation and cloning of odorant-binding proteins from a primitive termite species, the dampwood termite. Zootermopsis nevadensis nevadensis (Isoptera: Termopsidae). A major antennae-specific protein was detected by native PAGE along with four other minor proteins, which were also absent in the extract from control tissues (hindlegs). Multiple cDNA cloning led to the full characterization of the major antennae-specific protein (ZnevOBP1) and to the identification of two other antennae-specific cDNAs, encoding putative odorant-binding proteins (ZnevOBP2 and ZnevOBP3). N-terminal amino acid sequencing of the minor antennal bands and cDNA cloning showed that olfaction in Z. n. nevadensis may involve multiple odorant-binding proteins. Database searches suggest that the OBPs from this primitive termite are homologues of the pheromone-binding proteins from scarab beetles and antennal-binding proteins from moths.

La-related protein 1 (LARP1) binds the mRNA cap, blocking eIF4F assembly on TOP mRNAs.

PubMed

Lahr, Roni M; Fonseca, Bruno D; Ciotti, Gabrielle E; Al-Ashtal, Hiba A; Jia, Jian-Jun; Niklaus, Marius R; Blagden, Sarah P; Alain, Tommy; Berman, Andrea J

2017-04-07

The 5'terminal oligopyrimidine (5'TOP) motif is a cis -regulatory RNA element located immediately downstream of the 7-methylguanosine [m 7 G] cap of TOP mRNAs, which encode ribosomal proteins and translation factors. In eukaryotes, this motif coordinates the synchronous and stoichiometric expression of the protein components of the translation machinery. La-related protein 1 (LARP1) binds TOP mRNAs, regulating their stability and translation. We present crystal structures of the human LARP1 DM15 region in complex with a 5'TOP motif, a cap analog (m 7 GTP), and a capped cytidine (m 7 GpppC), resolved to 2.6, 1.8 and 1.7 Å, respectively. Our binding, competition, and immunoprecipitation data corroborate and elaborate on the mechanism of 5'TOP motif binding by LARP1. We show that LARP1 directly binds the cap and adjacent 5'TOP motif of TOP mRNAs, effectively impeding access of eIF4E to the cap and preventing eIF4F assembly. Thus, LARP1 is a specialized TOP mRNA cap-binding protein that controls ribosome biogenesis.

Computational search for aflatoxin binding proteins

NASA Astrophysics Data System (ADS)

Wang, Ying; Liu, Jinfeng; Zhang, Lujia; He, Xiao; Zhang, John Z. H.

2017-10-01

Aflatoxin is one of the mycotoxins that contaminate various food products. Among various aflatoxin types (B1, B2, G1, G2 and M1), aflatoxin B1 is the most important and the most toxic one. In this study, through computational screening, we found that several proteins may bind specifically with different type of aflatoxins. Combination of theoretical methods including target fishing, molecular docking, molecular dynamics (MD) simulation, MM/PBSA calculation were utilized to search for new aflatoxin B1 binding proteins. A recently developed method for calculating entropic contribution to binding free energy called interaction entropy (IE) was employed to compute the binding free energy between the protein and aflatoxin B1. Through comprehensive comparison, three proteins, namely, trihydroxynaphthalene reductase, GSK-3b, and Pim-1 were eventually selected as potent aflatoxin B1 binding proteins. GSK-3b and Pim-1 are drug targets of cancers or neurological diseases. GSK-3b is the strongest binder for aflatoxin B1.

The NS5A-binding heat shock proteins HSC70 and HSP70 play distinct roles in the hepatitis C viral life cycle.

PubMed

Khachatoorian, Ronik; Ganapathy, Ekambaram; Ahmadieh, Yasaman; Wheatley, Nicole; Sundberg, Christopher; Jung, Chun-Ling; Arumugaswami, Vaithilingaraja; Raychaudhuri, Santanu; Dasgupta, Asim; French, Samuel W

2014-04-01

We previously identified HSP70 and HSC70 in complex with NS5A in a proteomic screen. Here, coimmunoprecipitation studies confirmed NS5A/HSC70 complex formation during infection, and immunofluorescence studies showed NS5A and HSC70 to colocalize. Unlike HSP70, HSC70 knockdown did not decrease viral protein levels. Rather, intracellular infectious virion assembly was significantly impaired by HSC70 knockdown. We also discovered that both HSC70 nucleotide binding and substrate binding domains directly bind NS5A whereas only the HSP70 nucleotide binding domain does. Knockdown of both HSC70 and HSP70 demonstrated an additive reduction in virus production. This data suggests that HSC70 and HSP70 play discrete roles in the viral life cycle. Investigation of these different functions may facilitate developing of novel strategies that target host proteins to treat HCV infection. Copyright © 2014 Elsevier Inc. All rights reserved.

Simulation of Reversible Protein–Protein Binding and Calculation of Binding Free Energies Using Perturbed Distance Restraints

PubMed Central

2017-01-01

Virtually all biological processes depend on the interaction between proteins at some point. The correct prediction of biomolecular binding free-energies has many interesting applications in both basic and applied pharmaceutical research. While recent advances in the field of molecular dynamics (MD) simulations have proven the feasibility of the calculation of protein–protein binding free energies, the large conformational freedom of proteins and complex free energy landscapes of binding processes make such calculations a difficult task. Moreover, convergence and reversibility of resulting free-energy values remain poorly described. In this work, an easy-to-use, yet robust approach for the calculation of standard-state protein–protein binding free energies using perturbed distance restraints is described. In the binding process the conformations of the proteins were restrained, as suggested earlier. Two approaches to avoid end-state problems upon release of the conformational restraints were compared. The method was evaluated by practical application to a small model complex of ubiquitin and the very flexible ubiquitin-binding domain of human DNA polymerase ι (UBM2). All computed free energy differences were closely monitored for convergence, and the calculated binding free energies had a mean unsigned deviation of only 1.4 or 2.5 kJ·mol–1 from experimental values. Statistical error estimates were in the order of thermal noise. We conclude that the presented method has promising potential for broad applicability to quantitatively describe protein–protein and various other kinds of complex formation. PMID:28898077

Nuclear proteins that bind the human gamma-globin gene promoter: alterations in binding produced by point mutations associated with hereditary persistence of fetal hemoglobin.

PubMed Central

Gumucio, D L; Rood, K L; Gray, T A; Riordan, M F; Sartor, C I; Collins, F S

1988-01-01

The molecular mechanisms responsible for the human fetal-to-adult hemoglobin switch have not yet been elucidated. Point mutations identified in the promoter regions of gamma-globin genes from individuals with nondeletion hereditary persistence of fetal hemoglobin (HPFH) may mark cis-acting sequences important for this switch, and the trans-acting factors which interact with these sequences may be integral parts in the puzzle of gamma-globin gene regulation. We have used gel retardation and footprinting strategies to define nuclear proteins which bind to the normal gamma-globin promoter and to determine the effect of HPFH mutations on the binding of a subset of these proteins. We have identified five proteins in human erythroleukemia cells (K562 and HEL) which bind to the proximal promoter region of the normal gamma-globin gene. One factor, gamma CAAT, binds the duplicated CCAAT box sequences; the -117 HPFH mutation increases the affinity of interaction between gamma CAAT and its cognate site. Two proteins, gamma CAC1 and gamma CAC2, bind the CACCC sequence. These proteins require divalent cations for binding. The -175 HPFH mutation interferes with the binding of a fourth protein, gamma OBP, which binds an octamer sequence (ATGCAAAT) in the normal gamma-globin promoter. The HPFH phenotype of the -175 mutation indicates that the octamer-binding protein may play a negative regulatory role in this setting. A fifth protein, EF gamma a, binds to sequences which overlap the octamer-binding site. The erythroid-specific distribution of EF gamma a and its close approximation to an apparent repressor-binding site suggest that it may be important in gamma-globin regulation. Images PMID:2468996

Developmental regulation of collagenase-3 mRNA in normal, differentiating osteoblasts through the activator protein-1 and the runt domain binding sites

NASA Technical Reports Server (NTRS)

Winchester, S. K.; Selvamurugan, N.; D'Alonzo, R. C.; Partridge, N. C.

2000-01-01

Collagenase-3 mRNA is initially detectable when osteoblasts cease proliferation, increasing during differentiation and mineralization. We showed that this developmental expression is due to an increase in collagenase-3 gene transcription. Mutation of either the activator protein-1 or the runt domain binding site decreased collagenase-3 promoter activity, demonstrating that these sites are responsible for collagenase-3 gene transcription. The activator protein-1 and runt domain binding sites bind members of the activator protein-1 and core-binding factor family of transcription factors, respectively. We identified core-binding factor a1 binding to the runt domain binding site and JunD in addition to a Fos-related antigen binding to the activator protein-1 site. Overexpression of both c-Fos and c-Jun in osteoblasts or core-binding factor a1 increased collagenase-3 promoter activity. Furthermore, overexpression of c-Fos, c-Jun, and core-binding factor a1 synergistically increased collagenase-3 promoter activity. Mutation of either the activator protein-1 or the runt domain binding site resulted in the inability of c-Fos and c-Jun or core-binding factor a1 to increase collagenase-3 promoter activity, suggesting that there is cooperative interaction between the sites and the proteins. Overexpression of Fra-2 and JunD repressed core-binding factor a1-induced collagenase-3 promoter activity. Our results suggest that members of the activator protein-1 and core-binding factor families, binding to the activator protein-1 and runt domain binding sites are responsible for the developmental regulation of collagenase-3 gene expression in osteoblasts.

Calcium binding to Procambarus clarkii sarcoplasmic calcium binding protein splice variants.

PubMed

Rohrback, Suzanne E; Wheatly, Michele G; Gillen, Christopher M

2015-01-01

Sarcoplasmic calcium binding protein (SCP) is a high-affinity calcium buffering protein expressed in muscle of crayfish and other invertebrates. In previous work, we identified three splice variants of Procambarus clarkii SCP (pcSCP1a, pcSCP1b, and pcSCP1c) that differ in a 37 amino acid region that lies mainly between the 2nd and 3ed EF-hand calcium binding domain. To evaluate the function of the proteins encoded by the pcSCP1 transcripts, we produced recombinant pcSCP1 and used tryptophan fluorescence to characterize calcium binding. Tryptophan fluorescence of pcSCP1a decreased in response to increased calcium, while tryptophan fluorescence of the pcSCP1b and pcSCP1c variants increased. We estimated calcium binding constants and Hill coefficients with two different equations: the standard Hill equation and a modified Hill equation that accounts for contributions from two different tryptophans. The approaches gave similar results. Steady-state calcium binding constants (Kd) ranged from 2.7±0.7×10(-8)M to 5.6±0.1×10(-7)M, consistent with previous work. Variants displayed significantly different apparent calcium affinities, which were decreased in the presence of magnesium. Calcium Kd was lowest for pcSCP1a and highest for pcSCP1c. Site-directed mutagenesis of pcSCP1c residues to the amino acids of pcSCP1b decreased the calcium Kd, identifying residues outside the EF-hand domains that contribute to calcium binding in crayfish SCP. Copyright © 2014 Elsevier Inc. All rights reserved.

Insulin- like Growth Factor-Binding Protein Action in Bone Tissue: A Key Role for Pregnancy- Associated Plasma Protein-A.

PubMed

Beattie, James; Al-Khafaji, Hasanain; Noer, Pernille R; Alkharobi, Hanaa Esa; Alhodhodi, Aishah; Meade, Josephine; El-Gendy, Reem; Oxvig, Claus

2018-01-01

The insulin-like growth factor (IGF) axis is required for the differentiation, development, and maintenance of bone tissue. Accordingly, dysregulation of this axis is associated with various skeletal pathologies including growth abnormalities and compromised bone structure. It is becoming increasingly apparent that the action of the IGF axis must be viewed holistically taking into account not just the actions of the growth factors and receptors, but also the influence of soluble high affinity IGF binding proteins (IGFBPs).There is a recognition that IGFBPs exert IGF-dependent and IGF-independent effects in bone and other tissues and that an understanding of the mechanisms of action of IGFBPs and their regulation in the pericellular environment impact critically on tissue physiology. In this respect, a group of IGFBP proteinases (which may be considered as ancillary members of the IGF axis) play a crucial role in regulating IGFBP function. In this model, cleavage of IGFBPs by specific proteinases into fragments with lower affinity for growth factor(s) regulates the partition of IGFs between IGFBPs and cell surface IGF receptors. In this review, we examine the importance of IGFBP function in bone tissue with special emphasis on the role of pregnancy associated plasma protein-A (PAPP-A). We examine the function of PAPP-A primarily as an IGFBP-4 proteinase and present evidence that PAPP-A induced cleavage of IGFBP-4 is potentially a key regulatory step in bone metabolism. We also highlight some recent findings with regard to IGFBP-2 and IGFBP-5 (also PAPP-A substrates) function in bone tissue and briefly discuss the actions of the other three IGFBPs (-1, -3, and -6) in this tissue. Although our main focus will be in bone we will allude to IGFBP activity in other cells and tissues where appropriate.

Tighter Ligand Binding Can Compensate for Impaired Stability of an RNA-Binding Protein.

PubMed

Wallis, Christopher P; Richman, Tara R; Filipovska, Aleksandra; Rackham, Oliver

2018-06-15

It has been widely shown that ligand-binding residues, by virtue of their orientation, charge, and solvent exposure, often have a net destabilizing effect on proteins that is offset by stability conferring residues elsewhere in the protein. This structure-function trade-off can constrain possible adaptive evolutionary changes of function and may hamper protein engineering efforts to design proteins with new functions. Here, we present evidence from a large randomized mutant library screen that, in the case of PUF RNA-binding proteins, this structural relationship may be inverted and that active-site mutations that increase protein activity are also able to compensate for impaired stability. We show that certain mutations in RNA-protein binding residues are not necessarily destabilizing and that increased ligand-binding can rescue an insoluble, unstable PUF protein. We hypothesize that these mutations restabilize the protein via thermodynamic coupling of protein folding and RNA binding.

hPOC5 is a centrin-binding protein required for assembly of full-length centrioles.

PubMed

Azimzadeh, Juliette; Hergert, Polla; Delouvée, Annie; Euteneuer, Ursula; Formstecher, Etienne; Khodjakov, Alexey; Bornens, Michel

2009-04-06

Centrin has been shown to be involved in centrosome biogenesis in a variety of eukaryotes. In this study, we characterize hPOC5, a conserved centrin-binding protein that contains Sfi1p-like repeats. hPOC5 is localized, like centrin, in the distal portion of human centrioles. hPOC5 recruitment to procentrioles occurs during G2/M, a process that continues up to the full maturation of the centriole during the next cell cycle and is correlated with hyperphosphorylation of the protein. In the absence of hPOC5, RPE1 cells arrest in G1 phase, whereas HeLa cells show an extended S phase followed by cell death. We show that hPOC5 is not required for the initiation of procentriole assembly but is essential for building the distal half of centrioles. Interestingly, the hPOC5 family reveals an evolutionary divergence between vertebrates and organisms like Drosophila melanogaster or Caenorhabditis elegans, in which the loss of hPOC5 may correlate with the conspicuous differences in centriolar structure.

Potential use of sugar binding proteins in reactors for regeneration of CO2 fixation acceptor D-Ribulose-1,5-bisphosphate

PubMed Central

Mahato, Sourav; De, Debojyoti; Dutta, Debajyoti; Kundu, Moloy; Bhattacharya, Sumana; Schiavone, Marc T; Bhattacharya, Sanjoy K

2004-01-01

Sugar binding proteins and binders of intermediate sugar metabolites derived from microbes are increasingly being used as reagents in new and expanding areas of biotechnology. The fixation of carbon dioxide at emission source has recently emerged as a technology with potentially significant implications for environmental biotechnology. Carbon dioxide is fixed onto a five carbon sugar D-ribulose-1,5-bisphosphate. We present a review of enzymatic and non-enzymatic binding proteins, for 3-phosphoglycerate (3PGA), 3-phosphoglyceraldehyde (3PGAL), dihydroxyacetone phosphate (DHAP), xylulose-5-phosphate (X5P) and ribulose-1,5-bisphosphate (RuBP) which could be potentially used in reactors regenerating RuBP from 3PGA. A series of reactors combined in a linear fashion has been previously shown to convert 3-PGA, (the product of fixed CO2 on RuBP as starting material) into RuBP (Bhattacharya et al., 2004; Bhattacharya, 2001). This was the basis for designing reactors harboring enzyme complexes/mixtures instead of linear combination of single-enzyme reactors for conversion of 3PGA into RuBP. Specific sugars in such enzyme-complex harboring reactors requires removal at key steps and fed to different reactors necessitating reversible sugar binders. In this review we present an account of existing microbial sugar binding proteins and their potential utility in these operations. PMID:15175111

Extensive Use of RNA-Binding Proteins in Drosophila Sensory Neuron Dendrite Morphogenesis

PubMed Central

Olesnicky, Eugenia C.; Killian, Darrell J.; Garcia, Evelyn; Morton, Mary C.; Rathjen, Alan R.; Sola, Ismail E.; Gavis, Elizabeth R.

2013-01-01

The large number of RNA-binding proteins and translation factors encoded in the Drosophila and other metazoan genomes predicts widespread use of post-transcriptional regulation in cellular and developmental processes. Previous studies identified roles for several RNA-binding proteins in dendrite branching morphogenesis of Drosophila larval sensory neurons. To determine the larger contribution of post-transcriptional gene regulation to neuronal morphogenesis, we conducted an RNA interference screen to identify additional Drosophila proteins annotated as either RNA-binding proteins or translation factors that function in producing the complex dendritic trees of larval class IV dendritic arborization neurons. We identified 88 genes encoding such proteins whose knockdown resulted in aberrant dendritic morphology, including alterations in dendritic branch number, branch length, field size, and patterning of the dendritic tree. In particular, splicing and translation initiation factors were associated with distinct and characteristic phenotypes, suggesting that different morphogenetic events are best controlled at specific steps in post-transcriptional messenger RNA metabolism. Many of the factors identified in the screen have been implicated in controlling the subcellular distributions and translation of maternal messenger RNAs; thus, common post-transcriptional regulatory strategies may be used in neurogenesis and in the generation of asymmetry in the female germline and embryo. PMID:24347626

General Model for Retroviral Capsid Pattern Recognition by TRIM5 Proteins.

PubMed

Wagner, Jonathan M; Christensen, Devin E; Bhattacharya, Akash; Dawidziak, Daria M; Roganowicz, Marcin D; Wan, Yueping; Pumroy, Ruth A; Demeler, Borries; Ivanov, Dmitri N; Ganser-Pornillos, Barbie K; Sundquist, Wesley I; Pornillos, Owen

2018-02-15

Restriction factors are intrinsic cellular defense proteins that have evolved to block microbial infections. Retroviruses such as HIV-1 are restricted by TRIM5 proteins, which recognize the viral capsid shell that surrounds, organizes, and protects the viral genome. TRIM5α uses a SPRY domain to bind capsids with low intrinsic affinity ( K D of >1 mM) and therefore requires higher-order assembly into a hexagonal lattice to generate sufficient avidity for productive capsid recognition. TRIMCyp, on the other hand, binds HIV-1 capsids through a cyclophilin A domain, which has a well-defined binding site and higher affinity ( K D of ∼10 μM) for isolated capsid subunits. Therefore, it has been argued that TRIMCyp proteins have dispensed with the need for higher-order assembly to function as antiviral factors. Here, we show that, consistent with its high degree of sequence similarity with TRIM5α, the TRIMCyp B-box 2 domain shares the same ability to self-associate and facilitate assembly of a TRIMCyp hexagonal lattice that can wrap about the HIV-1 capsid. We also show that under stringent experimental conditions, TRIMCyp-mediated restriction of HIV-1 is indeed dependent on higher-order assembly. Both forms of TRIM5 therefore use the same mechanism of avidity-driven capsid pattern recognition. IMPORTANCE Rhesus macaques and owl monkeys are highly resistant to HIV-1 infection due to the activity of TRIM5 restriction factors. The rhesus macaque TRIM5α protein blocks HIV-1 through a mechanism that requires self-assembly of a hexagonal TRIM5α lattice around the invading viral core. Lattice assembly amplifies very weak interactions between the TRIM5α SPRY domain and the HIV-1 capsid. Assembly also promotes dimerization of the TRIM5α RING E3 ligase domain, resulting in synthesis of polyubiquitin chains that mediate downstream steps of restriction. In contrast to rhesus TRIM5α, the owl monkey TRIM5 homolog, TRIMCyp, binds isolated HIV-1 CA subunits much more tightly

Regulation of mRNA abundance by polypyrimidine tract-binding protein-controlled alternate 5' splice site choice.

PubMed

Hamid, Fursham M; Makeyev, Eugene V

2014-11-01

Alternative splicing (AS) provides a potent mechanism for increasing protein diversity and modulating gene expression levels. How alternate splice sites are selected by the splicing machinery and how AS is integrated into gene regulation networks remain important questions of eukaryotic biology. Here we report that polypyrimidine tract-binding protein 1 (Ptbp1/PTB/hnRNP-I) controls alternate 5' and 3' splice site (5'ss and 3'ss) usage in a large set of mammalian transcripts. A top scoring event identified by our analysis was the choice between competing upstream and downstream 5'ss (u5'ss and d5'ss) in the exon 18 of the Hps1 gene. Hps1 is essential for proper biogenesis of lysosome-related organelles and loss of its function leads to a disease called type 1 Hermansky-Pudlak Syndrome (HPS). We show that Ptbp1 promotes preferential utilization of the u5'ss giving rise to stable mRNAs encoding a full-length Hps1 protein, whereas bias towards d5'ss triggered by Ptbp1 down-regulation generates transcripts susceptible to nonsense-mediated decay (NMD). We further demonstrate that Ptbp1 binds to pyrimidine-rich sequences between the u5'ss and d5'ss and activates the former site rather than repressing the latter. Consistent with this mechanism, u5'ss is intrinsically weaker than d5'ss, with a similar tendency observed for other genes with Ptbp1-induced u5'ss bias. Interestingly, the brain-enriched Ptbp1 paralog Ptbp2/nPTB/brPTB stimulated the u5'ss utilization but with a considerably lower efficiency than Ptbp1. This may account for the tight correlation between Hps1 with Ptbp1 expression levels observed across mammalian tissues. More generally, these data expand our understanding of AS regulation and uncover a post-transcriptional strategy ensuring co-expression of a subordinate gene with its master regulator through an AS-NMD tracking mechanism.

Insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 mediate TGF-{beta}- and myostatin-induced suppression of proliferation in porcine embryonic myogenic cell cultures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kamanga-Sollo, E.; Pampusch, M.S.; White, M.E.

2005-11-15

We have previously shown that cultured porcine embryonic myogenic cells (PEMC) produce both insulin-like growth factor binding protein (IGFBP)-3 and IGFBP-5 and secrete these proteins into their media. Exogenously added recombinant porcine (rp) IGFBP-3 and rpIGFBP-5 act via IGF-dependent and IGF-independent mechanisms to suppress proliferation of PEMC cultures. Furthermore, immunoneutralization of endogenous IGFBP-3 and IGFBP-5 in the PEMC culture medium results in increased DNA synthesis rate suggesting that endogenous IGFBP-3 and IGFBP-5 suppress PEMC proliferation. TGF-{beta} superfamily members myostatin and TGF-{beta}{sub 1} have also been shown to suppress proliferation of myogenic cells, and treatment of cultured PEMC with either TGF-{beta}{submore » 1} or myostatin significantly (P < 0.01) increases levels of IGFBP-3 and -5 mRNA. We have previously shown that immunoneutralization of IGFBP-3 decreases the proliferation-suppressing activity of TGF-{beta}{sub 1} and myostatin. Here, we show that immunoneutralization of IGFBP-5 also significantly (P < 0.05) decreases the DNA synthesis-suppressing activity of these molecules. Simultaneous immunoneutralization of both IGFBP-3 and IGFBP-5 in TGF-{beta}{sub 1} or myostatin-treated PEMC cultures restores Long-R3-IGF-I-stimulated DNA synthesis rates to 90% of the levels observed in control cultures receiving no TGF-{beta}{sub 1} or myostatin treatment (P < 0.05). Even though immunoneutralization of IGFBP-3 and -5 increased DNA synthesis rates in TGF-{beta}{sub 1} or myostatin-treated PEMC cultures, phosphosmad2 levels in these cultures were not affected. These findings strongly suggest that IGFBP-3 and IGFBP-5 affect processes downstream from receptor-mediated Smad phosphorylation that facilitate the ability of TGF-{beta} and myostatin to suppress proliferation of PEMC.« less

SARNAclust: Semi-automatic detection of RNA protein binding motifs from immunoprecipitation data

PubMed Central

Dotu, Ivan; Adamson, Scott I.; Coleman, Benjamin; Fournier, Cyril; Ricart-Altimiras, Emma; Eyras, Eduardo

2018-01-01

RNA-protein binding is critical to gene regulation, controlling fundamental processes including splicing, translation, localization and stability, and aberrant RNA-protein interactions are known to play a role in a wide variety of diseases. However, molecular understanding of RNA-protein interactions remains limited; in particular, identification of RNA motifs that bind proteins has long been challenging, especially when such motifs depend on both sequence and structure. Moreover, although RNA binding proteins (RBPs) often contain more than one binding domain, algorithms capable of identifying more than one binding motif simultaneously have not been developed. In this paper we present a novel pipeline to determine binding peaks in crosslinking immunoprecipitation (CLIP) data, to discover multiple possible RNA sequence/structure motifs among them, and to experimentally validate such motifs. At the core is a new semi-automatic algorithm SARNAclust, the first unsupervised method to identify and deconvolve multiple sequence/structure motifs simultaneously. SARNAclust computes similarity between sequence/structure objects using a graph kernel, providing the ability to isolate the impact of specific features through the bulge graph formalism. Application of SARNAclust to synthetic data shows its capability of clustering 5 motifs at once with a V-measure value of over 0.95, while GraphClust achieves only a V-measure of 0.083 and RNAcontext cannot detect any of the motifs. When applied to existing eCLIP sets, SARNAclust finds known motifs for SLBP and HNRNPC and novel motifs for several other RBPs such as AGGF1, AKAP8L and ILF3. We demonstrate an experimental validation protocol, a targeted Bind-n-Seq-like high-throughput sequencing approach that relies on RNA inverse folding for oligo pool design, that can validate the components within the SLBP motif. Finally, we use this protocol to experimentally interrogate the SARNAclust motif predictions for protein ILF3. Our

Insulin-like growth factor binding proteins initiate cell death and extracellular matrix remodeling in the mammary gland.

PubMed

Flint, D J; Boutinaud, M; Tonner, E; Wilde, C J; Hurley, W; Accorsi, P A; Kolb, A F; Whitelaw, C B A; Beattie, J; Allan, G J

2005-08-01

We have demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) production by mammary epithelial cells increases dramatically during forced involution of the mammary gland in rats, mice and pigs. We proposed that growth hormone (GH) increases the survival factor IGF-I, whilst prolactin (PRL) enhances the effects of GH by decreasing the concentration of IGFBP-5, which would otherwise inhibit the actions of IGFs. To demonstrate a causal relationship between IGFBP-5 and cell death, we created transgenic mice expressing IGFBP-5, specifically, in the mammary gland. DNA content in the mammary glands of transgenic mice was decreased as early as day 10 of pregnancy. Mammary cell number and milk synthesis were both decreased by approximately 50% during the first 10 days of lactation. The concentrations of the pro-apoptotic molecule caspase-3 was increased in transgenic animals whilst the concentrations of two pro-survival molecules Bcl-2 and Bcl-x were both decreased. In order to examine whether IGFBP-5 acts by inhibiting the survival effect of IGF-I, we examined IGF receptor- and Akt-phoshorylation and showed that both were inhibited. These studies also indicated that the effects of IGFBP-5 could be mediated in part by IGF-independent effects involving potential interactions with components of the extracellular matrix involved in tissue remodeling, such as components of the plasminogen system, and the matrix metallo-proteinases (MMPs). Mammary development was normalised in transgenic mice by R3-IGF-I, an analogue of IGF-I which binds weakly to IGFBPs, although milk production was only partially restored. In contrast, treatment with prolactin was able to inhibit early involutionary processes in normal mice but was unable to prevent this in mice over-expressing IGFBP-5, although it was able to inhibit activation of MMPs. Thus, IGFBP-5 can simultaneously inhibit IGF action and activate the plasminogen system thereby coordinating cell death and tissue

Flexible DNA binding of the BTB/POZ-domain protein FBI-1.

PubMed

Pessler, Frank; Hernandez, Nouria

2003-08-01

POZ-domain transcription factors are characterized by the presence of a protein-protein interaction domain called the POZ or BTB domain at their N terminus and zinc fingers at their C terminus. Despite the large number of POZ-domain transcription factors that have been identified to date and the significant insights that have been gained into their cellular functions, relatively little is known about their DNA binding properties. FBI-1 is a BTB/POZ-domain protein that has been shown to modulate HIV-1 Tat trans-activation and to repress transcription of some cellular genes. We have used various viral and cellular FBI-1 binding sites to characterize the interaction of a POZ-domain protein with DNA in detail. We find that FBI-1 binds to inverted sequence repeats downstream of the HIV-1 transcription start site. Remarkably, it binds efficiently to probes carrying these repeats in various orientations and spacings with no particular rotational alignment, indicating that its interaction with DNA is highly flexible. Indeed, FBI-1 binding sites in the adenovirus 2 major late promoter, the c-fos gene, and the c-myc P1 and P2 promoters reveal variously spaced direct, inverted, and everted sequence repeats with the consensus sequence G(A/G)GGG(T/C)(C/T)(T/C)(C/T) for each repeat.

SECRET domain of variola virus CrmB protein can be a member of poxviral type II chemokine-binding proteins family

PubMed Central

2010-01-01

Background Variola virus (VARV) the causative agent of smallpox, eradicated in 1980, have wide spectrum of immunomodulatory proteins to evade host immunity. Recently additional biological activity was discovered for VARV CrmB protein, known to bind and inhibit tumour necrosis factor (TNF) through its N-terminal domain homologous to cellular TNF receptors. Besides binding TNF, this protein was also shown to bind with high affinity several chemokines which recruit B- and T-lymphocytes and dendritic cells to sites of viral entry and replication. Ability to bind chemokines was shown to be associated with unique C-terminal domain of CrmB protein. This domain named SECRET (Smallpox virus-Encoded Chemokine Receptor) is unrelated to the host proteins and lacks significant homology with other known viral chemokine-binding proteins or any other known protein. Findings De novo modelling of VARV-CrmB SECRET domain spatial structure revealed its apparent structural homology with cowpox virus CC-chemokine binding protein (vCCI) and vaccinia virus A41 protein, despite low sequence identity between these three proteins. Potential ligand-binding surface of modelled VARV-CrmB SECRET domain was also predicted to bear prominent electronegative charge which is characteristic to known orthopoxviral chemokine-binding proteins. Conclusions Our results suggest that SECRET should be included into the family of poxviral type II chemokine-binding proteins and that it might have been evolved from the vCCI-like predecessor protein. PMID:20979600

Fasting Induces Nuclear Factor E2-Related Factor 2 and ATP-Binding Cassette Transporters via Protein Kinase A and Sirtuin-1 in Mouse and Human

PubMed Central

Kulkarni, Supriya R.; Donepudi, Ajay C.; Xu, Jialin; Wei, Wei; Cheng, Qiuqiong C.; Driscoll, Maureen V.; Johnson, Delinda A.; Johnson, Jeffrey A.; Li, Xiaoling

2014-01-01

Abstract Aims: The purpose of this study was to determine whether 3′-5′-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) and Sirtuin-1 (SIRT1) dependent mechanisms modulate ATP-binding Cassette (ABC) transport protein expression. ABC transport proteins (ABCC2–4) are essential for chemical elimination from hepatocytes and biliary excretion. Nuclear factor-E2 related-factor 2 (NRF2) is a transcription factor that mediates ABCC induction in response to chemical inducers and liver injury. However, a role for NRF2 in the regulation of transporter expression in nonchemical models of liver perturbation is largely undescribed. Results: Here we show that fasting increased NRF2 target gene expression through NRF2- and SIRT1–dependent mechanisms. In intact mouse liver, fasting induces NRF2 target gene expression by at least 1.5 to 5-fold. In mouse and human hepatocytes, treatment with 8-Bromoadenosine-cAMP, a cAMP analogue, increased NRF2 target gene expression and antioxidant response element activity, which was decreased by the PKA inhibitor, H-89. Moreover, fasting induced NRF2 target gene expression was decreased in liver and hepatocytes of SIRT1 liver-specific null mice and NRF2-null mice. Lastly, NRF2 and SIRT1 were recruited to MAREs and Antioxidant Response Elements (AREs) in the human ABCC2 promoter. Innovation: Oxidative stress mediated NRF2 activation is well described, yet the influence of basic metabolic processes on NRF2 activation is just emerging. Conclusion: The current data point toward a novel role of nutrient status in regulation of NRF2 activity and the antioxidant response, and indicates that cAMP/PKA and SIRT1 are upstream regulators for fasting-induced activation of the NRF2-ARE pathway. Antioxid. Redox Signal. 20, 15–30. PMID:23725046

Ginsenoside F2 reduces hair loss by controlling apoptosis through the sterol regulatory element-binding protein cleavage activating protein and transforming growth factor-β pathways in a dihydrotestosterone-induced mouse model.

PubMed

Shin, Heon-Sub; Park, Sang-Yong; Hwang, Eun-Son; Lee, Don-Gil; Mavlonov, Gafurjon Turdalievich; Yi, Tae-Hoo

2014-01-01

This study was conducted to test whether ginsenoside F2 can reduce hair loss by influencing sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) and the transforming growth factor beta (TGF-β) pathway of apoptosis in dihydrotestosterone (DHT)-treated hair cells and in a DHT-induced hair loss model in mice. Results for ginsenoside F2 were compared with finasteride. DHT inhibits proliferation of hair cells and induces androgenetic alopecia and was shown to activate an apoptosis signal pathway both in vitro and in vivo. The cell-based 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that the proliferation rates of DHT-treated human hair dermal papilla cells (HHDPCs) and HaCaTs increased by 48% in the ginsenoside F2-treated group and by 12% in the finasteride-treated group. Western blot analysis showed that ginsenoside F2 decreased expression of TGF-β2 related factors involved in hair loss. The present study suggested a hair loss related pathway by changing SCAP related apoptosis pathway, which has been known to control cholesterol metabolism. SCAP, sterol regulatory element-binding protein (SREBP) and caspase-12 expression in the ginsenoside F2-treated group were decreased compared to the DHT and finasteride-treated group. C57BL/6 mice were also prepared by injection with DHT and then treated with ginsenoside F2 or finasteride. Hair growth rate, density, thickness measurements and tissue histotological analysis in these groups suggested that ginsenoside F2 suppressed hair cell apoptosis and premature entry to catagen more effectively than finasteride. Our results indicated that ginsenoside F2 decreased the expression of TGF-β2 and SCAP proteins, which have been suggested to be involved in apoptosis and entry into catagen. This study provides evidence those factors in the SCAP pathway could be targets for hair loss prevention drugs.

Identification of transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) as a novel factor for TNF-α expression upon lipopolysaccharide stimulation in human monocytes.

PubMed

Murata, H; Hattori, T; Maeda, H; Takashiba, S; Takigawa, M; Kido, J; Nagata, T

2015-08-01

Tumor necrosis factor alpha (TNF-α) is a major cytokine implicated in various inflammatory diseases. The nature of the nuclear factors associated with human TNF-α gene regulation is not well elucidated. We previously identified a novel region located from -550 to -487 in human TNF-α promoter that did not contain the reported binding sites for nuclear factor kappa B (NF-κB) but showed lipopolysaccharide (LPS)-induced transcriptional activity. The purpose of this study is to identify novel factors that bind to the promoter region and regulate TNF-α expression. To identify DNA-binding proteins that bound to the target region of TNF-α promoter, a cDNA library from LPS-stimulated human monocytic cell line THP-1 was screened using a yeast one-hybrid system. Cellular localizations of the DNA-binding protein in the cells were examined by subcellular immunocytochemistry. Nuclear amounts of the protein in LPS-stimulated THP-1 cells were identified by western blot analysis. Expression of mRNA of the protein in the cells was quantified by real-time polymerase chain reaction. Electrophoretic mobility shift assays were performed to confirm the DNA-binding profile. Overexpression of the protein and knockdown of the gene were also performed to investigate the role for TNF-α expression. Several candidates were identified from the cDNA library and transactivation-responsive DNA-binding protein 43 (TARDBP43; TDP-43) was focused on. Western blot analysis revealed that nuclear TDP-43 protein was increased in the LPS-stimulated THP-1 cells. Expression of TDP-43 mRNA was already enhanced before TNF-α induction by LPS. Electrophoretic mobility shift assay analysis showed that nuclear extracts obtained by overexpressing FLAG-tagged TDP-43 bound to the -550 to -487 TNF-α promoter fragments. Overexpression of TDP-43 in THP-1 cells resulted in an increase of TNF-α expression. Knockdown of TDP-43 in THP-1 cells downregulated TNF-α expression. We identified TDP-43 as one of the novel

Multivalent Binding of Formin-binding Protein 21 (FBP21)-Tandem-WW Domains Fosters Protein Recognition in the Pre-spliceosome*

PubMed Central

Klippel, Stefan; Wieczorek, Marek; Schümann, Michael; Krause, Eberhard; Marg, Berenice; Seidel, Thorsten; Meyer, Tim; Knapp, Ernst-Walter; Freund, Christian

2011-01-01

The high abundance of repetitive but nonidentical proline-rich sequences in spliceosomal proteins raises the question of how these known interaction motifs recruit their interacting protein domains. Whereas complex formation of these adaptors with individual motifs has been studied in great detail, little is known about the binding mode of domains arranged in tandem repeats and long proline-rich sequences including multiple motifs. Here we studied the interaction of the two adjacent WW domains of spliceosomal protein FBP21 with several ligands of different lengths and composition to elucidate the hallmarks of multivalent binding for this class of recognition domains. First, we show that many of the proteins that define the cellular proteome interacting with FBP21-WW1-WW2 contain multiple proline-rich motifs. Among these is the newly identified binding partner SF3B4. Fluorescence resonance energy transfer (FRET) analysis reveals the tandem-WW domains of FBP21 to interact with splicing factor 3B4 (SF3B4) in nuclear speckles where splicing takes place. Isothermal titration calorimetry and NMR shows that the tandem arrangement of WW domains and the multivalency of the proline-rich ligands both contribute to affinity enhancement. However, ligand exchange remains fast compared with the NMR time scale. Surprisingly, a N-terminal spin label attached to a bivalent ligand induces NMR line broadening of signals corresponding to both WW domains of the FBP21-WW1-WW2 protein. This suggests that distinct orientations of the ligand contribute to a delocalized and semispecific binding mode that should facilitate search processes within the spliceosome. PMID:21917930

Discovery and information-theoretic characterization of transcription factor binding sites that act cooperatively.

PubMed

Clifford, Jacob; Adami, Christoph

2015-09-02

Transcription factor binding to the surface of DNA regulatory regions is one of the primary causes of regulating gene expression levels. A probabilistic approach to model protein-DNA interactions at the sequence level is through position weight matrices (PWMs) that estimate the joint probability of a DNA binding site sequence by assuming positional independence within the DNA sequence. Here we construct conditional PWMs that depend on the motif signatures in the flanking DNA sequence, by conditioning known binding site loci on the presence or absence of additional binding sites in the flanking sequence of each site's locus. Pooling known sites with similar flanking sequence patterns allows for the estimation of the conditional distribution function over the binding site sequences. We apply our model to the Dorsal transcription factor binding sites active in patterning the Dorsal-Ventral axis of Drosophila development. We find that those binding sites that cooperate with nearby Twist sites on average contain about 0.5 bits of information about the presence of Twist transcription factor binding sites in the flanking sequence. We also find that Dorsal binding site detectors conditioned on flanking sequence information make better predictions about what is a Dorsal site relative to background DNA than detection without information about flanking sequence features.

The ELAV RNA-stability factor HuR binds the 5′-untranslated region of the human IGF-IR transcript and differentially represses cap-dependent and IRES-mediated translation

PubMed Central

Meng, Zheng; King, Peter H.; Nabors, L. Burt; Jackson, Nateka L.; Chen, Ching-Yi; Emanuel, Peter D.; Blume, Scott W.

2005-01-01

The type I insulin-like growth factor receptor (IGF-IR) is an integral component in the control of cell proliferation, differentiation and apoptosis. The IGF-IR mRNA contains an extraordinarily long (1038 nt) 5′-untranslated region (5′-UTR), and we have characterized a diverse series of proteins interacting with this RNA sequence which may provide for intricate regulation of IGF-IR gene expression at the translational level. Here, we report the purification and identification of one of these IGF-IR 5′-UTR-binding proteins as HuR, using a novel RNA crosslinking/RNase elution strategy. Because HuR has been predominantly characterized as a 3′-UTR-binding protein, enhancing mRNA stability and generally increasing gene expression, we sought to determine whether HuR might serve a different function in the context of its binding the IGF-IR 5′-UTR. We found that HuR consistently repressed translation initiation through the IGF-IR 5′-UTR. The inhibition of translation by HuR was concentration dependent, and could be reversed in trans by addition of a fragment of the IGF-IR 5′-UTR containing the HuR binding sites as a specific competitor, or abrogated by deletion of the third RNA recognition motif of HuR. We determined that HuR repressed translation initiation through the IGF-IR 5′-UTR in cells as well, and that siRNA knockdown of HuR markedly increased IGF-IR protein levels. Interestingly, we also found that HuR potently inhibited IGF-IR translation mediated through internal ribosome entry. Kinetic assays were performed to investigate the mechanism of translation repression by HuR and the dynamic interplay between HuR and the translation apparatus. We found that HuR, occupying a cap-distal position, significantly delayed translation initiation mediated by cap-dependent scanning, but was eventually displaced from its binding site, directly or indirectly, as a consequence of ribosomal scanning. However, HuR perpetually blocked the activity of the IGF-IR IRES

The Human Splicing Factor ASF/SF2 can Specifically Recognize Pre-mRNA 5' Splice Sites

NASA Astrophysics Data System (ADS)

Zuo, Ping; Manley, James L.

1994-04-01

ASF/SF2 is a human protein previously shown to function in in vitro pre-mRNA splicing as an essential factor necessary for all splices and also as an alternative splicing factor, capable of switching selection of 5' splice sites. To begin to study the protein's mechanism of action, we have investigated the RNA binding properties of purified recombinant ASF/SF2. Using UV crosslinking and gel shift assays, we demonstrate that the RNA binding region of ASF/SF2 can interact with RNA in a sequence-specific manner, recognizing the 5' splice site in each of two different pre-mRNAs. Point mutations in the 5' splice site consensus can reduce binding by as much as a factor of 100, with the largest effects observed in competition assays. These findings support a model in which ASF/SF2 aids in the recognition of pre-mRNA 5' splice sites.

Temporal expression of the human alcohol dehydrogenase gene family during liver development correlates with differential promoter activation by hepatocyte nuclear factor 1, CCAAT/enhancer-binding protein alpha, liver activator protein, and D-element-binding protein.

PubMed Central

van Ooij, C; Snyder, R C; Paeper, B W; Duester, G

1992-01-01

The human class I alcohol dehydrogenase (ADH) gene family consists of ADH1, ADH2, and ADH3, which are sequentially activated in early fetal, late fetal, and postnatal liver, respectively. Analysis of ADH promoters revealed differential activation by several factors previously shown to control liver transcription. In cotransfection assays, the ADH1 promoter, but not the ADH2 or ADH3 promoter, was shown to respond to hepatocyte nuclear factor 1 (HNF-1), which has previously been shown to regulate transcription in early liver development. The ADH2 promoter, but not the ADH1 or ADH3 promoter, was shown to respond to CCAAT/enhancer-binding protein alpha (C/EBP alpha), a transcription factor particularly active during late fetal liver and early postnatal liver development. The ADH1, ADH2, and ADH3 promoters all responded to the liver transcription factors liver activator protein (LAP) and D-element-binding protein (DBP), which are most active in postnatal liver. For all three promoters, the activation by LAP or DBP was higher than that seen by HNF-1 or C/EBP alpha, and a significant synergism between C/EBP alpha and LAP was noticed for the ADH2 and ADH3 promoters when both factors were simultaneously cotransfected. A hierarchy of ADH promoter responsiveness to C/EBP alpha and LAP homo- and heterodimers is suggested. In all three ADH genes, LAP bound to the same four sites previously reported for C/EBP alpha (i.e., -160, -120, -40, and -20 bp), but DBP bound strongly only to the site located at -40 bp relative to the transcriptional start. Mutational analysis of ADH2 indicated that the -40 bp element accounts for most of the promoter regulation by the bZIP factors analyzed. These studies suggest that HNF-1 and C/EBP alpha help establish ADH gene family transcription in fetal liver and that LAP and DBP help maintain high-level ADH gene family transcription in postnatal liver. Images PMID:1620113

The pathogen-related yeast protein Pry1, a member of the CAP protein superfamily, is a fatty acid-binding protein

PubMed Central

Darwiche, Rabih; Mène-Saffrané, Laurent; Gfeller, David; Asojo, Oluwatoyin A.; Schneiter, Roger

2017-01-01

Members of the CAP superfamily (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins), also known as SCP superfamily (sperm-coating proteins), have been implicated in many physiological processes, including immune defenses, venom toxicity, and sperm maturation. Their mode of action, however, remains poorly understood. Three proteins of the CAP superfamily, Pry1, -2, and -3 (pathogen related in yeast), are encoded in the Saccharomyces cerevisiae genome. We have shown previously that Pry1 binds cholesterol in vitro and that Pry function is required for sterol secretion in yeast cells, indicating that members of this superfamily may generally bind sterols or related small hydrophobic compounds. On the other hand, tablysin-15, a CAP protein from the horsefly Tabanus yao, has been shown to bind leukotrienes and free fatty acids in vitro. Therefore, here we assessed whether the yeast Pry1 protein binds fatty acids. Computational modeling and site-directed mutagenesis indicated that the mode of fatty acid binding is conserved between tablysin-15 and Pry1. Pry1 bound fatty acids with micromolar affinity in vitro, and its function was essential for fatty acid export in cells lacking the acyl-CoA synthetases Faa1 and Faa4. Fatty acid binding of Pry1 is independent of its capacity to bind sterols, and the two sterol- and fatty acid-binding sites are nonoverlapping. These results indicate that some CAP family members, such as Pry1, can bind different lipids, particularly sterols and fatty acids, at distinct binding sites, suggesting that the CAP domain may serve as a stable, secreted protein domain that can accommodate multiple ligand-binding sites. PMID:28365570

A novel mechanism of RNase L inhibition: Theiler's virus L* protein prevents 2-5A from binding to RNase L

PubMed Central

Drappier, Melissa; Elliott, Ruth; Zhang, Rong; Weiss, Susan R.; Silverman, Robert H.

2018-01-01

The OAS/RNase L pathway is one of the best-characterized effector pathways of the IFN antiviral response. It inhibits the replication of many viruses and ultimately promotes apoptosis of infected cells, contributing to the control of virus spread. However, viruses have evolved a range of escape strategies that act against different steps in the pathway. Here we unraveled a novel escape strategy involving Theiler’s murine encephalomyelitis virus (TMEV) L* protein. Previously we found that L* was the first viral protein binding directly RNase L. Our current data show that L* binds the ankyrin repeats R1 and R2 of RNase L and inhibits 2’-5’ oligoadenylates (2-5A) binding to RNase L. Thereby, L* prevents dimerization and oligomerization of RNase L in response to 2-5A. Using chimeric mouse hepatitis virus (MHV) expressing TMEV L*, we showed that L* efficiently inhibits RNase L in vivo. Interestingly, those data show that L* can functionally substitute for the MHV-encoded phosphodiesterase ns2, which acts upstream of L* in the OAS/RNase L pathway, by degrading 2-5A. PMID:29652922

Downregulation of insulin-like growth factor binding protein 3 and 5 in nitrofen-induced pulmonary hypoplasia.

PubMed

Ruttenstock, Elke; Doi, Takashi; Dingemann, Jens; Puri, Prem

2010-01-01

The high mortality in congenital diaphragmatic hernia (CDH) is mainly attributed to pulmonary hypoplasia. Recent studies suggest that retinoid signaling pathway (RSP) is inhibited in the nitrofen-induced hypoplastic lung. The insulin-like growth factor (IGF) system plays a crucial role in fetal lung development by interaction of IGFBP-3 and IGFBP-5 with RSP. We hypothesized that pulmonary IGFBP-3 and IGFBP-5 gene expression levels are downregulated in the nitrofen-induced pulmonary hypoplasia. Pregnant rats were exposed to either olive oil or 100 mg nitrofen on day 9.5 (D9.5) of gestation. Fetal lungs were harvested on D18 and D21 and divided into control and nitrofen groups. IGFBP-3 and IGFBP-5 pulmonary gene and protein expression were determined using real-time RT-PCR and immunohistochemistry. Relative levels of IGFBP-3 mRNA were significantly decreased in the nitrofen group (8.00 +/- 14.44) in D21 compared to controls (14.81 +/- 16.11; p < 0.05). Expression levels of IGFBP-5 mRNA were also significantly decreased in nitrofen group (10.66 +/- 4.83) on D18 compared to controls (17.92 +/- 4.77). Immunohistochemistry showed decreased IGFBP-3 expression on D21 and decreased IGFBP-5 immunoreactivity on D18 in hypoplastic lungs compared to controls. Downregulation of IGFBP-3 and IGFBP-5 gene expression may cause pulmonary hypoplasia in the nitrofen-induced CDH model by interfering with retinoid signaling pathway.

Esophageal cancer alters the expression of nuclear pore complex binding protein Hsc70 and eIF5A-1.

PubMed

Moghanibashi, Mehdi; Rastgar Jazii, Ferdous; Soheili, Zahra-Soheila; Zare, Maryam; Karkhane, Aliasghar; Parivar, Kazem; Mohamadynejad, Parisa

2013-06-01

Nuclear pore complex (NPC) is the only corridor for macromolecules exchange between nucleus and cytoplasm. NPC and its components, nucleoporins, play important role in the diverse physiological processes including macromolecule exchange, chromosome segregation, apoptosis and gene expression. Recent reports also suggest involvement of nucleoporins in carcinogenesis. Applying proteomics, we analyzed expression pattern of the NPC components in a newly established esophageal cancer cell line from Persia (Iran), the high-risk region for esophageal cancer. Our results indicate overexpression of Hsc70 and downregulation of subunit alpha type-3 of proteasome, calpain small subunit 1, and eIF5A-1. Among these proteins, Hsc70 and eIF5A-1 are in direct interaction with NPC and involved in the nucleocytoplasmic exchange. Hsc70 plays a critical role as a chaperone in the formation of a cargo-receptor complex in nucleocytoplasmic transport. On the other hand, it is an NPC-associated protein that binds to nucleoporins and contributes in recycling of the nucleocytoplasmic transport receptors in mammals and affects transport of proteins between nucleus and cytoplasm. The other nuclear pore interacting protein: eIF5A-1 binds to the several nucleoporins and participates in nucleocytoplasmic transport. Altered expression of Hsc70 and eIF5A-1 may cause defects in nucleocytoplasmic transport and play a role in esophageal carcinogenesis.

Depletion of elongation initiation factor 4E binding proteins by CRISPR/Cas9 genome editing enhances antiviral response in porcine cells

USDA-ARS?s Scientific Manuscript database

Type I interferons (IFN) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF7), the master regulator of IFN transcription. The role of 4EBPs in the negat...

Characterization of the dextran-binding domain in the glucan-binding protein C of Streptococcus mutans.

PubMed

Takashima, Y; Fujita, K; Ardin, A C; Nagayama, K; Nomura, R; Nakano, K; Matsumoto-Nakano, M

2015-10-01

Streptococcus mutans produces multiple glucan-binding proteins (Gbps), among which GbpC encoded by the gbpC gene is known to be a cell-surface-associated protein involved in dextran-induced aggregation. The purpose of the present study was to characterize the dextran-binding domain of GbpC using bioinformatics analysis and molecular techniques. Bioinformatics analysis specified five possible regions containing molecular binding sites termed GB1 through GB5. Next, truncated recombinant GbpC (rGbpC) encoding each region was produced using a protein expression vector and five deletion mutant strains were generated, termed CDGB1 through CDGB5 respectively. The dextran-binding rates of truncated rGbpC that included the GB1, GB3, GB4 and GB5 regions in the upstream sequences were higher than that of the construct containing GB2 in the downstream region. In addition, the rates of dextran-binding for strains CDGB4 and CD1, which was entire gbpC deletion mutant, were significantly lower than for the other strains, while those of all other deletion mutants were quite similar to that of the parental strain MT8148. Biofilm structures formed by CDGB4 and CD1 were not as pronounced as that of MT8148, while those formed by other strains had greater density as compared to that of CD1. Our results suggest that the dextran-binding domain may be located in the GB4 region in the interior of the gbpC gene. Bioinformatics analysis is useful for determination of functional domains in many bacterial species. © 2015 The Society for Applied Microbiology.

Protein-Binding RNA Aptamers Affect Molecular Interactions Distantly from Their Binding Sites

PubMed Central

Dupont, Daniel M.; Thuesen, Cathrine K.; Bøtkjær, Kenneth A.; Behrens, Manja A.; Dam, Karen; Sørensen, Hans P.; Pedersen, Jan S.; Ploug, Michael; Jensen, Jan K.; Andreasen, Peter A.

2015-01-01

Nucleic acid aptamer selection is a powerful strategy for the development of regulatory agents for molecular intervention. Accordingly, aptamers have proven their diligence in the intervention with serine protease activities, which play important roles in physiology and pathophysiology. Nonetheless, there are only a few studies on the molecular basis underlying aptamer-protease interactions and the associated mechanisms of inhibition. In the present study, we use site-directed mutagenesis to delineate the binding sites of two 2´-fluoropyrimidine RNA aptamers (upanap-12 and upanap-126) with therapeutic potential, both binding to the serine protease urokinase-type plasminogen activator (uPA). We determine the subsequent impact of aptamer binding on the well-established molecular interactions (plasmin, PAI-1, uPAR, and LRP-1A) controlling uPA activities. One of the aptamers (upanap-126) binds to the area around the C-terminal α-helix in pro-uPA, while the other aptamer (upanap-12) binds to both the β-hairpin of the growth factor domain and the kringle domain of uPA. Based on the mapping studies, combined with data from small-angle X-ray scattering analysis, we construct a model for the upanap-12:pro-uPA complex. The results suggest and highlight that the size and shape of an aptamer as well as the domain organization of a multi-domain protein such as uPA, may provide the basis for extensive sterical interference with protein ligand interactions considered distant from the aptamer binding site. PMID:25793507

Mitogenicity of M5 protein extracted from Streptococcus pyogenes cells is due to streptococcal pyrogenic exotoxin C and mitogenic factor MF.

PubMed Central

Schmidt, K H; Gerlach, D; Wollweber, L; Reichardt, W; Mann, K; Ozegowski, J H; Fleischer, B

1995-01-01

M proteins of Streptococcus pyogenes are virulence factors which impede phagocytosis, bind to many plasma proteins, and induce formation of cross-reactive autoimmune antibodies. Recently, it has been reported that some M proteins, extracted with pepsin from streptococci (pep M), are superantigens. One of these, pep M5, was investigated in detail and was shown to stimulate human T cells bearing V beta 2, V beta 4, and V beta 8. In the present study, we extracted and purified M5 protein by different biochemical methods from two M type 5 group A streptococcal strains. The crude extracts were fractionated by affinity chromatography and ion-exchange chromatography. All fractions were tested in parallel for M protein by immunoblotting and for T-cell-stimulating activity. Although several crude preparations of M5 protein were associated with mitogenicity for V beta 2 and V beta 8 T cells, the M5 proteins, irrespective of the extraction method, could be purified to the extent that they were no longer mitogenic. The mitogenic activity was not destroyed during the purification procedures but was found in fractions separated from M protein. In these fractions, streptococcal pyrogenic exotoxin C and mitogenic factor MF could be detected by protein blotting and enzyme-linked immunosorbent assay. Moreover, anti-M protein sera did not inhibit the mitogenic activity of crude extracts, but antisera which contained anti-streptococcal pyrogenic exotoxin C antibodies showed inhibition. The inability of M5 protein to stimulate T cells was confirmed with recombinant pep M5 produced in Escherichia coli. Our data strongly suggest that the mitogenic activity in M protein preparations is caused by traces of streptococcal superantigens different from M protein. PMID:7591107

The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

PubMed Central

Gong, Wei; He, Kun; Covington, Mike; Dinesh-Kumar, S. P.; Snyder, Michael; Harmer, Stacey L.; Zhu, Yu-Xian; Deng, Xing Wang

2009-01-01

We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and protein-protein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale. PMID:19802365

Human granulocyte/pollen-binding protein. Recognition and identification as transferrin.

PubMed Central

Sass-Kuhn, S P; Moqbel, R; Mackay, J A; Cromwell, O; Kay, A B

1984-01-01

Normal human serum was found to contain a heat-stable protein which promoted the binding of granulocytes to timothy grass pollen (granulocyte/pollen-binding protein [GPBP]). GPBP was purified by gel filtration, anion exchange, and affinity chromatography. Virtually all of the granulocyte/pollen-binding activity was associated with a beta-1-protein having a molecular mass of approximately 77,000 D and an isoelectric point of between 5.5 and 6.1. By immunoelectrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the protein was identified as transferrin. Monospecific antisera raised against either GPBP or transferrin removed biological activity from GPBP preparations, and GPBP and transferrin gave lines of identity with these two antisera. The apparent heterogeneity in the molecular size and charge of GPBP observed during progressive purification was minimal when GPBP was saturated with ferric ions before the separation procedures. These experiments indicate that granulocyte/pollen binding is a hitherto unrecognized property of transferrin which appears to be unrelated to iron transport and raises the possibility that transferrin might have a physiological role in the removal of certain organic matter. Images PMID:6690479

Novel fluorescent labelled affinity probes for diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A)-binding studies.

PubMed

Wright, Michael; Miller, Andrew D

2006-02-15

Tandem synthetic-biosynthetic procedures were used to prepare two novel fluorescent labelled affinity probes for diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A)-binding studies. These compounds (dial-mant-Ap4A and azido-mant-Ap4A) are shown to clearly distinguish known Ap4A-binding proteins from Escherichia coli (LysU and GroEL) and a variety of other control proteins. Successful labelling of chaperonin GroEL appears to be allosteric with respect to the well-characterized adenosine 5'-triphosphate (ATP)-binding site, suggesting that GroEL possesses a distinct Ap4A-binding site.

The RNA-binding protein Spo5 promotes meiosis II by regulating cyclin Cdc13 in fission yeast.

PubMed

Arata, Mayumi; Sato, Masamitsu; Yamashita, Akira; Yamamoto, Masayuki

2014-03-01

Meiosis comprises two consecutive nuclear divisions, meiosis I and II. Despite this unique progression through the cell cycle, little is known about the mechanisms controlling the sequential divisions. In this study, we carried out a genetic screen to identify factors that regulate the initiation of meiosis II in the fission yeast Schizosaccharomyces pombe. We identified mutants deficient in meiosis II progression and repeatedly isolated mutants defective in spo5, which encodes an RNA-binding protein. Using fluorescence microscopy to visualize YFP-tagged protein, we found that spo5 mutant cells precociously lost Cdc13, the major B-type cyclin in fission yeast, before meiosis II. Importantly, the defect in meiosis II was rescued by increasing CDK activity. In wild-type cells, cdc13 transcripts increased during meiosis II, but this increase in cdc13 expression was weaker in spo5 mutants. Thus, Spo5 is a novel regulator of meiosis II that controls the level of cdc13 expression and promotes de novo synthesis of Cdc13. We previously reported that inhibition of Cdc13 degradation is necessary to initiate meiosis II; together with the previous information, the current findings indicate that the dual control of Cdc13 by de novo synthesis and suppression of proteolysis ensures the progression of meiosis II. © 2014 The Authors Genes to Cells © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

a Migration Well Model for the Binding of Ligands to Heme Proteins.

NASA Astrophysics Data System (ADS)

Beece, Daniel Kenneth

The binding of carbon monoxide and dioxygen to heme proteins can be viewed as occurring in distinct stages: diffusion in the solvent, migration through the matrix, and occupation of the pocket before the final binding step. A model is presented which can explain the dominant kinetic behavior of several different heme protein-ligand systems. The model assumes that a ligand molecule in the solvent sequentially encounters discrete energy barriers on the way to the binding site. The rate to surmount each barrier is distributed, except for the pseudofirst order rate corresponding to the step into the protein from the solvent. The migration through the matrix is equivalent to a small number of distinct jumps. Quantitative analysis of the data permit estimates of the barrier heights, preexponentials and solvent coupling factors for each rate. A migration coefficient and a matrix occupation factor are defined.