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Genetic Deletion of Mst1 Alters T Cell Function and Protects against Autoimmunity  [PDF]
Konstantin V. Salojin, Brian D. Hamman, Wei Chun Chang, Kanchan G. Jhaver, Amin Al-Shami, Jeannette Crisostomo, Carrie Wilkins, Ann Marie Digeorge-Foushee, Jason Allen, Nita Patel, Suma Gopinathan, Julia Zhou, Amr Nouraldeen, Theodore C. Jessop, Jeffrey T. Bagdanoff, David J. Augeri, Robert Read, Peter Vogel, Jonathan Swaffield, Alan Wilson, Kenneth A. Platt, Kenneth G. Carson, Alan Main, Brian P. Zambrowicz, Tamas Oravecz
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0098151
Abstract: Mammalian sterile 20-like kinase 1 (Mst1) is a MAPK kinase kinase kinase which is involved in a wide range of cellular responses, including apoptosis, lymphocyte adhesion and trafficking. The contribution of Mst1 to Ag-specific immune responses and autoimmunity has not been well defined. In this study, we provide evidence for the essential role of Mst1 in T cell differentiation and autoimmunity, using both genetic and pharmacologic approaches. Absence of Mst1 in mice reduced T cell proliferation and IL-2 production in vitro, blocked cell cycle progression, and elevated activation-induced cell death in Th1 cells. Mst1 deficiency led to a CD4+ T cell development path that was biased toward Th2 and immunoregulatory cytokine production with suppressed Th1 responses. In addition, Mst1?/? B cells showed decreased stimulation to B cell mitogens in vitro and deficient Ag-specific Ig production in vivo. Consistent with altered lymphocyte function, deletion of Mst1 reduced the severity of experimental autoimmune encephalomyelitis (EAE) and protected against collagen-induced arthritis development. Mst1?/? CD4+ T cells displayed an intrinsic defect in their ability to respond to encephalitogenic antigens and deletion of Mst1 in the CD4+ T cell compartment was sufficient to alleviate CNS inflammation during EAE. These findings have prompted the discovery of novel compounds that are potent inhibitors of Mst1 and exhibit desirable pharmacokinetic properties. In conclusion, this report implicates Mst1 as a critical regulator of adaptive immune responses, Th1/Th2-dependent cytokine production, and as a potential therapeutic target for immune disorders.
Cotranslational Protein Folding and Terminus Hydrophobicity  [PDF]
Sheenal Srivastava,Yumi Patton,David W. Fisher,Graham R. Wood
Advances in Bioinformatics , 2011, DOI: 10.1155/2011/176813
Abstract: Peptides fold on a time scale that is much smaller than the time required for synthesis, whence all proteins potentially fold cotranslationally to some degree (followed by additional folding events after release from the ribosome). In this paper, in three different ways, we find that cotranslational folding success is associated with higher hydrophobicity at the N-terminus than at the C-terminus. First, we fold simple HP models on a square lattice and observe that HP sequences that fold better cotranslationally than from a fully extended state exhibit a positive difference (N?C) in terminus hydrophobicity. Second, we examine real proteins using a previously established measure of potential cotranslationality known as ALR (Average Logarithmic Ratio of the extent of previous contacts) and again find a correlation with the difference in terminus hydrophobicity. Finally, we use the cotranslational protein structure prediction program SAINT and again find that such an approach to folding is more successful for proteins with higher N-terminus than C-terminus hydrophobicity. All results indicate that cotranslational folding is promoted in part by a hydrophobic start and a less hydrophobic finish to the sequence. 1. Background An understanding of protein folding is keenly sought for a variety of oft-stated reasons. From a theoretician’s perspective, hydrophobic collapse of the string of residues is often conjectured to be a key driver of protein folding [1]. Under such collapse, the manner of folding will be to some extent determined by the hydrophobicity profile. On the other hand, from an experimentalist’s perspective, cotranslational folding is acknowledged to occur for certain proteins. Marrying these two perspectives causes us to ask whether there is a hydrophobicity profile that is compatible with and that may even assist in driving cotranslational folding. In this paper, we find evidence in three independent ways that greater hydrophobicity at the N-terminus than at the C-terminus is associated with cotranslational folding. That cotranslational folding occurs (and may be supported by an associated hydrophobicity pattern) underpins this paper, so we now review this process, together with evidence of asymmetry in protein folding from other sources. Phillips [2] noted over forty years ago, in expounding the structure of the hen egg-white lysozyme molecule, evidence of the nonuniform distribution of hydrophobic residues in a protein and had the bravery to suggest that this may be an indicator of cotranslational folding. The first 40 residues of the
Recognition of protein complexation based on hydrophobicity distribution  [cached]
Irena Roterman,Mateusz Banach
Bioinformation , 2009,
Abstract: The identification of the surface area able to generate the protein-protein complexation ligand and ion ligation is critical for the recognition of the biological function of particular proteins. The technique based on the analysis of the irregularity of hydrophobicity distribution is used as the criterion for the recognition of the interaction regions. Particularly, the exposure of hydrophobic residues on the surface of protein as well as the localization of the hydrophilic residues in the hydrophobic core is treated as potential area ready to interact with external molecules. The model based on the “fuzzy oil drop” approach treating the protein molecule as the drop of hydrophobicity concentrated in the central part of structure with the hydrophobicity close to zero on the surface according to 3-dimensional Gauss function. The comparison with the observed hydrophobicy in particular protein reveals some irregularities. These irregularities seem to represent the aim-oriented localization.
Structure–Function Studies Using Deletion Mutants Identify Domains of gC1qR/p33 as Potential Therapeutic Targets for Vascular Permeability and Inflammation  [PDF]
Berhane Ghebrehiwet,Allen P. Kaplan,Ellinor I. B. Peerschke
Frontiers in Immunology , 2011, DOI: 10.3389/fimmu.2011.00058
Abstract: The endothelial cell receptor complex for kininogen (HK) comprises gC1qR, cytokeratin 1, and urokinase-type plasminogen activator receptor and is essential for activation of the kinin system that leads to bradykinin (BK) generation. Of these, gC1qR/p33 constitutes a high affinity site for HK – the BK precursor – and is therefore critical for the assembly of the kinin-generating cascade. Previous studies have identified a putative HK site within the C-terminal domain (residues 204–218) of gC1qR recognized by mAb 74.5.2. In these studies, we used information from the crystal structure of gC1qR, to engineer several deletion (Δ) mutants and test their ability to bind and/or support BK generation. While deletion of residues 204–218 (gC1qRΔ204–218), showed significantly reduced binding to HK, BK generation was not affected when tested by a sensitive bradykinin immunoassay. In fact, all of the gC1qR deletion mutants supported BK generation with the exception of gC1qRΔ154–162 and a point mutation in which Trp 233 was substituted with Gly. Binding studies also identified the existence of two additional sites at residues 144–162 and 190–202. Moreover, binding of HK to a synthetic peptide 190–202 was inhibited by mAbs 48 and 83, but not by mAb 74.5.2. Since a single residue separates domains 190–202 and 204–218, they may be part of a highly stable HK binding pocket and therefore a potential target for drug design to prevent vascular permeability and inflammation.
Deletion of Lipoprotein PG0717 in Porphyromonas gingivalis W83 Reduces Gingipain Activity and Alters Trafficking in and Response by Host Cells  [PDF]
Leticia Reyes, Eileen Eiler-McManis, Paulo H. Rodrigues, Amandeep S. Chadda, Shannon M. Wallet, Myriam Bélanger, Amanda G. Barrett, Sophie Alvarez, Debra Akin, William A. Dunn, Ann Progulske-Fox
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0074230
Abstract: P. gingivalis (Pg), a causative agent of chronic generalized periodontitis, has been implicated in promoting cardiovascular disease. Expression of lipoprotein gene PG0717 of Pg strain W83 was found to be transiently upregulated during invasion of human coronary artery endothelial cells (HCAEC), suggesting this protein may be involved in virulence. We characterized the virulence phenotype of a PG0717 deletion mutant of pg W83. There were no differences in the ability of W83Δ717 to adhere and invade HCAEC. However, the increased proportion of internalized W83 at 24 hours post-inoculation was not observed with W83?717. Deletion of PG0717 also impaired the ability of W83 to usurp the autophagic pathway in HCAEC and to induce autophagy in Saos-2 sarcoma cells. HCAEC infected with W83Δ717 also secreted significantly greater amounts of MCP-1, IL-8, IL-6, GM-CSF, and soluble ICAM-1, VCAM-1, and E-selectin when compared to W83. Further characterization of W83Δ717 revealed that neither capsule nor lipid A structure was affected by deletion of PG0717. Interestingly, the activity of both arginine (Rgp) and lysine (Kgp) gingipains was reduced in whole-cell extracts and culture supernatant of W83Δ717. RT-PCR revealed a corresponding decrease in transcription of rgpB but not rgpA or kgp. Quantitative proteome studies of the two strains revealed that both RgpA and RgpB, along with putative virulence factors peptidylarginine deiminase and Clp protease were significantly decreased in the W83Δ717. Our results suggest that PG0717 has pleiotropic effects on W83 that affect microbial induced manipulation of host responses important for microbial clearance and infection control.
Correlation between sequence hydrophobicity and surface-exposure pattern of database proteins  [PDF]
Susanne Moelbert,Eldon Emberly,Chao Tang
Quantitative Biology , 2003,
Abstract: Hydrophobicity is thought to be one of the primary forces driving the folding of proteins. On average, hydrophobic residues occur preferentially in the core, whereas polar residues tends to occur at the surface of a folded protein. By analyzing the known protein structures, we quantify the degree to which the hydrophobicity sequence of a protein correlates with its pattern of surface exposure. We have assessed the statistical significance of this correlation for several hydrophobicity scales in the literature, and find that the computed correlations are significant but far from optimal. We show that this less than optimal correlation arises primarily from the large degree of mutations that naturally occurring proteins can tolerate. Lesser effects are due in part to forces other than hydrophobicity and we quantify this by analyzing the surface exposure distributions of all amino acids. Lastly we show that our database findings are consistent with those found from an off-lattice hydrophobic-polar model of protein folding.
Deletion of Ubiquitin Fold Modifier Protein Ufm1 Processing Peptidase Ufsp in L. donovani Abolishes Ufm1 Processing and Alters Pathogenesis  [PDF]
Sreenivas Gannavaram ,Sonya Davey,Ines Lakhal-Naouar,Robert Duncan,Hira L. Nakhasi
PLOS Neglected Tropical Diseases , 2014, DOI: 10.1371/journal.pntd.0002707
Abstract: Previously, we showed Leishmania donovani Ufm1 has a Gly residue conserved at the C-terminal region with a unique 17 amino acid residue extension that must be processed prior to conjugation to target proteins. In this report, we describe for the first time the isolation and characterization of the Leishmania Ufm1-specific protease Ufsp. Biochemical analysis of L. donovani Ufsp showed that this protein possesses the Ufm1 processing activity using sensitive FRET based activity probes. The Ufm1 cleavage activity was absent in a mutant Ufsp in which the active site cysteine is altered to a serine. To examine the effects of abolition of Ufm1 processing activity, we generated a L. donovani null mutant of Ufsp (LdUfsp?/?). Ufm1 processing activity was abolished in LdUfsp?/? mutant, and the processing defect was reversed by re-expression of wild type but not the cys>ser mutant in the LdUfsp?/? parasites. Further LdUfsp?/? mutants showed reduced survival as amastigotes in infected human macrophages but not as promastigotes. This growth defect in the amastigotes was reversed by re-expression of wild type but not the cys>ser mutant in the Ufsp?/? indicating the essential nature of this protease for Leishmania pathogenesis. Further, mouse infection experiments showed deletion of Ufsp results in reduced virulence of the parasites. Additionally, Ufsp activity was inhibited by an anti-leishmanial drug Amphotericin B. These studies provide an opportunity to test LdUfsp?/? parasites as drug and vaccine targets.
On Hydrophobicity Correlations in Protein Chains  [PDF]
Anders Irb?ck,Erik Sandelin
Quantitative Biology , 2000, DOI: 10.1016/S0006-3495(00)76472-1
Abstract: We study the statistical properties of hydrophobic/polar model sequences with unique native states on the square lattice. It is shown that this ensemble of sequences differs from random sequences in significant ways in terms of both the distribution of hydrophobicity along the chains and total hydrophobicity. Whenever statistically feasible, the analogous calculations are performed for a set of real enzymes, too.
Deletion of the Vaccinia Virus Gene A46R, Encoding for an Inhibitor of TLR Signalling, Is an Effective Approach to Enhance the Immunogenicity in Mice of the HIV/AIDS Vaccine Candidate NYVAC-C  [PDF]
Beatriz Perdiguero, Carmen Elena Gómez, Mauro Di Pilato, Carlos Oscar S. Sorzano, Julie Delaloye, Thierry Roger, Thierry Calandra, Giuseppe Pantaleo, Mariano Esteban
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0074831
Abstract: Viruses have developed strategies to counteract signalling through Toll-like receptors (TLRs) that are involved in the detection of viruses and induction of proinflammatory cytokines and IFNs. Vaccinia virus (VACV) encodes A46 protein which disrupts TLR signalling by interfering with TLR: adaptor interactions. Since the innate immune response to viruses is critical to induce protective immunity, we studied whether deletion of A46R gene in a NYVAC vector expressing HIV-1 Env, Gag, Pol and Nef antigens (NYVAC-C) improves immune responses against HIV-1 antigens. This question was examined in human macrophages and in mice infected with a single A46R deletion mutant of the vaccine candidate NYVAC-C (NYVAC-C-ΔA46R). The viral gene A46R is not required for virus replication in primary chicken embryo fibroblast (CEF) cells and its deletion in NYVAC-C markedly increases TNF, IL-6 and IL-8 secretion by human macrophages. Analysis of the immune responses elicited in BALB/c mice after DNA prime/NYVAC boost immunization shows that deletion of A46R improves the magnitude of the HIV-1-specific CD4 and CD8 T cell immune responses during adaptive and memory phases, maintains the functional profile observed with the parental NYVAC-C and enhances anti-gp120 humoral response during the memory phase. These findings establish the immunological role of VACV A46R on innate immune responses of macrophages in vitro and antigen-specific T and B cell immune responses in vivo and suggest that deletion of viral inhibitors of TLR signalling is a useful approach for the improvement of poxvirus-based vaccine candidates.
Deletion of 12/15-Lipoxygenase Alters Macrophage and Islet Function in NOD-Alox15null Mice, Leading to Protection against Type 1 Diabetes Development  [PDF]
Shamina M. Green-Mitchell, Sarah A. Tersey, Banumathi K. Cole, Kaiwen Ma, Norine S. Kuhn, Tina Duong Cunningham, Nelly A. Maybee, Swarup K. Chakrabarti, Marcia McDuffie, David A. Taylor-Fishwick, Raghavendra G. Mirmira, Jerry L. Nadler, Margaret A. Morris
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0056763
Abstract: Aims Type 1 diabetes (T1D) is characterized by autoimmune depletion of insulin-producing pancreatic beta cells. We showed previously that deletion of the 12/15-lipoxygenase enzyme (12/15-LO, Alox15 gene) in NOD mice leads to nearly 100 percent protection from T1D. In this study, we test the hypothesis that cytokines involved in the IL-12/12/15-LO axis affect both macrophage and islet function, which contributes to the development of T1D. Methods 12/15-LO expression was clarified in immune cells by qRT-PCR, and timing of expression was tested in islets using qRT-PCR and Western blotting. Expression of key proinflammatory cytokines and pancreatic transcription factors was studied in NOD and NOD-Alox15null macrophages and islets using qRT-PCR. The two mouse strains were also assessed for the ability of splenocytes to transfer diabetes in an adoptive transfer model, and beta cell mass. Results 12/15-LO is expressed in macrophages, but not B and T cells of NOD mice. In macrophages, 12/15-LO deletion leads to decreased proinflammatory cytokine mRNA and protein levels. Furthermore, splenocytes from NOD-Alox15null mice are unable to transfer diabetes in an adoptive transfer model. In islets, expression of 12/15-LO in NOD mice peaks at a crucial time during insulitis development. The absence of 12/15-LO results in maintenance of islet health with respect to measurements of islet-specific transcription factors, markers of islet health, proinflammatory cytokines, and beta cell mass. Conclusions These results suggest that 12/15-LO affects islet and macrophage function, causing inflammation, and leading to autoimmunity and reduced beta cell mass.
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