Critical Role of a Loop at C-Terminal Domain on the Conformational Stability and Catalytic Efficiency of Chondroitinase ABC I

Misfolding of prion protein (PrP) into amyloid aggregates is the central feature of prion diseases. PrP has an amyloidogenic C-terminal domain with three α-helices and a flexible tail in the N-terminal domain in which multiple octapeptide repeats are present in most mammals. The role of the octapeptides in prion diseases has previously been underestimated because the octapeptides are not located in the amyloidogenic domain. Correlation between the number of octapeptide repeats and age of onset suggests the critical role of octapeptide repeats in prion diseases. In this study, we have investigated four PrP variants without any octapeptides and with 1, 5 and 8 octapeptide repeats. From the comparison of the protein structure and the thermal stability of these proteins, as well as the characterization of amyloids converted from these PrP variants, we found that octapeptide repeats affect both folding and misfolding of PrP creating amyloid fibrils with distinct structures. Deletion of octapeptides forms fewer twisted fibrils and weakens the cytotoxicity. Insertion of octapeptides enhances the formation of typical silk-like fibrils but it does not increase the cytotoxicity. There might be some threshold effect and increasing the number of peptides beyond a certain limit has no further effect on the cell viability, though the reasons are unclear at this stage. Overall, the results of this study elucidate the molecular mechanism of octapeptides at the onset of prion diseases.

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Molecular basis of the dual role of the Mlh1-Mlh3 endonuclease in MMR and in meiotic crossover formation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2022704118 ◽

2021 ◽

Vol 118 (23) ◽

pp. e2022704118

Author(s):

Jingqi Dai ◽

Aurore Sanchez ◽

Céline Adam ◽

Lepakshi Ranjha ◽

Giordano Reginato ◽

...

Keyword(s):

Meiotic Recombination ◽

Molecular Basis ◽

Crystal Packing ◽

Critical Role ◽

Holliday Junctions ◽

Dual Roles ◽

Terminal Domain ◽

C Terminus ◽

Meiotic Crossover

In budding yeast, the MutL homolog heterodimer Mlh1-Mlh3 (MutLγ) plays a central role in the formation of meiotic crossovers. It is also involved in the repair of a subset of mismatches besides the main mismatch repair (MMR) endonuclease Mlh1-Pms1 (MutLα). The heterodimer interface and endonuclease sites of MutLγ and MutLα are located in their C-terminal domain (CTD). The molecular basis of MutLγ’s dual roles in MMR and meiosis is not known. To better understand the specificity of MutLγ, we characterized the crystal structure of Saccharomyces cerevisiae MutLγ(CTD). Although MutLγ(CTD) presents overall similarities with MutLα(CTD), it harbors some rearrangement of the surface surrounding the active site, which indicates altered substrate preference. The last amino acids of Mlh1 participate in the Mlh3 endonuclease site as previously reported for Pms1. We characterized mlh1 alleles and showed a critical role of this Mlh1 extreme C terminus both in MMR and in meiotic recombination. We showed that the MutLγ(CTD) preferentially binds Holliday junctions, contrary to MutLα(CTD). We characterized Mlh3 positions on the N-terminal domain (NTD) and CTD that could contribute to the positioning of the NTD close to the CTD in the context of the full-length MutLγ. Finally, crystal packing revealed an assembly of MutLγ(CTD) molecules in filament structures. Mutation at the corresponding interfaces reduced crossover formation, suggesting that these superstructures may contribute to the oligomer formation proposed for MutLγ. This study defines clear divergent features between the MutL homologs and identifies, at the molecular level, their specialization toward MMR or meiotic recombination functions.

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GPIHBP1 C89F Neomutation and Hydrophobic C-Terminal Domain G175R Mutation in Two Pedigrees with Severe Hyperchylomicronemia

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2011-1444 ◽

2011 ◽

Vol 96 (10) ◽

pp. E1675-E1679 ◽

Cited By ~ 49

Author(s):

Sybil Charrière ◽

Noël Peretti ◽

Sophie Bernard ◽

Mathilde Di Filippo ◽

Agnès Sassolas ◽

...

Keyword(s):

Critical Role ◽

Phenotypic Expression ◽

Missense Mutations ◽

Terminal Domain ◽

Apoa5 Gene ◽

Key Enzyme ◽

History Of ◽

Functional Consequences ◽

Signal Peptide Polymorphism

Abstract Context: GPIHBP1 is a new endothelial binding site for lipoprotein lipase (LPL), the key enzyme for intravascular lipolysis of triglyceride-rich lipoproteins (TGRL). We have identified two new missense mutations of the GPIHBP1 gene, C89F and G175R, by systematic sequencing in a cohort of 376 hyperchylomicronemic patients without mutations on the LPL, APOC2, or APOA5 gene. Objective: Phenotypic expression and functional consequences of these two mutations were studied. Design: We performed clinical and genotypic studies of probands and their families. GPIHBP1 functional alterations were studied in CHO pgsA-745 transfected cells. Results: Probands are an adult with a homozygous G175R mutation and a child with a hemizygous C89F neomutation and a deletion of the second allele. C89F mutation was associated with a C14F signal peptide polymorphism on the same haplotype. Both patients had resistant hyperchylomicronemia, low LPL activity, and history of acute pancreatitis. In CHO pgsA-745 cells, both G175R and C14F variants reduce the expression of GPIHBP1 at the cell surface. C89F mutation is responsible for a drastic LPL-binding defect to GPIHBP1. C14F may further potentiate C89F effect. Conclusions: The emergence of hyperchylomicronemia in the generation after a neomutation further establishes a critical role for GPIHBP1 in TGRL physiopathology in humans. Our results highlight the crucial role of C65-C89 disulfide bond in LPL binding by GPIHBP1 Ly6 domain. Furthermore, we first report a mutation of the hydrophobic C-terminal domain that impairs GPIHBP1 membrane targeting.

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Critical Role of Helix 4 of HIV-1 Capsid C-terminal Domain in Interactions with Human Lysyl-tRNA Synthetase

Journal of Biological Chemistry ◽

10.1074/jbc.m706256200 ◽

2007 ◽

Vol 282 (44) ◽

pp. 32274-32279 ◽

Cited By ~ 27

Author(s):

Brandie J. Kovaleski ◽

Robert Kennedy ◽

Ahmad Khorchid ◽

Lawrence Kleiman ◽

Hiroshi Matsuo ◽

...

Keyword(s):

Critical Role ◽

Trna Synthetase ◽

Terminal Domain ◽

Hiv 1

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Critical Role of the PA-X C-Terminal Domain of Influenza A Virus in Its Subcellular Localization and Shutoff Activity

Journal of Virology ◽

10.1128/jvi.00954-16 ◽

2016 ◽

Vol 90 (16) ◽

pp. 7131-7141 ◽

Cited By ~ 27

Author(s):

Tsuyoshi Hayashi ◽

Chutikarn Chaimayo ◽

James McGuinness ◽

Toru Takimoto

Keyword(s):

Amino Acids ◽

Subcellular Localization ◽

Nuclear Localization ◽

Influenza A ◽

Critical Role ◽

Terminal Region ◽

Terminal Deletion ◽

Link Type ◽

Terminal Domain

ABSTRACTPA-X is a recently identified influenza virus protein that is composed of the PA N-terminal 191 amino acids and unique C-terminal 41 or 61 residues. We and others showed that PA-X has a strong ability to suppress host protein synthesis via host mRNA decay, which is mediated by endonuclease activity in its N-terminal domain (B. W. Jagger, H. M. Wise, J. C. Kash, K. A. Walters, N. M. Wills, Y. L. Xiao, R. L. Dunfee, L. M. Schwartzman, A. Ozinsky, G. L. Bell, R. M. Dalton, A. Lo, S. Efstathiou, J. F. Atkins, A. E. Firth, J. K. Taubenberger, and P. Digard, 2012, Science337:199–204,http://dx.doi.org/10.1126/science.1222213, and E. A. Desmet, K. A. Bussey, R. Stone, and T. Takimoto, 2013, J Virol87:3108–3118,http://dx.doi.org/10.1128/JVI.02826-12). However, the mechanism of host mRNA degradation, especially where and how PA-X targets mRNAs, has not been analyzed. In this study, we determined the localization of PA-X and the role of the C-terminal unique region in shutoff activity. Quantitative subcellular localization analysis revealed that PA-X was located equally in both cytoplasm and nucleus. By characterizing a series of PA-X C-terminal deletion mutants, we found that the first 9 amino acids were sufficient for nuclear localization, but an additional 6 residues were required to induce the maximum shutoff activity observed with intact PA-X. Importantly, forced nuclear localization of the PA-X C-terminal deletion mutant enhanced shutoff activity, highlighting the ability of nuclear PA-X to degrade host mRNAs more efficiently. However, PA-X also inhibited luciferase expression from transfected mRNAs synthesizedin vitro, suggesting that PA-X also degrades mRNAs in the cytoplasm. Among the basic amino acids in the PA-X C-terminal region, 3 residues, 195K, 198K, and 199R, were identified as key residues for inducing host shutoff and nuclear localization. Overall, our data indicate a critical role for the 15 residues in the PA-X C-terminal domain in degrading mRNAs in both the cytoplasm and nucleus.IMPORTANCEInfluenza A viruses express PA-X proteins to suppress global host gene expression, including host antiviral genes, to allow efficient viral replication in infected cells. However, little is known about how PA-X induces host shutoff. In this study, we showed that PA-X localized equally in both the cytoplasm and nucleus of the cells, but the nuclear localization of PA-X mediated by its C-terminal region has a significant impact on shutoff activity. Three basic residues at the C-terminal region play a critical role in nuclear localization, but additional basic residues were required for maximum shutoff activity. Our findings indicate that PA-X targets and degrades mRNAs in both the nucleus and cytoplasm, and that the first 15 residues of the PA-X unique C-terminal region play a critical role in shutoff activity.

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Critical role of tryptophanyl residues in the conformational stability of goose δ-crystallin

Experimental Eye Research ◽

10.1016/j.exer.2006.03.001 ◽

2006 ◽

Vol 83 (3) ◽

pp. 658-666 ◽

Cited By ~ 3

Author(s):

Hwei-Jen Lee ◽

Young-Hsang Lai ◽

Yu-Ting Huang ◽

Chih-Wei Huang ◽

Yu-Hou Chen ◽

...

Keyword(s):

Conformational Stability ◽

Critical Role

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Catalytic activation of mitogen-activated protein (MAP) kinase phosphatase-1 by binding to p38 MAP kinase: critical role of the p38 C-terminal domain in its negative regulation

Biochemical Journal ◽

10.1042/0264-6021:3520155 ◽

2000 ◽

Vol 352 (1) ◽

pp. 155 ◽

Cited By ~ 32

Author(s):

Dorothy HUTTER ◽

Peili CHEN ◽

Janice BARNES ◽

Yusen LIU

Keyword(s):

Map Kinase ◽

Critical Role ◽

P38 Map Kinase ◽

Negative Regulation ◽

Catalytic Activation ◽

Terminal Domain ◽

Map Kinase Phosphatase ◽

Protein Map ◽

Mitogen Activated Protein

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Dual role of strigolactone receptor signaling partner in inhibiting substrate hydrolysis

10.1101/2021.12.01.470725 ◽

2021 ◽

Author(s):

Briana L Sobecks ◽

Jiming Chen ◽

Diwakar Shukla

Keyword(s):

Active Site ◽

Conformational Stability ◽

Critical Role ◽

Md Simulations ◽

Binding Pocket ◽

Conformational Ensemble ◽

Downstream Signaling ◽

F Box Protein ◽

Substrate Hydrolysis

Plant branch and root growth relies on metabolism of the strigolactone (SL) hormone. The interaction between the SL molecule, Oryza sativa DWARF14 (D14) SL receptor, and D3 F-box protein has been shown to play a critical role in SL perception. Previously, it was believed that D3 only interacts with the closed form of D14 to induce downstream signaling, but recent experiments indicate that D3, as well as its C-terminal helix (CTH), can interact with the open form as well to inhibit strigolactone signaling. Two hypotheses for the CTH induced inhibition are that either the CTH affects the conformational ensemble of D14 by stabilizing catalytically inactive states, or the CTH interacts with SLs in a way that prevents them from entering the binding pocket. In this study, we have performed molecular dynamics (MD) simulations to assess the validity of these hypotheses. We used an apo system with only D14 and the CTH to test the active site conformational stability and a holo system with D14, the CTH, and an SL molecule to test the interaction between the SL and CTH. Our simulations show that the CTH affects both active site conformation and the ability of SLs to move into the binding pocket. In the apo system, the CTH allosterically stabilized catalytic residues into their inactive conformation. In the holo system, significant interactions between SLs and the CTH hindered the ability of SLs to enter the D14 binding pocket. These two mechanisms account for the observed decrease in SL binding to D14 and subsequent ligand hydrolysis in the presence of the CTH.

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