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

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.

scholarly journals Catalytic effectiveness of human aldose reductase. Critical role of C-terminal domain.

1992 ◽  
Vol 267 (29) ◽  
pp. 20965-20970
Author(s):  
K.M. Bohren ◽  
C.E. Grimshaw ◽  
K.H. Gabbay
Keyword(s):  
Aldose Reductase ◽  
Critical Role ◽  
Terminal Domain

scholarly journals Dual roles for the Drosophila PI 4-kinase Four wheel drive in localizing Rab11 during cytokinesis

2009 ◽  
Vol 187 (6) ◽  
pp. 847-858 ◽  
Author(s):  
Gordon Polevoy ◽  
Ho-Chun Wei ◽  
Raymond Wong ◽  
Zsofia Szentpetery ◽  
Yeun Ju Kim ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Critical Role ◽  
Successful Completion ◽  
Dual Roles ◽  
Recycling Endosome ◽  
Golgi Membranes ◽  
Wheel Drive ◽  
Dividing Cells ◽  
Four Wheel Drive

Successful completion of cytokinesis relies on addition of new membrane, and requires the recycling endosome regulator Rab11, which localizes to the midzone. Despite the critical role of Rab11 in this process, little is known about the formation and composition of Rab11-containing organelles. Here, we identify the phosphatidylinositol (PI) 4-kinase III β Four wheel drive (Fwd) as a key regulator of Rab11 during cytokinesis in Drosophila melanogaster spermatocytes. We show Fwd is required for synthesis of PI 4-phosphate (PI4P) on Golgi membranes and for formation of PI4P-containing secretory organelles that localize to the midzone. Fwd binds and colocalizes with Rab11 on Golgi membranes, and is required for localization of Rab11 in dividing cells. A kinase-dead version of Fwd also binds Rab11 and partially restores cytokinesis to fwd mutant flies. Moreover, activated Rab11 partially suppresses loss of fwd. Our data suggest Fwd plays catalytic and noncatalytic roles in regulating Rab11 during cytokinesis.

scholarly journals The Effect of Octapeptide Repeats on Prion Folding and Misfolding

2021 ◽  
Vol 22 (4) ◽  
pp. 1800
Author(s):  
Kun-Hua Yu ◽  
Mei-Yu Huang ◽  
Yi-Ru Lee ◽  
Yu-Kie Lin ◽  
Hau-Ren Chen ◽  
...  
Keyword(s):  
Amyloid Fibrils ◽  
Age Of Onset ◽  
Prion Diseases ◽  
Critical Role ◽  
Threshold Effect ◽  
Central Feature ◽  
Terminal Domain ◽  
Amyloid Aggregates

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.

scholarly journals CTCF cooperates with CtIP to drive homologous recombination repair of double-strand breaks

2019 ◽  
Vol 47 (17) ◽  
pp. 9160-9179 ◽  
Author(s):  
Soon Young Hwang ◽  
Mi Ae Kang ◽  
Chul Joon Baik ◽  
Yejin Lee ◽  
Ngo Thanh Hang ◽  
...  
Keyword(s):  
Homologous Recombination ◽  
Critical Role ◽  
Control Point ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Strand Breaks ◽  
C Terminus ◽  
Dna End Resection ◽  
End Resection

Abstract The pleiotropic CCCTC-binding factor (CTCF) plays a role in homologous recombination (HR) repair of DNA double-strand breaks (DSBs). However, the precise mechanistic role of CTCF in HR remains largely unclear. Here, we show that CTCF engages in DNA end resection, which is the initial, crucial step in HR, through its interactions with MRE11 and CtIP. Depletion of CTCF profoundly impairs HR and attenuates CtIP recruitment at DSBs. CTCF physically interacts with MRE11 and CtIP and promotes CtIP recruitment to sites of DNA damage. Subsequently, CTCF facilitates DNA end resection to allow HR, in conjunction with MRE11–CtIP. Notably, the zinc finger domain of CTCF binds to both MRE11 and CtIP and enables proficient CtIP recruitment, DNA end resection and HR. The N-terminus of CTCF is able to bind to only MRE11 and its C-terminus is incapable of binding to MRE11 and CtIP, thereby resulting in compromised CtIP recruitment, DSB resection and HR. Overall, this suggests an important function of CTCF in DNA end resection through the recruitment of CtIP at DSBs. Collectively, our findings identify a critical role of CTCF at the first control point in selecting the HR repair pathway.

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

2011 ◽  
Vol 96 (10) ◽  
pp. E1675-E1679 ◽  
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.

scholarly journals Differential HspBP1 expression accounts for the greater vulnerability of neurons than astrocytes to misfolded proteins

2017 ◽  
Vol 114 (37) ◽  
pp. E7803-E7811 ◽  
Author(s):  
Ting Zhao ◽  
Yan Hong ◽  
Peng Yin ◽  
Shihua Li ◽  
Xiao-Jiang Li
Keyword(s):  
Neurodegenerative Diseases ◽  
Critical Role ◽  
Misfolded Proteins ◽  
Inhibitory Protein ◽  
Interacting Protein ◽  
C Terminus ◽  
Differential Vulnerability ◽  
Disease Protein ◽  
Knockin Mouse

Although it is well known that astrocytes are less vulnerable than neurons in neurodegenerative diseases, the mechanism behind this differential vulnerability is unclear. Here we report that neurons and astrocytes show markedly different activities in C terminus of Hsp70-interacting protein (CHIP), a cochaperone of Hsp70. In astrocytes, CHIP is more actively monoubiquitinated and binds to mutant huntingtin (mHtt), the Huntington’s disease protein, more avidly, facilitating its K48-linked polyubiquitination and degradation. Astrocytes also show the higher level and heat-shock induction of Hsp70 and faster CHIP-mediated degradation of various misfolded proteins than neurons. In contrast to astrocytes, neurons express abundant HspBP1, a CHIP inhibitory protein, resulting in the low activity of CHIP. Silencing HspBP1 expression via CRISPR-Cas9 in neurons ameliorated mHtt aggregation and neuropathology in HD knockin mouse brains. Our findings indicate a critical role of HspBP1 in differential CHIP/Hsp70 activities in neuronal and glial cells and the greater neuronal vulnerability to misfolded proteins in neurodegenerative diseases.

scholarly journals Specificity of Baculovirus P6.9 Basic DNA-Binding Proteins and Critical Role of the C Terminus in Virion Formation

2010 ◽  
Vol 84 (17) ◽  
pp. 8821-8828 ◽  
Author(s):  
Manli Wang ◽  
Era Tuladhar ◽  
Shu Shen ◽  
Hualin Wang ◽  
Monique M. van Oers ◽  
...  
Keyword(s):  
Virus Assembly ◽  
Critical Role ◽  
Nucleocapsid Proteins ◽  
Double Stranded Dna ◽  
C Terminus ◽  
Sequence Elements ◽  
Eukaryotic Organisms ◽  
Dsdna Viruses ◽  
Virion Formation

ABSTRACT The majority of double-stranded DNA (dsDNA) viruses infecting eukaryotic organisms use host- or virus-expressed histones or protamine-like proteins to condense their genomes. In contrast, members of the Baculoviridae family use a protamine-like protein named P6.9. The dephosphorylated form of P6.9 binds to DNA in a non-sequence-specific manner. By using a p6.9-null mutant of Autographa californica multiple nucleopolyhedrovirus (AcMNPV), we demonstrate that P6.9 is not required for viral DNA replication but is essential for the production of infectious virus. Virion production was rescued by P6.9 homologs from a number of Alpha baculovirus species and one Gammabaculovirus species but not from the genus Betabaculovirus, comprising the granuloviruses, or by the P6.9 homolog VP15 from the unrelated white spot syndrome virus of shrimp. Mutational analyses demonstrated that AcMNPV P6.9 with a conserved 11-residue deletion of the C terminus was not capable of rescuing p6.9-null AcMNPV, while a chimeric Betabaculovirus P6.9 containing the P6.9 C-terminal region of an Alphabaculovirus strain was able to do so. This implies that the C terminus of baculovirus P6.9 contains sequence elements essential for virion formation. Such elements may possibly interact with species- or genus-specific domains of other nucleocapsid proteins during virus assembly.

Withaferin A binds covalently to the N-terminal domain of annexin A2

2012 ◽  
Vol 393 (10) ◽  
pp. 1151-1163 ◽  
Author(s):  
Gabriel Ozorowski ◽  
Christopher M. Ryan ◽  
Julian P. Whitelegge ◽  
Hartmut Luecke
Keyword(s):  
Actin Polymerization ◽  
Inhibitory Effect ◽  
Annexin A2 ◽  
Actin Dynamics ◽  
Withaferin A ◽  
Core Domain ◽  
Cancer Pathways ◽  
Terminal Domain ◽  
C Terminus

Abstract Annexin A2 (AnxA2), a 38-kDa member of the Ca2+-binding annexin family, has been implicated in numerous cancer pathways. Withaferin A (WithfA), a natural plant compound, has been reported previously to bind covalently to Cys133 of the AnxA2 core domain leading to a reduction of the invasive capabilities of cancer cells by altering their cytoskeleton. We show here that AnxA2 has an inhibitory effect on actin polymerization, and a modification with WithfA significantly increases this inhibitory role of AnxA2. Using mass spectrometry and single-site mutants, we localized the WithfA-AnxA2 interaction to the N-terminal domain of AnxA2 where WithfA binds covalently to Cys9. Whereas binding to F-actin filaments has been mapped to the C terminus of AnxA2, our results suggest that the N-terminal domain modified by WithfA may also play a role in the AnxA2-actin interaction. The binding of WithfA may regulate the AnxA2-mediated actin dynamics in two distinct ways: (i) the increase of F-actin bundling activity by the Anx2/p11 heterotetramer and (ii) the decrease of actin polymerization as a result of the increased affinity of AnxA2 to the barbed end of actin microfilaments. We demonstrate the susceptibility of Cys9 of AnxA2 to chemical modifications and exclude Cys133 as a binding site for WithfA.

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

2007 ◽  
Vol 282 (44) ◽  
pp. 32274-32279 ◽  
Author(s):  
Brandie J. Kovaleski ◽  
Robert Kennedy ◽  
Ahmad Khorchid ◽  
Lawrence Kleiman ◽  
Hiroshi Matsuo ◽  
...  
Keyword(s):  
Critical Role ◽  
Trna Synthetase ◽  
Terminal Domain ◽  
Hiv 1

scholarly journals Structure of APP-C991-99 and Implications for Role of Extra-Membrane Domains in Function and Oligomerization

2018 ◽  
Author(s):  
George A. Pantelopulos ◽  
John E. Straub ◽  
D. Thirumalai ◽  
Yuji Sugita
Keyword(s):  
Transmembrane Domain ◽  
Chemical Shifts ◽  
Membrane Surface ◽  
Critical Role ◽  
Amyloid Formation ◽  
Cytoplasmic Proteins ◽  
C Terminus ◽  
N Terminus ◽  
Thin Membranes

AbstractThe 99 amino acid C-terminal fragment of Amyloid Precursor Protein APP-C99 (C99) is cleaved by γ-secretase to form Aβ peptide, which plays a critical role in the etiology of Alzheimer’s Disease (AD). The structure of C99 consists of a single transmembrane domain flanked by intra and intercellular domains. While the structure of the transmembrane domain has been well characterized, little is known about the structure of the flanking domains and their role in C99 processing by γ-secretase. To gain insight into the structure of full-length C99, REMD simulations were performed for monomeric C99 in model membranes of varying thickness. We find equilibrium ensembles of C99 from simulation agree with experimentally-inferred residue insertion depths and protein backbone chemical shifts. In thin membranes, the transmembrane domain structure is correlated with extra-membrane structural states. Mean and variance of the transmembrane and G37G38 hinge angles are found to increase with thinning membrane. The N-terminus of C99 forms β-strands that may seed aggregation of Aβ on the membrane surface, promoting amyloid formation. The N-terminus, which forms α-helices that interact with the nicastrin domain of γ-secretase. The C-terminus of C99 becomes more α-helical as the membrane thickens, forming structures that may be suitable for binding by cytoplasmic proteins, while C-terminal residues essential to cytotoxic function become α-helical as the membrane thins. The heterogeneous but discrete extra-membrane domain states analyzed here open the path to new investigations of the role of C99 structure and membrane in amyloidogenesis.

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