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ACS Chemical Neuroscience Jun 2023Mutations in a microglia-associated gene 2 increase the risk of Alzheimer's disease. Currently, structural and functional studies of TREM2 mainly rely on recombinant...
Mutations in a microglia-associated gene 2 increase the risk of Alzheimer's disease. Currently, structural and functional studies of TREM2 mainly rely on recombinant TREM2 proteins expressed from mammalian cells. However, using this method, it is difficult to achieve site-specific labeling. Here, we present the total chemical synthesis of the 116 amino acid TREM2 ectodomain. Rigorous structural analysis ensured correct structural fold after refolding. Treating microglial cells with refolded synthetic TREM2 enhanced microglial phagocytosis, proliferation, and survival. We also prepared TREM2 constructs with defined glycosylation patterns and found that glycosylation at N79 is critical to the thermal stability of TREM2. This method will provide access to TREM2 constructs with site-specific labeling, such as fluorescent labeling, reactive chemical handles, and enrichment handles, to further advance our understanding of TREM2 in Alzheimer's disease.
Topics: Animals; Humans; Alzheimer Disease; Glycosylation; Phagocytosis; Microglia; Mutation; Mammals; Membrane Glycoproteins; Receptors, Immunologic
PubMed: 37235776
DOI: 10.1021/acschemneuro.3c00257 -
The Journal of Physical Chemistry. B Jun 2023The folding of proteins into their native conformation is a complex process that has been extensively studied over the past half-century. The ribosome, the molecular...
The folding of proteins into their native conformation is a complex process that has been extensively studied over the past half-century. The ribosome, the molecular machine responsible for protein synthesis, is known to interact with nascent proteins, adding further complexity to the protein folding landscape. Consequently, it is unclear whether the folding pathways of proteins are conserved on and off the ribosome. The main question remains: to what extent does the ribosome help proteins fold? To address this question, we used coarse-grained molecular dynamics simulations to compare the mechanisms by which the proteins dihydrofolate reductase, type III chloramphenicol acetyltransferase, and d-alanine-d-alanine ligase B fold during and after vectorial synthesis on the ribosome to folding from the full-length unfolded state in bulk solution. Our results reveal that the influence of the ribosome on protein folding mechanisms varies depending on the size and complexity of the protein. Specifically, for a small protein with a simple fold, the ribosome facilitates efficient folding by helping the nascent protein avoid misfolded conformations. However, for larger and more complex proteins, the ribosome does not promote folding and may contribute to the formation of intermediate misfolded states cotranslationally. These misfolded states persist posttranslationally and do not convert to the native state during the 6 μs runtime of our coarse-grain simulations. Overall, our study highlights the complex interplay between the ribosome and protein folding and provides insight into the mechanisms of protein folding on and off the ribosome.
Topics: Protein Biosynthesis; Protein Folding; Ribosomes; Proteins; Molecular Dynamics Simulation
PubMed: 37200608
DOI: 10.1021/acs.jpcb.3c01694 -
Channels (Austin, Tex.) Dec 2023Structural evidence and much experimental data have demonstrated the presence of non-canonical helical substructures (π and 3) in regions of great functional relevance...
Structural evidence and much experimental data have demonstrated the presence of non-canonical helical substructures (π and 3) in regions of great functional relevance both in TRP as in Kv channels. Through an exhaustive compositional analysis of the sequences underlying these substructures, we find that each of them is associated with characteristic local flexibility profiles, which in turn are implicated in significant conformational rearrangements and interactions with specific ligands. We found that α-to-π helical transitions are associated with patterns of local rigidity whereas α-to-3 transitions are mainly leagued with high local flexibility profiles. We also study the relationship between flexibility and protein disorder in the transmembrane domain of these proteins. By contrasting these two parameters, we located regions showing a sort of structural discrepancy between these similar but not identical protein attributes. Notably, these regions are presumably implicated in important conformational rearrangements during the gating in those channels. In that sense, finding these regions where flexibility and disorder are not proportional allows us to detect regions with potential functional dynamism. From this point of view, we highlighted some conformational rearrangements that occur during ligand binding events, the compaction, and refolding of the outer pore loops in several TRP channels, as well as the well-known S4 motion in Kv channels.
Topics: Molecular Conformation; Protein Domains
PubMed: 37196183
DOI: 10.1080/19336950.2023.2212349 -
Protein Science : a Publication of the... Jul 2023Hsp90 is a molecular chaperone involved in the refolding and activation of numerous protein substrates referred to as clients. While the molecular determinants of Hsp90...
Hsp90 is a molecular chaperone involved in the refolding and activation of numerous protein substrates referred to as clients. While the molecular determinants of Hsp90 client specificity are poorly understood and limited to a handful of client proteins, strong clients are thought to be destabilized and conformationally extended. Here, we measured the phosphotransferase activity of 3929 variants of the tyrosine kinase Src in both the presence and absence of an Hsp90 inhibitor. We identified 84 previously unknown functionally dependent client variants. Unexpectedly, many destabilized or extended variants were not functionally dependent on Hsp90. Instead, functionally dependent client variants were clustered in the αF pocket and β1-β2 strand regions of Src, which have yet to be described in driving Hsp90 dependence. Hsp90 dependence was also strongly correlated with kinase activity. We found that a combination of activation, global extension, and general conformational flexibility, primarily induced by variants at the αF pocket and β1-β2 strands, was necessary to render Src functionally dependent on Hsp90. Moreover, the degree of activation and flexibility required to transform Src into a functionally dependent client varied with variant location, suggesting that a combination of regulatory domain disengagement and catalytic domain flexibility are required for chaperone dependence. Thus, by studying the chaperone dependence of a massive number of variants, we highlight factors driving Hsp90 client specificity and propose a model of chaperone-kinase interactions.
Topics: Humans; src-Family Kinases; Protein Conformation; HSP90 Heat-Shock Proteins; Molecular Chaperones; Mutation; Protein Binding
PubMed: 37167432
DOI: 10.1002/pro.4656 -
Science Advances May 2023Ring-forming AAA chaperones solubilize protein aggregates and protect organisms from proteostatic stress. In metazoans, the AAA chaperone Skd3 in the mitochondrial...
Ring-forming AAA chaperones solubilize protein aggregates and protect organisms from proteostatic stress. In metazoans, the AAA chaperone Skd3 in the mitochondrial intermembrane space (IMS) is critical for human health and efficiently refolds aggregated proteins, but its underlying mechanism is poorly understood. Here, we show that Skd3 harbors both disaggregase and protein refolding activities enabled by distinct assembly states. High-resolution structures of Skd3 hexamers in distinct conformations capture ratchet-like motions that mediate substrate extraction. Unlike previously described disaggregases, Skd3 hexamers further assemble into dodecameric cages in which solubilized substrate proteins can attain near-native states. Skd3 mutants defective in dodecamer assembly retain disaggregase activity but are impaired in client refolding, linking the disaggregase and refolding activities to the hexameric and dodecameric states of Skd3, respectively. We suggest that Skd3 is a combined disaggregase and foldase, and this property is particularly suited to meet the complex proteostatic demands in the mitochondrial IMS.
Topics: Animals; Humans; Molecular Chaperones; Protein Refolding
PubMed: 37163603
DOI: 10.1126/sciadv.adf5336 -
Frontiers in Immunology 2023Cartilaginous fishes are the most evolutionary-distant vertebrates from mammals and possess an immunoglobulin (Ig)- and T cell-mediated adaptive immunity. CD8 is the...
INTRODUCTION
Cartilaginous fishes are the most evolutionary-distant vertebrates from mammals and possess an immunoglobulin (Ig)- and T cell-mediated adaptive immunity. CD8 is the hallmark receptor of cytotoxic T cells and is required for the formation of T cell receptor-major histocompatibility complex (TCR-MHC) class I complexes.
METHODS
RACE PCR was used to obtain gene sequences. Direct dilution was applied for the refolding of denatured recombinant CD8 protein. Hanging-drop vapor diffusion method was performed for protein crystallization.
RESULTS
In this study, CD8α and CD8β orthologues (termed ScCD8α and ScCD8β) were identified in small-spotted catshark (). Both ScCD8α and ScCD8β possess an extracellular immunoglobulin superfamily (IgSF) V domain as in previously identified CD8 proteins. The genes encoding CD8α and CD8β are tandemly linked in the genomes of all jawed vertebrates studied, suggesting that they were duplicated from a common ancestral gene before the divergence of cartilaginous fishes and other vertebrates. We determined the crystal structure of the ScCD8α ectodomain homodimer at a resolution of 1.35 Å and show that it exhibits the typical topological structure of CD8α from endotherms. As in mammals, the homodimer formation of ScCD8αα relies upon interactions within a hydrophobic core although this differs in position and amino acid composition. Importantly, ScCD8αα shares the canonical cavity required for interaction with peptide-loaded MHC I in mammals. Furthermore, it was found that ScCD8α can co-immunoprecipitate with ScCD8β, indicating that it can form both homodimeric and heterodimeric complexes.
CONCLUSION
Our results expand the current knowledge of vertebrate CD8 dimerization and the interaction between CD8α with p/MHC I from an evolutionary perspective.
Topics: Animals; CD8 Antigens; Fishes; Vertebrates; Histocompatibility Antigens Class I; Biological Evolution; Recombinant Proteins; Mammals
PubMed: 37122697
DOI: 10.3389/fimmu.2023.1156219 -
Genes Mar 2023Misfolded proteins after stress-induced denaturation can regain their functions through correct re-folding with the aid of molecular chaperones. As a molecular... (Review)
Review
Misfolded proteins after stress-induced denaturation can regain their functions through correct re-folding with the aid of molecular chaperones. As a molecular chaperone, heat shock proteins (HSPs) can help client proteins fold correctly. During viral infection, HSPs are involved with replication, movement, assembly, disassembly, subcellular localization, and transport of the virus via the formation of macromolecular protein complexes, such as the viral replicase complex. Recent studies have indicated that HSP inhibitors can inhibit viral replication by interfering with the interaction of the virus with the HSP. In this review, we describe the function and classification of HSPs, the transcriptional mechanism of HSPs promoted by heat shock factors (HSFs), discuss the interaction between HSPs and viruses, and the mode of action of HSP inhibitors at two aspects of inhibiting the expression of HSPs and targeting the HSPs, and elaborate their potential use as antiviral agents.
Topics: Humans; Heat-Shock Proteins; Molecular Chaperones; Heat-Shock Response; Viruses
PubMed: 37107550
DOI: 10.3390/genes14040792 -
STAR Protocols Apr 2023Single-molecule fluorescence microscopy (SMFM) has been shown to be informative in understanding the interaction of chromatin-associated factors with nucleosomes, the...
Single-molecule fluorescence microscopy (SMFM) has been shown to be informative in understanding the interaction of chromatin-associated factors with nucleosomes, the basic building unit of chromatin. Here, we present a protocol for preparing doubly labeled fluorescent nucleosomes for SMFM. We describe steps for over-expression in E. coli and purification of recombinant human core histones. We then detail fluorescent labeling of histones and nucleosomal double-stranded DNA followed by octamer refolding and nucleosome reconstitution.
PubMed: 37083320
DOI: 10.1016/j.xpro.2023.102229 -
BBA Advances 2023Permeabilization of the mitochondrial outer membrane-a point of no return in apoptotic regulation-is tightly controlled by proteins of the Bcl-2 family. Apoptotic...
Permeabilization of the mitochondrial outer membrane-a point of no return in apoptotic regulation-is tightly controlled by proteins of the Bcl-2 family. Apoptotic inhibitor Bcl-xL is an important member of this family, responsible for blocking the permeabilization, and is also a promising target for anti-cancer drugs. Bcl-xL exists in the following conformations, each believed to play a role in the inhibition of apoptosis: (i) a soluble folded conformation, (ii) a membrane-anchored (by its C-terminal α8 helix) form, which retains the same fold as in solution and (iii) refolded membrane-inserted conformations, for which no structural data are available. In this review, we present the summary of the application of various methods of fluorescence spectroscopy for studying membrane interaction of Bcl-xL, and specifically the formation of the refolded inserted conformation. We discuss the application of environment-sensitive probes, Förster resonance energy transfer, fluorescence correlation spectroscopy, and fluorescent quenching for structural, thermodynamic, and functional characterization of protein-lipid interactions, which can benefit studies of other members of Bcl-2 (e.g., Bax, BAK, Bid). The conformational switching between various conformations of Bcl-xL depends on the presence of divalent cations, pH and lipid composition. This insertion-refolding transition also results in the release of the BH4 regulatory domain from the folded structure of Bcl-xL, which is relevant to the lipid-regulated conversion between canonical and non-canonical modes of apoptotic inhibition.
PubMed: 37082264
DOI: 10.1016/j.bbadva.2023.100076 -
ACS Omega Apr 2023γ-Secretase activating protein (GSAP) is known to play an important role in the β-amyloid pathway. It acts as a modulator and accentuates the truncation of the amyloid...
γ-Secretase activating protein (GSAP) is known to play an important role in the β-amyloid pathway. It acts as a modulator and accentuates the truncation of the amyloid precursor protein C-99 fragment through the γ-secretase complex. GSAP has four isoforms, out of which canonical isoform 1, a 16 kDa C-terminal portion, has been extensively studied, whereas the function of other three isoforms remains unknown. Here, we explore the GSAP isoform 4 (GSAP_I4) expression and purification from inclusion bodies followed by the refolding of the protein. The secondary structure of GSAP_I4 is predicted using circular dichroism. The protein is further characterized by western blotting and mass spectroscopy analysis. Additionally, biochemical assays and in silico molecular docking and molecular simulation are performed to investigate the binding of GSAP_I4 and APP-C99 peptide fragments. The results reflect that although GSAP_I1 and GSAP_I4 share high sequence similarity, the isoform 4 does not show any affinity toward APP-C99 peptide fragments. This hints toward the fact that GSAP_I4 might have a different role in the living system that is yet unexplored.
PubMed: 37065030
DOI: 10.1021/acsomega.3c01117