Membrane Protein Chaperone Research Articles

Membrane protein chaperone and sodium chloride modulate the kinetics and morphology of amyloid beta aggregation.

Protein aggregation is a biological phenomenon caused by the accumulation of misfolded proteins. Amyloid beta (Aβ) peptides are derived from the cleavage of a larger membrane protein molecule and accumulate to form plaques extracellularly. According to the amyloid hypothesis, accumulation of Aβ aggregates in the brain is primarily responsible for the pathogenesis of Alzheimer's disease (AD). Therefore, the disassembly of Aβ aggregates may provide opportunities for alleviating or treating AD. Here, we show that the novel protein targeting machinery from chloroplast, chloroplast signal recognition particle 43 (cpSRP43), is an ATP-independent membrane protein chaperone that can both prevent and reverse Aβ aggregation effectively. Using of thioflavin T dye, we obtained the aggregation kinetics of Aβ aggregation and determined that the chaperone prevents Aβ aggregation in a concentration-dependent manner. Size exclusion chromatography and sedimentation assays showed that 10-fold excess of cpSRP43 can keep Aβ in the soluble monomeric form. Electron microscopy showed that the fibril structure was disrupted in the presence of this chaperone. Importantly, cpSRP43 utilizes the binding energy to actively remodel the preformed Aβ aggregates without assistance by a co-chaperone and ATP, emphasizing its unique function among protein chaperones. Moreover, when sodium chloride concentration is higher than 25 mm, the Aβ aggregation rate increases drastically to form tightly associated aggregates and generate more oligomers. Our results demonstrate that the presence of cpSRP43 and low NaCl levels inhibit or retard Aβ peptide aggregation, potentially opening new avenues to strategically develop an effective treatment for AD.

Membrane protein chaperone affects the kinetics and morphology of amyloid beta aggregation.

Protein aggregation is a biological phenomenon caused by the accumulation of misfolded proteins which correlates with diseases like Alzheimer's disease (AD). Amyloid beta (Aβ) peptides are derived from the cleavage of larger membrane protein molecule and accumulates to form plaques extracellularly around nerve endings. According to the amyloid hypothesis, accumulation of misfolded Aβ in the brain is primarily responsible for AD. Therefore, the disassembly of Aβ aggregates may provide opportunities for alleviating or treating AD. We show that the novel protein targeting machinery from chloroplast signal recognition particle (cpSRP43) is an effective ATP-independent membrane protein chaperone that can prevent and reverse Aβ aggregation. Thioflavin T dye determines the kinetics of Aβ aggregation and shows that the chaperone prevents Aβ aggregation in a concentration-dependent manner. Electron microscopy reveals the fibril structure is disrupted in the presence of chaperone. Fluorescence anisotropy and light scattering show that the disaggregation effect of cpSRP43 on Aβ aggregation correlates with urea denaturation. Importantly, cpSRP43 utilizes the binding energy to active remodeling the preformed Aβ aggregates without assistance by a co-chaperone and ATP, emphasizing its unique function among protein chaperones. Our results demonstrate that the presence of cpSRP43 will inhibit or disrupt Aβ peptide aggregation, potentially opening new avenues to develop an effective treatment for AD.

De novo variants in EMC1 lead to neurodevelopmental delay and cerebellar degeneration and affect glial function in Drosophila.

The endoplasmic reticulum (ER)-membrane protein complex (EMC) is a multi-protein transmembrane complex composed of 10 subunits that functions as a membrane-protein chaperone. Variants in EMC1 lead to neurodevelopmental delay and cerebellar degeneration. Multiple families with biallelic variants have been published, yet to date, only a single report of a monoallelic variant has been described, and functional evidence is sparse. Exome sequencing was used to investigate the genetic cause underlying severe developmental delay in three unrelated children. EMC1 variants were modeled in Drosophila, using loss-of-function (LoF) and overexpression studies. Glial-specific and neuronal-specific assays were used to determine whether the dysfunction was specific to one cell type. Exome sequencing identified de novo variants in EMC1 in three individuals affected by global developmental delay, hypotonia, seizures, visual impairment and cerebellar atrophy. All variants were located at Pro582 or Pro584. Drosophila studies indicated that imbalance of EMC1-either overexpression or knockdown-results in pupal lethality and suggest that the tested homologous variants are LoF alleles. In addition, glia-specific gene dosage, overexpression or knockdown, of EMC1 led to lethality, whereas neuron-specific alterations were tolerated. We establish de novo monoallelic EMC1 variants as causative of a neurological disease trait by providing functional evidence in a Drosophila model. The identified variants failed to rescue the lethality of a null allele. Variations in dosage of the wild-type EMC1, specifically in glia, lead to pupal lethality, which we hypothesize results from the altered stoichiometry of the multi-subunit protein complex EMC.

Membrane Protein Biogenesis: PAT Complex Pats Membrane Proteins into Shape

Membrane proteins with multiple transmembrane domains play essential roles in the cell, but little is known about the machinery involved in the assembly of these domains into functional proteins. Two recent studies report the discovery of novel membrane protein chaperone complexes for the biogenesis of multi-pass membrane proteins.

Two distinct sites of client protein interaction with the chaperone cpSRP43

Integral membrane proteins are prone to aggregation and misfolding in aqueous environments and therefore require binding by molecular chaperones during their biogenesis. Chloroplast signal recognition particle 43 (cpSRP43) is an ATP-independent chaperone required for the biogenesis of the most abundant class of membrane proteins, the light-harvesting chlorophyll a/b-binding proteins (LHCPs). Previous work has shown that cpSRP43 specifically recognizes an L18 loop sequence conserved among LHCP paralogs. However, how cpSRP43 protects the transmembrane domains (TMDs) of LHCP from aggregation was unclear. In this work, alkylation-protection and site-specific cross-linking experiments found that cpSRP43 makes extensive contacts with all the TMDs in LHCP. Site-directed mutagenesis identified a class of cpSRP43 mutants that bind tightly to the L18 sequence but are defective in chaperoning full-length LHCP. These mutations mapped to hydrophobic surfaces on or near the bridging helix and the β-hairpins lining the ankyrin repeat motifs of cpSRP43, suggesting that these regions are potential sites for interaction with the client TMDs. Our results suggest a working model for client protein interactions in this membrane protein chaperone.

Interactions of the Membrane Protein Chaperone Skp with BamD of the β-Barrel Assembly Machinery Complex from E. coli

Interactions of the Membrane Protein Chaperone Skp with BamD of the β-Barrel Assembly Machinery Complex from E. coli

Conformational dynamics of a membrane protein chaperone enables spatially regulated substrate capture and release

Membrane protein biogenesis poses enormous challenges to cellular protein homeostasis and requires effective molecular chaperones. Compared with chaperones that promote soluble protein folding, membrane protein chaperones require tight spatiotemporal coordination of their substrate binding and release cycles. Here we define the chaperone cycle for cpSRP43, which protects the largest family of membrane proteins, the light harvesting chlorophyll a/b-binding proteins (LHCPs), during their delivery. Biochemical and NMR analyses demonstrate that cpSRP43 samples three distinct conformations. The stromal factor cpSRP54 drives cpSRP43 to the active state, allowing it to tightly bind substrate in the aqueous compartment. Bidentate interactions with the Alb3 translocase drive cpSRP43 to a partially inactive state, triggering selective release of LHCP's transmembrane domains in a productive unloading complex at the membrane. Our work demonstrates how the intrinsic conformational dynamics of a chaperone enables spatially coordinated substrate capture and release, which may be general to other ATP-independent chaperone systems.

Sigma-1 receptor directly interacts with Rac1-GTPase in the brain mitochondria.

BackgroundSmall Rho-GTPases are critical mediators of neuronal plasticity and are involved in the pathogenesis of several psychiatric and neurological disorders. Rac-GTPase forms a multiprotein complex with upstream and downstream regulators that are essential for the spatiotemporal transmission of Rac signaling. The sigma-1 receptor (Sig1R) is a ligand-regulated membrane protein chaperone, and multiprotein complex assembly is essential to sigma-receptor function.ResultsUsing immunoprecipitation techniques, we have shown that in mitochondrial membranes Sig1R could directly interact with Rac1. Besides Rac1, the Sig1R forms complexes with inositol 1,4,5-trisphosphate receptor and Bcl2, suggesting that mitochondrial associated membranes (MAM) are involved in this macromolecular complex formation. Assembly of this complex is ligand-specific and depends on the presence of sigma agonist/antagonist, as well as on the presence of GTP/GDP. Treatment of mitochondrial membranes with (+)-pentazocine leads to the (+)-pentazocine-sensitive phosphorylation of Bad and the pentazocine-sensitive NADPH-dependent production of ROS.ConclusionWe suggest that Sig1R through Rac1 signaling induces mild oxidative stress that possibly is involved in the regulation of neuroplasticity, as well as in the prevention of apoptosis and autophagy.

A Structural Model of the Ribosome-Bound Protein Insertase YidC Reveals Lateral Translocation of the Nascent Chain

The integration of membrane proteins into the cytoplasmic membrane of bacteria usually occurs co-translationally. The universally conserved YidC protein mediates this process either individually as a membrane protein insertase, or as a membrane protein chaperone in concert with the canonical protein-conducting channel, the SecY complex. However, little is known about the structural basis of YidCs interaction with ribosome, and its co-translational insertion activity. Here, we present a structural model of YidC based on evolutionary co-variation analysis, lipid versus protein exposure and molecular dynamics simulations. The model suggests a distinct arrangement of the conserved five transmembrane domains and an amphipathic helical hairpin between TM2 and TM3 on the cytoplasmic surface of the bilayer. The model was used for docking into a cryo-electron microscopy reconstruction of a translating YidC-ribosome complex carrying the YidC substrate FOc. This structure revealed how a single copy of YidC interacts with the ribosome at the ribosomal tunnel exit and suggests a site for membrane protein insertion at the YidC protein-lipid interphase. This site was confirmed by chemical crosslinking of FOc to TM3 of YidC. Together, these data suggest a mechanism for the co-translational mode of YidC-mediated membrane protein insertion.

Crystal structure of Escherichia coli YidC, a membrane protein chaperone and insertase.

Bacterial YidC, an evolutionally conserved membrane protein, functions as a membrane protein chaperone in cooperation with the Sec translocon and as an independent insertase for membrane proteins. In Gram-negative bacteria, the transmembrane and periplasmic regions of YidC interact with the Sec proteins, forming a multi-protein complex for Sec-dependent membrane protein integration. Here, we report the crystal structure of full-length Escherichia coli YidC. The structure reveals that a hydrophilic groove, formed by five transmembrane helices, is a conserved structural feature of YidC, as compared to the previous YidC structure from Bacillus halodurans, which lacks a periplasmic domain. Structural mapping of the substrate- or Sec protein-contact sites suggested the importance of the groove for the YidC functions as a chaperone and an insertase, and provided structural insight into the multi-protein complex.

Structural Mapping of a Chaperone–Substrate Interaction Surface

AbstractNMR spectroscopy is used to detect site‐specific intermolecular short‐range contacts in a membrane–protein–chaperone complex. This is achieved by an “orthogonal” isotope‐labeling scheme that permits the unambiguous detection of intermolecular NOEs between the well‐folded chaperone and the unfolded substrate ensemble. The residues involved in these contacts are part of the chaperone–substrate contact interface. The approach is demonstrated for the 70 kDa bacterial Skp‐tOmpA complex.

The Activity and Specificity of the Outer Membrane Protein Chaperone SurA Are Modulated by a Proline Isomerase Domain

ABSTRACTSurA is a component of the periplasmic chaperone network that plays a central role in biogenesis of integral outer membrane β-barrel proteins (OMPs) in Escherichia coli. Although SurA contains two well-conserved proline isomerase (PPIase) domains, the contribution of these domains to SurA function is unclear. In the present work, we show that defects in OMP assembly caused by mutation of the β-barrel assembly factors BamA or BamB can be corrected by gain-of-function mutations in SurA that map to the first PPIase domain. These mutations apparently bypass the requirement for a stable interaction between SurA and the Bam complex and enhance SurA chaperone activity in vivo despite destabilization of the protein in vitro. Our findings suggest an autoinhibitory mechanism for regulation of SurA chaperone activity through interdomain interactions involving a PPIase domain. We propose a model in which SurA activity is modulated by an interaction between SurA and the Bam complex that alters the substrate specificity of the chaperone.

Characterization of the Human Sigma-1 Receptor Chaperone Domain Structure and Binding Immunoglobulin Protein (BiP) Interactions

The sigma-1 receptor (S1R) is a ligand-regulated membrane protein chaperone involved in the ER stress response. S1R activity is implicated in diseases of the central nervous system including amnesia, schizophrenia, depression, Alzheimer disease, and addiction. S1R has been shown previously to regulate the Hsp70 binding immunoglobulin protein (BiP) and the inositol triphosphate receptor calcium channel through a C-terminal domain. We have developed methods for bacterial expression and reconstitution of the chaperone domain of human S1R into detergent micelles that enable its study by solution NMR spectroscopy. The chaperone domain is found to contain a helix at the N terminus followed by a largely dynamic region and a structured, helical C-terminal region that encompasses a membrane associated domain containing four helices. The helical region at residues ∼198-206 is strongly amphipathic and proposed to anchor the chaperone domain to micelles and membranes. Three of the helices in the C-terminal region closely correspond to previously identified cholesterol and drug recognition sites. In addition, it is shown that the chaperone domain interacts with full-length BiP or the isolated nucleotide binding domain of BiP, but not the substrate binding domain, suggesting that the nucleotide binding domain is sufficient for S1R interactions.

Binding Regions of Outer Membrane Protein A in Complexes with the Periplasmic Chaperone Skp. A Site-Directed Fluorescence Study

Periplasmic Skp facilitates folding and membrane insertion of many outer membrane proteins (OMPs) into the outer membrane of Gram-negative bacteria. We have examined the binding sites of outer membrane protein A (OmpA) from Escherichia coli in its complexes with the membrane protein chaperone Skp and with Skp and lipopolysaccharide (LPS) by site-directed fluorescence spectroscopy. Single-Trp OmpA mutants, W(n)-OmpA, with tryptophan at position n in the polypeptide chain were isolated in the unfolded form in 8 M urea. In five beta(x)W(n)-OmpA mutants, the tryptophan was located in beta-strand x, in four l(y)W(n)-OmpA mutants, in outer loop y, and in three t(z)W(n)-OmpA mutants in turn z of the beta-barrel transmembrane domain (TMD) of OmpA. PDW(286)-OmpA contained tryptophan in the periplasmic domain (PD). After dilution of the denaturant urea in aqueous solution, spectra indicated a more hydrophobic environment of the tryptophans in beta(x)W(n) mutants in comparison to l(y)W(n)-OmpA and t(z)W(n)-OmpA, indicating that the loops and turns form the surface of hydrophobically collapsed OmpA, while the strand regions are less exposed to water. Addition of Skp increased the fluorescence of all OmpA mutants except PDW(286)-OmpA, demonstrating binding of Skp to the entire beta-barrel domain but not to the PD of OmpA. Skp bound the TMD of OmpA asymmetrically, displaying much stronger interactions with strands beta(1) to beta(3) in the N-terminus than with strands beta(5) to beta(7) in the C-terminus. This asymmetry was not observed for the outer loops and the periplasmic turns of the TMD of OmpA. The fluorescence results demonstrated that all turns and loops l(1), l(2), and l(4) were as strongly bound to Skp as the N-terminal beta-strands. Addition of five negatively charged LPS per one preformed Skp.W(n)-OmpA complex released the C-terminal loops l(2), l(3), and l(4) of the TMD of OmpA from the complex, while its periplasmic turn regions remained bound to Skp. Our results demonstrate that interactions of Skp.OmpA complexes with LPS change the conformation of OmpA in the Skp complex for facilitated insertion and folding into membranes.

Functional domain mapping of peroxin Pex19p: interaction with Pex3p is essential for function and translocation

The peroxin Pex19p functions in peroxisomal membrane assembly. Here we mapped functional domains of human Pex19p comprising 299 amino acids. Pex19p mutants deleted in the C-terminal CAAx farnesylation motif, the C-terminal 38 amino acid residues and the N-terminal 11 residues, maintained peroxisome-restoring activity in pex19 cells. The sequence 12-261 was essential for re-establishing peroxisome activity. Pex19p was partly localized to peroxisomes but mostly localized in the cytosol. Pex19p interacted with multiple membrane proteins, including the other two membrane biogenesis peroxins, Pex3p and Pex16p, those involved in matrix protein import such as Pex14p, Pex13p, Pex10p, and Pex26p, peroxisome morphogenesis factor Pex11pbeta, and a PMP70 peroxisome-targeting signal region at residues 1-123. In yeast two-hybrid assays, Pex10p and Pex11pbeta interacted only with full-length Pex19p. Of various truncated Pex19p variants active in translocating to peroxisomes, the mutants with the shortest sequence (residues 12-73 and 40-131) were localized to peroxisomes and competent in binding to Pex3p. Furthermore, membrane peroxins were initially discernible in a cytosolic staining pattern in pex19 cells only when co-expressed with Pex19p and were then localized to peroxisomes in a temporally differentiated manner. Pex19p probably functions as a chaperone for membrane proteins and transports them to peroxisomes by anchoring to Pex3p using residues 12-73 and 40-131.