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1.
Sci Adv ; 8(17): eabl5394, 2022 04 29.
Article in English | MEDLINE | ID: mdl-35486722

ABSTRACT

Understanding peptide presentation by specific MHC alleles is fundamental for controlling physiological functions of T cells and harnessing them for therapeutic use. However, commonly used in silico predictions and mass spectroscopy have their limitations in precision, sensitivity, and throughput, particularly for MHC class II. Here, we present MEDi, a novel mammalian epitope display that allows an unbiased, affordable, high-resolution mapping of MHC peptide presentation capacity. Our platform provides a detailed picture by testing every antigen-derived peptide and is scalable to all the MHC II alleles. Given the urgent need to understand immune evasion for formulating effective responses to threats such as SARS-CoV-2, we provide a comprehensive analysis of the presentability of all SARS-CoV-2 peptides in the context of several HLA class II alleles. We show that several mutations arising in viral strains expanding globally resulted in reduced peptide presentability by multiple HLA class II alleles, while some increased it, suggesting alteration of MHC II presentation landscapes as a possible immune escape mechanism.


Subject(s)
COVID-19 , Histocompatibility Antigens Class II , Animals , Antigen Presentation , CD4-Positive T-Lymphocytes , Histocompatibility Antigens Class II/genetics , Mammals , Peptides , SARS-CoV-2
2.
Cell Rep ; 12(6): 1032-41, 2015 Aug 11.
Article in English | MEDLINE | ID: mdl-26235618

ABSTRACT

In the axial channels of ClpX and related hexameric AAA+ protein-remodeling rings, the pore-1 loops are thought to play important roles in engaging, mechanically unfolding, and translocating protein substrates. How these loops perform these functions and whether they also prevent substrate dissociation to ensure processive degradation by AAA+ proteases are open questions. Using ClpX pore-1-loop variants, single-molecule force spectroscopy, and ensemble assays, we find that the six pore-1 loops function synchronously to grip and unfold protein substrates during a power stroke but are not important in preventing substrate slipping between power strokes. The importance of grip strength is task dependent. ClpX variants with multiple mutant pore-1 loops translocate substrates as well as the wild-type enzyme against a resisting force but show unfolding defects and a higher frequency of substrate release. These problems are magnified for more mechanically stable target proteins, supporting a threshold model of substrate gripping.


Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Models, Biological , Mutation , Protein Folding , Rhodococcus/metabolism
4.
Nat Chem Biol ; 11(3): 201-6, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25599533

ABSTRACT

Hexameric ATP-dependent proteases and protein remodeling machines use conserved loops that line the axial pore to apply force to substrates during the mechanical processes of protein unfolding and translocation. Whether loops from multiple subunits act independently or coordinately in these processes is a critical aspect of the mechanism but is currently unknown for any AAA+ machine. By studying covalently linked hexamers of the Escherichia coli ClpX unfoldase bearing different numbers and configurations of wild-type and mutant pore loops, we show that loops function synergistically, and the number of wild-type loops required for efficient degradation is dependent on the stability of the protein substrate. Our results support a mechanism in which a power stroke initiated in one subunit of the ClpX hexamer results in the concurrent movement of all six pore loops, which coordinately grip and apply force to the substrate.


Subject(s)
ATP-Dependent Proteases/chemistry , Peptide Hydrolases/chemistry , ATPases Associated with Diverse Cellular Activities , Adenosine Triphosphatases/chemistry , Adenosine Triphosphate/metabolism , Endopeptidase Clp/chemistry , Escherichia coli/enzymology , Escherichia coli/genetics , Escherichia coli Proteins/chemistry , Molecular Chaperones/chemistry , Mutation , Protein Unfolding , Substrate Specificity , Translocation, Genetic
5.
Nat Struct Mol Biol ; 21(10): 871-5, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25195048

ABSTRACT

Molecular machines containing double or single AAA+ rings power energy-dependent protein degradation and other critical cellular processes, including disaggregation and remodeling of macromolecular complexes. How the mechanical activities of double-ring and single-ring AAA+ enzymes differ is unknown. Using single-molecule optical trapping, we determine how the double-ring ClpA enzyme from Escherichia coli, in complex with the ClpP peptidase, mechanically degrades proteins. We demonstrate that ClpA unfolds some protein substrates substantially faster than does the single-ring ClpX enzyme, which also degrades substrates in collaboration with ClpP. We find that ClpA is a slower polypeptide translocase and that it moves in physical steps that are smaller and more regular than steps taken by ClpX. These direct measurements of protein unfolding and translocation define the core mechanochemical behavior of a double-ring AAA+ machine and provide insight into the degradation of proteins that unfold via metastable intermediates.


Subject(s)
Endopeptidase Clp/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Proteolysis , Adenosine Triphosphate/metabolism , Endopeptidase Clp/chemistry , Escherichia coli Proteins/chemistry , Hydrolysis , Optical Tweezers , Protein Unfolding
6.
J Biol Chem ; 289(21): 15014-22, 2014 May 23.
Article in English | MEDLINE | ID: mdl-24719326

ABSTRACT

Mortalin/GRP75, the mitochondrial heat shock protein 70, plays a role in cell protection from complement-dependent cytotoxicity (CDC). As shown here, interference with mortalin synthesis enhances sensitivity of K562 erythroleukemia cells to CDC, whereas overexpression of mortalin leads to their resistance to CDC. Quantification of the binding of the C5b-9 membrane attack complex to cells during complement activation shows an inverse correlation between C5b-9 deposition and the level of mortalin in the cell. Following transfection, mortalin-enhanced GFP (EGFP) is located primarily in mitochondria, whereas mortalinΔ51-EGFP lacking the mitochondrial targeting sequence is distributed throughout the cytoplasm. Overexpressed cytosolic mortalinΔ51-EGFP has a reduced protective capacity against CDC relative to mitochondrial mortalin-EGFP. Mortalin was previously shown by us to bind to components of the C5b-9 complex. Two functional domains of mortalin, the N-terminal ATPase domain and the C-terminal substrate-binding domain, were purified after expression in bacteria. Similar to intact mortalin, the ATPase domain, but not the substrate-binding domain, was found to bind to complement proteins C8 and C9 and to inhibit zinc-induced polymerization of C9. Binding of mortalin to complement C9 and C8 occurs through an ionic interaction that is nucleotide-sensitive. We suggest that to express its full protective effect from CDC, mortalin must first reach the mitochondria. In addition, mortalin can potentially target the C8 and C9 complement components through its ATPase domain and inhibit C5b-9 assembly and stability.


Subject(s)
Complement C9/immunology , Complement System Proteins/immunology , Cytotoxicity, Immunologic/immunology , HSP70 Heat-Shock Proteins/immunology , Adenosine Diphosphate/immunology , Adenosine Diphosphate/pharmacology , Adenosine Triphosphatases/immunology , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/immunology , Adenosine Triphosphate/pharmacology , Binding Sites/genetics , Binding Sites/immunology , Blotting, Western , Complement C9/metabolism , Complement Membrane Attack Complex/immunology , Complement Membrane Attack Complex/metabolism , Complement System Proteins/metabolism , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/metabolism , Humans , K562 Cells , Microscopy, Confocal , Protein Binding/drug effects , Protein Binding/immunology , RNA Interference , Sodium Chloride/immunology , Sodium Chloride/pharmacology
7.
Cell Stress Chaperones ; 17(1): 57-66, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21811887

ABSTRACT

The mitochondrial 70-kDa heat shock protein (mtHsp70), also known in humans as mortalin, is a central component of the mitochondrial protein import motor and plays a key role in the folding of matrix-localized mitochondrial proteins. MtHsp70 is assisted by a member of the 40-kDa heat shock protein co-chaperone family named Tid1 and a nucleotide exchange factor. Whereas, yeast mtHsp70 has been extensively studied in the context of protein import in the mitochondria, and the bacterial 70-kDa heat shock protein was recently shown to act as an ATP-fuelled unfolding enzyme capable of detoxifying stably misfolded polypeptides into harmless natively refolded proteins, little is known about the molecular functions of the human mortalin in protein homeostasis. Here, we developed novel and efficient purification protocols for mortalin and the two spliced versions of Tid1, Tid1-S, and Tid1-L and showed that mortalin can mediate the in vitro ATP-dependent reactivation of stable-preformed heat-denatured model aggregates, with the assistance of Mge1 and either Tid1-L or Tid1-S co-chaperones or yeast Mdj1. Thus, in addition of being a central component of the protein import machinery, human mortalin together with Tid1, may serve as a protein disaggregating machine which, for lack of Hsp100/ClpB disaggregating co-chaperones, may carry alone the scavenging of toxic protein aggregates in stressed, diseased, or aging human mitochondria.


Subject(s)
HSP40 Heat-Shock Proteins/metabolism , HSP70 Heat-Shock Proteins/metabolism , Mitochondria/metabolism , Acetyltransferases/metabolism , Adenosine Triphosphate/metabolism , Fatty Acid Elongases , HSP40 Heat-Shock Proteins/genetics , HSP40 Heat-Shock Proteins/isolation & purification , HSP70 Heat-Shock Proteins/genetics , HSP70 Heat-Shock Proteins/isolation & purification , Humans , Mitochondrial Membrane Transport Proteins/metabolism , Molecular Chaperones/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism
8.
FEBS Lett ; 584(6): 1080-4, 2010 Mar 19.
Article in English | MEDLINE | ID: mdl-20153329

ABSTRACT

Previous studies have shown that the mammalian mitochondrial 70 kDa heat-shock protein (mortalin) can also be detected in the cytosol. Cytosolic mortalin binds p53 and by doing so, prevents translocation of the tumor suppressor into the nucleus. In this study, we developed a novel binding assay, using purified proteins, for tracking the interaction between p53 and mortalin. Our results reveal that: (i) P53 binds to the peptide-binding site of mortalin which enhances the ability of the former to bind DNA. (ii) An additional previously unknown binding site for mortalin exists within the C-terminal domain of p53.


Subject(s)
HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/metabolism , Protein Interaction Mapping , Tumor Suppressor Protein p53/chemistry , Tumor Suppressor Protein p53/metabolism , Binding Sites , DNA/metabolism , HSP70 Heat-Shock Proteins/isolation & purification , Humans , Mitochondria/chemistry , Mitochondria/metabolism , Nucleotides/pharmacology , Protein Binding/drug effects , Protein Interaction Domains and Motifs/drug effects , Protein Interaction Mapping/methods , Protein Multimerization/drug effects , Proteins/isolation & purification , Proteins/metabolism , Tumor Suppressor Protein p53/isolation & purification
9.
Int J Mol Sci ; 10(5): 2041-2053, 2009 May 06.
Article in English | MEDLINE | ID: mdl-19564938

ABSTRACT

Most of our knowledge regarding the process of protein import into mitochondria has come from research employing Saccharomyces cerevisiae as a model system. Recently, several mammalian homologues of the mitochondrial motor proteins were identified. Of particular interest for us is the human Tim14/Pam18-Tim16/Pam16 complex. We chose a structural approach in order to examine the evolutionary conservation between yeast Tim14/Pam18-Tim16/Pam16 proteins and their human homologues. For this purpose, we examined the structural properties of the purified human proteins and their interaction with their yeast homologues, in vitro. Our results show that the soluble domains of the human Tim14/Pam18 and Tim16/Pam16 proteins interact with their yeast counterparts, forming heterodimeric complexes and that these complexes interact with yeast mtHsp70.


Subject(s)
HSP70 Heat-Shock Proteins/metabolism , Membrane Transport Proteins/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Mitochondrial Proteins/pharmacokinetics , Saccharomyces cerevisiae Proteins/metabolism , Humans , Mitochondria/metabolism , Mitochondrial Membranes/metabolism , Mitochondrial Precursor Protein Import Complex Proteins , Mitochondrial Proteins/chemistry , Mitochondrial Proteins/metabolism , Mitochondrial Proton-Translocating ATPases/metabolism , Protein Structure, Secondary , Protein Transport , Saccharomyces cerevisiae/metabolism
10.
J Biol Chem ; 282(47): 33935-42, 2007 Nov 23.
Article in English | MEDLINE | ID: mdl-17881357

ABSTRACT

The final step of protein translocation across the mitochondrial inner membrane is mediated by a translocation motor composed of 1) the matrix-localized, ATP-hydrolyzing, 70-kDa heat shock protein mHsp70; 2) its anchor to the import channel, Tim44; 3) the nucleotide exchange factor Mge1; and 4) a J-domain-containing complex of co-chaperones, Tim14/Pam18-Tim16/Pam16. Despite its essential role in the biogenesis of mitochondria, the mechanism by which the translocation motor functions is still largely unknown. The goal of this work was to carry out a structure-function analysis of the mitochondrial translocation motor utilizing purified components, with an emphasis on the formation of the Tim44-mHsp70 complex. To this end, we purified Tim44 and monitored its interaction with other components of the motor using cross-linking with bifunctional reagents. The effects of nucleotides, the J-domain-containing components, and the P5 peptide (CALLSAPRR, representing part of the mitochondrial targeting signal of aspartate aminotransferase) on the formation of the translocation motor were examined. Our results show that only the peptide and nucleotides, but not J-domain-containing proteins, affect the Tim44-mHsp70 interaction. Additionally, binding of Tim44 to mHsp70 prevents the formation of a complex between the latter and Tim14/Pam18-Tim16/Pam16. Thus, mutually exclusive interactions between various components of the motor with mHsp70 regulate its functional cycle. The results are discussed in light of known models for the function of the mitochondrial translocation motor.


Subject(s)
HSP70 Heat-Shock Proteins/metabolism , Heat-Shock Proteins/metabolism , Membrane Transport Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , HSP70 Heat-Shock Proteins/chemistry , HSP70 Heat-Shock Proteins/isolation & purification , Heat-Shock Proteins/chemistry , Heat-Shock Proteins/isolation & purification , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/isolation & purification , Mitochondria/chemistry , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Membrane Transport Proteins/isolation & purification , Mitochondrial Precursor Protein Import Complex Proteins , Models, Biological , Molecular Chaperones , Multiprotein Complexes/chemistry , Multiprotein Complexes/isolation & purification , Multiprotein Complexes/metabolism , Nucleotides/chemistry , Nucleotides/metabolism , Peptides/chemistry , Peptides/metabolism , Protein Binding/physiology , Protein Transport/physiology , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/chemistry , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/isolation & purification
11.
Protein Sci ; 16(2): 316-22, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17242434

ABSTRACT

The vast majority of mitochondrial proteins are imported from the cytosol. For matrix-localized proteins, the final step of translocation across the inner membrane is mediated by the mitochondrial translocation motor, of which mhsp70 is a key component. The ATP-dependent function of mhsp70 is regulated by a complex, composed of a J-protein (called Pam18 or Tim14) and a J-like protein (called Pam16 or Tim16), and the nucleotide exchange factor Mge1. In this study, we investigated the structural properties of a recombinant purified Pam18/Tim14-Pam16/Tim16 complex using cross-linking with the bifunctional reagent DSS and CD-spectroscopy. The results of the study show that both Pam18/Tim14 and Pam16/Tim16 are thermally unstable proteins that unfold at very low temperatures (T(m) values of 16.5 degrees C and 29 degrees C, respectively). Upon mixing the proteins in vitro, or when both proteins are co-overexpressed in bacteria, Pam18/Tim14 and Pam16/Tim16 form a heterodimer that is thermally more stable than the individual proteins (T(m) = 41 degrees C). Analysis of the properties of the complex in GdnHCl shows that dissociation of the heterodimer is the limiting step in achieving full denaturation.


Subject(s)
Membrane Transport Proteins/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Circular Dichroism , Cloning, Molecular , Dimerization , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/genetics , Mitochondrial Membrane Transport Proteins/chemistry , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Precursor Protein Import Complex Proteins , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Thermodynamics
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