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2.
Cell ; 183(2): 347-362.e24, 2020 10 15.
Article in English | MEDLINE | ID: mdl-33064988

ABSTRACT

Neoantigens arise from mutations in cancer cells and are important targets of T cell-mediated anti-tumor immunity. Here, we report the first open-label, phase Ib clinical trial of a personalized neoantigen-based vaccine, NEO-PV-01, in combination with PD-1 blockade in patients with advanced melanoma, non-small cell lung cancer, or bladder cancer. This analysis of 82 patients demonstrated that the regimen was safe, with no treatment-related serious adverse events observed. De novo neoantigen-specific CD4+ and CD8+ T cell responses were observed post-vaccination in all of the patients. The vaccine-induced T cells had a cytotoxic phenotype and were capable of trafficking to the tumor and mediating cell killing. In addition, epitope spread to neoantigens not included in the vaccine was detected post-vaccination. These data support the safety and immunogenicity of this regimen in patients with advanced solid tumors (Clinicaltrials.gov: NCT02897765).


Subject(s)
Cancer Vaccines/immunology , Immunotherapy/methods , Precision Medicine/methods , Aged , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/genetics , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/immunology , Female , Humans , Kaplan-Meier Estimate , Male , Melanoma/drug therapy , Melanoma/immunology , Middle Aged , Mutation , Nivolumab/therapeutic use , Programmed Cell Death 1 Receptor/immunology , Programmed Cell Death 1 Receptor/metabolism , Urinary Bladder Neoplasms/drug therapy , Urinary Bladder Neoplasms/immunology
3.
Immunity ; 51(4): 766-779.e17, 2019 10 15.
Article in English | MEDLINE | ID: mdl-31495665

ABSTRACT

Increasing evidence indicates CD4+ T cells can recognize cancer-specific antigens and control tumor growth. However, it remains difficult to predict the antigens that will be presented by human leukocyte antigen class II molecules (HLA-II), hindering efforts to optimally target them therapeutically. Obstacles include inaccurate peptide-binding prediction and unsolved complexities of the HLA-II pathway. To address these challenges, we developed an improved technology for discovering HLA-II binding motifs and conducted a comprehensive analysis of tumor ligandomes to learn processing rules relevant in the tumor microenvironment. We profiled >40 HLA-II alleles and showed that binding motifs were highly sensitive to HLA-DM, a peptide-loading chaperone. We also revealed that intratumoral HLA-II presentation was dominated by professional antigen-presenting cells (APCs) rather than cancer cells. Integrating these observations, we developed algorithms that accurately predicted APC ligandomes, including peptides from phagocytosed cancer cells. These tools and biological insights will enable improved HLA-II-directed cancer therapies.


Subject(s)
Antigen-Presenting Cells/immunology , CD4-Positive T-Lymphocytes/immunology , Cancer Vaccines/immunology , Epitope Mapping/methods , HLA Antigens/metabolism , Histocompatibility Antigens Class II/genetics , Immunotherapy/methods , Mass Spectrometry/methods , Neoplasms/therapy , Algorithms , Alleles , Antigen Presentation , Antigens, Neoplasm/immunology , Antigens, Neoplasm/metabolism , Datasets as Topic , HLA Antigens/genetics , HLA-D Antigens/metabolism , Humans , Neoplasms/immunology , Protein Binding , Protein Interaction Domains and Motifs/genetics , Software
4.
Proteomics ; 18(12): e1700259, 2018 06.
Article in English | MEDLINE | ID: mdl-29314742

ABSTRACT

A challenge in developing personalized cancer immunotherapies is the prediction of putative cancer-specific antigens. Currently, predictive algorithms are used to infer binding of peptides to human leukocyte antigen (HLA) heterodimers to aid in the selection of putative epitope targets. One drawback of current epitope prediction algorithms is that they are trained on datasets containing biochemical HLA-peptide binding data that may not completely capture the rules associated with endogenous processing and presentation. The field of MS has made great improvements in instrumentation speed and sensitivity, chromatographic resolution, and proteogenomic database search strategies to facilitate the identification of HLA-ligands from a variety of cell types and tumor tissues. As such, these advances have enabled MS profiling of HLA-binding peptides to be a tractable, orthogonal approach to lower throughput biochemical assays for generating comprehensive datasets to train epitope prediction algorithms. In this review, we will highlight the progress made in the field of HLA-ligand profiling enabled by MS and its impact on current and future epitope prediction strategies.


Subject(s)
Computational Biology/methods , Epitopes/immunology , HLA Antigens/immunology , Mass Spectrometry/methods , Proteogenomics/methods , Epitopes/metabolism , HLA Antigens/metabolism , Humans
5.
ACS Infect Dis ; 3(2): 176-181, 2017 02 10.
Article in English | MEDLINE | ID: mdl-28183185

ABSTRACT

The 20S core particle of the proteasome in Mycobacterium tuberculosis (Mtb) is a promising, yet unconventional, drug target. This multimeric peptidase is not essential, yet degrades proteins that have become damaged and toxic via reactions with nitric oxide (and/or the associated reactive nitrogen intermediates) produced during the host immune response. Proteasome inhibitors could render Mtb susceptible to the immune system, but they would only be therapeutically viable if they do not inhibit the essential 20S counterpart in humans. Selective inhibitors of the Mtb 20S were designed and synthesized on the bases of both its unique substrate preferences and the structures of substrate-mimicking covalent inhibitors of eukaryotic proteasomes called syringolins. Unlike the parent syringolins, the designed analogues weakly inhibit the human 20S (Hs 20S) proteasome and preferentially inhibit Mtb 20S over the human counterpart by as much as 74-fold. Moreover, they can penetrate the mycobacterial cell envelope and render Mtb susceptible to nitric oxide-mediated stress. Importantly, they do not inhibit the growth of human cell lines in vitro and thus may be starting points for tuberculosis drug development.


Subject(s)
Mycobacterium tuberculosis/enzymology , Peptides, Cyclic/chemical synthesis , Proteasome Inhibitors/chemical synthesis , Cell Line , Drug Design , Humans , Models, Molecular , Mycobacterium tuberculosis/drug effects , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/chemistry , Proteasome Inhibitors/pharmacology , Protein Binding , Substrate Specificity
6.
J Mol Biol ; 428(9 Pt B): 1861-9, 2016 05 08.
Article in English | MEDLINE | ID: mdl-26608813

ABSTRACT

The AAA+ Cdc48 ATPase (alias p97 or VCP) is a key player in multiple ubiquitin-dependent cell signaling, degradation, and quality control pathways. Central to these broad biological functions is the ability of Cdc48 to interact with a large number of adaptor proteins and to remodel macromolecular proteins and their complexes. Different models have been proposed to explain how Cdc48 might couple ATP hydrolysis to forcible unfolding, dissociation, or remodeling of cellular clients. In this review, we provide an overview of possible mechanisms for substrate unfolding/remodeling by this conserved and essential AAA+ protein machine and their adaption and possible biological function throughout evolution.


Subject(s)
Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Macromolecular Substances/metabolism , Protein Unfolding , Adenosine Triphosphate/metabolism , Evolution, Molecular , Hydrolysis , Models, Biological , Models, Molecular , Valosin Containing Protein
7.
Bioorg Med Chem ; 23(18): 6218-22, 2015 Sep 15.
Article in English | MEDLINE | ID: mdl-26296913

ABSTRACT

Natural products that inhibit the proteasome have been fruitful starting points for the development of drug candidates. Those of the syringolin family have been underexploited in this context. Using the published model for substrate mimicry by the syringolins and knowledge about the substrate preferences of the proteolytic subunits of the human proteasome, we have designed, synthesized, and evaluated syringolin analogs. As some of our analogs inhibit the activity of the proteasome with second-order rate constants 5-fold greater than that of the methyl ester of syringolin B, we conclude that the substrate mimicry model for the syringolins is valid. The improvements in in vitro potency and the activities of particular analogs against leukemia cell lines are strong bases for further development of the syringolins as anti-cancer drugs.


Subject(s)
Antineoplastic Agents/chemistry , Peptides, Cyclic/chemistry , Proteasome Inhibitors/chemistry , Antineoplastic Agents/metabolism , Antineoplastic Agents/toxicity , Biological Products/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Humans , Peptides, Cyclic/chemical synthesis , Peptides, Cyclic/toxicity , Proteasome Endopeptidase Complex/chemistry , Proteasome Endopeptidase Complex/metabolism , Proteasome Inhibitors/metabolism , Proteasome Inhibitors/toxicity , Protein Binding , Structure-Activity Relationship , Substrate Specificity
8.
Protein Sci ; 24(9): 1521-7, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26134898

ABSTRACT

Cdc48 (also known as p97 or VCP) is an essential and highly abundant, double-ring AAA+ ATPase, which is ubiquitous in archaea and eukaryotes. In archaea, Cdc48 ring hexamers play a direct role in quality control by unfolding and translocating protein substrates into the degradation chamber of the 20S proteasome. Whether Cdc48 and 20S cooperate directly in protein degradation in eukaryotic cells is unclear. Two regions of Cdc48 are important for 20S binding, the pore-2 loop at the bottom of the D2 AAA+ ring and a C-terminal tripeptide. Here, we identify an aspartic acid in the pore-2 loop as an important element in 20S recognition. Importantly, mutation of this aspartate in human Cdc48 has been linked to familial amyotrophic lateral sclerosis (ALS). In archaeal or human Cdc48 variants, we find that mutation of this pore-2 residue impairs 20S binding and proteolytic communication but does not affect the stability of the hexamer or rates of ATP hydrolysis and protein unfolding. These results suggest that human Cdc48 interacts functionally with the 20S proteasome.


Subject(s)
Adenosine Triphosphatases/genetics , Amyotrophic Lateral Sclerosis/enzymology , Amyotrophic Lateral Sclerosis/genetics , Cell Cycle Proteins/genetics , Proteasome Endopeptidase Complex/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/metabolism , Aspartic Acid/chemistry , Aspartic Acid/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/metabolism , Humans , Hydrolysis , Models, Molecular , Proteasome Endopeptidase Complex/chemistry , Protein Binding , Protein Transport , Protein Unfolding , Proteolysis , Valosin Containing Protein
9.
Proc Natl Acad Sci U S A ; 111(17): E1687-94, 2014 Apr 29.
Article in English | MEDLINE | ID: mdl-24711419

ABSTRACT

ATP-dependent proteases maintain protein quality control and regulate diverse intracellular functions. Proteasomes are primarily responsible for these tasks in the archaeal and eukaryotic domains of life. Even the simplest of these proteases function as large complexes, consisting of the 20S peptidase, a barrel-like structure composed of four heptameric rings, and one or two AAA+ (ATPase associated with a variety of cellular activities) ring hexamers, which use cycles of ATP binding and hydrolysis to unfold and translocate substrates into the 20S proteolytic chamber. Understanding how the AAA+ and 20S components of these enzymes interact and collaborate to execute protein degradation is important, but the highly dynamic nature of prokaryotic proteasomes has hampered structural characterization. Here, we use electron microscopy to determine the architecture of an archaeal Cdc48 ⋅ 20S proteasome, which we stabilized by site-specific cross-linking. This complex displays coaxial alignment of Cdc48 and 20S and is enzymatically active, demonstrating that AAA+ unfoldase wobbling with respect to 20S is not required for function. In the complex, the N-terminal domain of Cdc48, which regulates ATP hydrolysis and degradation, packs against the D1 ring of Cdc48 in a coplanar fashion, constraining mechanisms by which the N-terminal domain alters 20S affinity and degradation activity.


Subject(s)
Adenosine Triphosphatases/chemistry , Cell Cycle Proteins/chemistry , Proteasome Endopeptidase Complex/chemistry , Thermoplasma/enzymology , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/ultrastructure , Adenosine Triphosphate/metabolism , Animals , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/ultrastructure , Cross-Linking Reagents/metabolism , Enzyme Stability , Hydrolysis , Mice , Models, Molecular , Negative Staining , Proteasome Endopeptidase Complex/metabolism , Proteasome Endopeptidase Complex/ultrastructure , Protein Binding , Protein Structure, Tertiary , Valosin Containing Protein
10.
Proc Natl Acad Sci U S A ; 110(9): 3327-32, 2013 Feb 26.
Article in English | MEDLINE | ID: mdl-23401548

ABSTRACT

Proteasomes are essential and ubiquitous ATP-dependent proteases that function in eukarya, archaea, and some bacteria. These destructive but critically important proteolytic machines use a 20S core peptidase and a hexameric ATPase associated with a variety of cellular activities (AAA+) unfolding ring that unfolds and spools substrates into the peptidase chamber. In archaea, 20S can function with the AAA+ Cdc48 or proteasome-activating nucleotidase (PAN) unfoldases. Both interactions are stabilized by C-terminal tripeptides in AAA+ subunits that dock into pockets on the 20S periphery. Here, we provide evidence that archaeal Cdc48 also uses a distinct set of near-axial interactions to bind 20S and propose that similar dual determinants mediate PAN-20S interactions and Rpt(1-6)-20S interactions in the 26S proteasome. Current dogma holds that the Rpt(1-6) unfolding ring of the 19S regulatory particle is the only AAA+ partner of eukaryotic 20S. By contrast, we show that mammalian Cdc48, a key player in cell-cycle regulation, membrane fusion, and endoplasmic-reticulum-associated degradation, activates mammalian 20S and find that a mouse Cdc48 variant supports protein degradation in combination with 20S. Our results suggest that eukaryotic Cdc48 orthologs function directly with 20S to maintain intracellular protein quality control.


Subject(s)
Adenosine Triphosphatases/metabolism , Cell Cycle Proteins/metabolism , Evolution, Molecular , Proteasome Endopeptidase Complex/metabolism , Adenosine Triphosphatases/chemistry , Amino Acid Motifs , Amino Acid Sequence , Animals , Archaeal Proteins/chemistry , Archaeal Proteins/metabolism , Cell Cycle Proteins/chemistry , Mice , Models, Biological , Molecular Sequence Data , Mutant Proteins/metabolism , Proteasome Endopeptidase Complex/chemistry , Protein Binding , Protein Structure, Secondary , Proteolysis , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae Proteins , Substrate Specificity , Thermoplasma/metabolism , Valosin Containing Protein
11.
Science ; 337(6096): 843-6, 2012 Aug 17.
Article in English | MEDLINE | ID: mdl-22837385

ABSTRACT

Proteasomes are the major energy-dependent proteolytic machines in the eukaryotic and archaeal domains of life. To execute protein degradation, the 20S core peptidase combines with the AAA+ ring of the 19S regulatory particle in eukarya or with the AAA+ proteasome-activating nucleotidase ring in some archaea. Here, we find that Cdc48 and 20S from the archaeon Thermoplasma acidophilum interact to form a functional proteasome. Cdc48 is an abundant and essential double-ring AAA+ molecular machine ubiquitously present in archaea, where its function has been uncertain, and in eukarya where Cdc48 participates by largely unknown mechanisms in diverse cellular processes, including multiple proteolytic pathways. Thus, proteolysis in collaboration with the 20S peptidase may represent an ancestral function of the Cdc48 family.


Subject(s)
Adenosine Triphosphatases/metabolism , Archaeal Proteins/metabolism , Cell Cycle Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Proteolysis , Thermoplasma/enzymology , Adenosine Triphosphatases/chemistry , Cell Cycle Proteins/chemistry , Evolution, Molecular , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/metabolism , Proteasome Endopeptidase Complex/chemistry , Protein Folding , Valosin Containing Protein
12.
Proc Natl Acad Sci U S A ; 108(8): 3228-33, 2011 Feb 22.
Article in English | MEDLINE | ID: mdl-21292982

ABSTRACT

Despite some appealing similarities of protein synthesis across all phyla of life, the final phase of mRNA translation has yet to be captured. Here, we reveal the ancestral role and mechanistic principles of the newly identified twin-ATPase ABCE1 in ribosome recycling. We demonstrate that the unique iron-sulfur cluster domain and an ATP-dependent conformational switch of ABCE1 are essential both for ribosome binding and recycling. By direct (11) interaction, the peptide release factor aRF1 is shown to synergistically promote ABCE1 function in posttermination ribosome recycling. Upon ATP binding, ABCE1 undergoes a conformational switch from an open to a closed ATP-occluded state, which drives ribosome dissociation as well as the disengagement of aRF1. ATP hydrolysis is not required for a single round of ribosome splitting but for ABCE1 release from the 30S subunit to reenter a new cycle. These results provide a mechanistic understanding of final phases in mRNA translation.


Subject(s)
ATP-Binding Cassette Transporters/chemistry , Iron-Sulfur Proteins/chemistry , Ribosomes/metabolism , ADP-Ribosylation Factor 1/metabolism , ATP-Binding Cassette Transporters/metabolism , Archaea , Phase Transition , Protein Biosynthesis , Protein Conformation
13.
J Biol Chem ; 282(19): 14598-607, 2007 May 11.
Article in English | MEDLINE | ID: mdl-17355973

ABSTRACT

The ABC protein ABCE1, formerly named RNase L inhibitor RLI1, is one of the most conserved proteins in evolution and is expressed in all organisms except eubacteria. Because of its fundamental role in translation initiation and/or ribosome biosynthesis, ABCE1 is essential for life. Its molecular mechanism has, however, not been elucidated. In addition to two ABC ATPase domains, ABCE1 contains a unique N-terminal region with eight conserved cysteines, predicted to coordinate iron-sulfur clusters. Here we present detailed information on the type and on the structural organization of the Fe-S clusters in ABCE1. Based on biophysical, biochemical, and yeast genetic analyses, ABCE1 harbors two essential diamagnetic [4Fe-4S](2+) clusters with different electronic environments, one ferredoxin-like (CPX(n)CX(2)CX(2)C; Cys at positions 4-7) and one unique ABCE1-type cluster (CXPX(2)CX(3)CX(n)CP; Cys at positions 1, 2, 3, and 8). Strikingly, only seven of the eight conserved cysteines coordinating the Fe-S clusters are essential for cell viability. Mutagenesis of the cysteine at position 6 yielded a functional ABCE1 with the ferredoxin-like Fe-S cluster in a paramagnetic [3Fe-4S](+) state. Notably, a lethal mutation of the cysteine at position 4 can be rescued by ligand swapping with an adjacent, extra cysteine conserved among all eukaryotes.


Subject(s)
ATP-Binding Cassette Transporters/chemistry , Evolution, Molecular , Iron-Sulfur Proteins/chemistry , Iron/chemistry , Sulfur/chemistry , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Amino Acid Sequence , Cysteine/chemistry , Cysteine/metabolism , Electron Spin Resonance Spectroscopy , Ferredoxins/chemistry , Ferredoxins/metabolism , Genetic Complementation Test , Iron-Sulfur Proteins/genetics , Iron-Sulfur Proteins/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Saccharomyces cerevisiae , Sequence Homology, Amino Acid , Spectrophotometry, Ultraviolet , Spectroscopy, Mossbauer , Sulfolobus solfataricus/genetics , Sulfolobus solfataricus/metabolism
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