Mesoscale DNA features impact APOBEC3A and APOBEC3B deaminase activity and shape tumor mutational landscapes.

Antiviral DNA cytosine deaminases APOBEC3A and APOBEC3B are major sources of mutations in cancer by catalyzing cytosine-to-uracil deamination. APOBEC3A preferentially targets single-stranded DNAs, with a noted affinity for DNA regions that adopt stem-loop secondary structures. However, the detailed substrate preferences of APOBEC3A and APOBEC3B have not been fully established, and the specific influence of the DNA sequence on APOBEC3A and APOBEC3B deaminase activity remains to be investigated. Here, we find that APOBEC3B also selectively targets DNA stem-loop structures, and they are distinct from those subjected to deamination by APOBEC3A. We develop Oligo-seq, an in vitro sequencing-based method to identify specific sequence contexts promoting APOBEC3A and APOBEC3B activity. Through this approach, we demonstrate that APOBEC3A and APOBEC3B deaminase activity is strongly regulated by specific sequences surrounding the targeted cytosine. Moreover, we identify the structural features of APOBEC3B and APOBEC3A responsible for their substrate preferences. Importantly, we determine that APOBEC3B-induced mutations in hairpin-forming sequences within tumor genomes differ from the DNA stem-loop sequences mutated by APOBEC3A. Together, our study provides evidence that APOBEC3A and APOBEC3B can generate distinct mutation landscapes in cancer genomes, driven by their unique substrate selectivity.

Structural and biochemical insights into the association between ERAP1 polymorphism and autoimmune diseases.

Autoimmune diseases afflict nearly 10% of the world's population and have a serious impact on survival and quality of life. Unfortunately, the specific pathogenesis of almost all autoimmune diseases is still unclear, with more research findings identifying some key pathogenic genes at the genetic level and several pathogenic inflammatory factor phenotypes. ERAP1 has been suggested as a potential therapeutic target for several autoimmune diseases, especially MHC-Ⅰ related. How the structure and antigenic peptide processing function of ERAP1 affect the pathogenesis of these autoimmune diseases needs to be elucidated more clearly. Genetic studies on single nucleotide polymorphism of ERAP1 provide a good bridge to better understand the relationship and pattern between ERAP1 structure, function, and disease. However, existing reviews have focused on the genetic association of ERAP1 SNPs with autoimmune diseases, and no one has specifically addressed how ERAP1 gene polymorphisms embodied at the protein level specifically mediate antigenic peptide editing and the development of multiple autoimmune diseases. In this paper, we present a comprehensive review of these ERAP1 SNPs associated with multiple autoimmune diseases, in particular the polymorphisms affecting their protein structure and enzyme function, and attempt to unravel the underlying structural and biochemical mechanisms by which ERAP1 affects the pathogenesis of multiple autoimmune diseases through the SNP-protein structure-function-disease relationship. This study will provide theoretical help and ideas for understanding the relationship between ERAP1 and autoimmune diseases and for drug design targeting wild-type and mutant proteins with different polymorphisms.

Cysteine 467 of the ASCT2 Amino Acid Transporter Is a Molecular Determinant of the Antiport Mechanism.

The plasma membrane transporter ASCT2 is a well-known Na+-dependent obligatory antiporter of neutral amino acids. The crucial role of the residue C467 in the recognition and binding of the ASCT2 substrate glutamine, has been highlighted by structure/function relationship studies. The reconstitution in proteoliposomes of the human ASCT2 produced in P. pastoris is here employed to unveil another role of the C467 residue in the transport reaction. Indeed, the site-directed mutant C467A displayed a novel property of the transporter, i.e., the ability of mediating a low but measurable unidirectional transport of [3H]-glutamine. This reaction conforms to the main features of the ASCT2-mediated transport, namely the Na+-dependence, the pH dependence, the stimulation by cholesterol included in the proteoliposome membrane, and the specific inhibition by other common substrates of the reconstituted human ASCT2. Interestingly, the WT protein cannot catalyze the unidirectional transport of [3H]-glutamine, demonstrating an unspecific phenomenon. This difference is in favor of a structural conformational change between a WT and C467A mutant that triggers the appearance of the unidirectional flux; this feature has been investigated by comparing the available 3D structures in two different conformations, and two homology models built on the basis of hEAAT1 and GLTPh.

Recognition of the antigen-presenting molecule MR1 by a Vδ3+ γδ T cell receptor.

Unlike conventional αß T cells, γδ T cells typically recognize nonpeptide ligands independently of major histocompatibility complex (MHC) restriction. Accordingly, the γδ T cell receptor (TCR) can potentially recognize a wide array of ligands; however, few ligands have been described to date. While there is a growing appreciation of the molecular bases underpinning variable (V)δ1+ and Vδ2+ γδ TCR-mediated ligand recognition, the mode of Vδ3+ TCR ligand engagement is unknown. MHC class I-related protein, MR1, presents vitamin B metabolites to αß T cells known as mucosal-associated invariant T cells, diverse MR1-restricted T cells, and a subset of human γδ T cells. Here, we identify Vδ1/2- γδ T cells in the blood and duodenal biopsy specimens of children that showed metabolite-independent binding of MR1 tetramers. Characterization of one Vδ3Vγ8 TCR clone showed MR1 reactivity was independent of the presented antigen. Determination of two Vδ3Vγ8 TCR-MR1-antigen complex structures revealed a recognition mechanism by the Vδ3 TCR chain that mediated specific contacts to the side of the MR1 antigen-binding groove, representing a previously uncharacterized MR1 docking topology. The binding of the Vδ3+ TCR to MR1 did not involve contacts with the presented antigen, providing a basis for understanding its inherent MR1 autoreactivity. We provide molecular insight into antigen-independent recognition of MR1 by a Vδ3+ γδ TCR that strengthens an emerging paradigm of antibody-like ligand engagement by γδ TCRs.

CaMKK2 facilitates Golgi-associated vesicle trafficking to sustain cancer cell proliferation.

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) regulates cell and whole-body metabolism and supports tumorigenesis. The cellular impacts of perturbing CAMKK2 expression are, however, not yet fully characterised. By knocking down CAMKK2 levels, we have identified a number of significant subcellular changes indicative of perturbations in vesicle trafficking within the endomembrane compartment. To determine how they might contribute to effects on cell proliferation, we have used proteomics to identify Gemin4 as a direct interactor, capable of binding CAMKK2 and COPI subunits. Prompted by this, we confirmed that CAMKK2 knockdown leads to concomitant and significant reductions in δ-COP protein. Using imaging, we show that CAMKK2 knockdown leads to Golgi expansion, the induction of ER stress, abortive autophagy and impaired lysosomal acidification. All are phenotypes of COPI depletion. Based on our findings, we hypothesise that CAMKK2 sustains cell proliferation in large part through effects on organelle integrity and membrane trafficking.

Allotypic variation in antigen processing controls antigenic peptide generation from SARS-CoV-2 S1 spike glycoprotein.

Population genetic variability in immune system genes can often underlie variability in immune responses to pathogens. Cytotoxic T-lymphocytes are emerging as critical determinants of both severe acute respiratory syndrome coronavirus 2 infection severity and long-term immunity, after either recovery or vaccination. A hallmark of coronavirus disease 2019 is its highly variable severity and breadth of immune responses between individuals. To address the underlying mechanisms behind this phenomenon, we analyzed the proteolytic processing of S1 spike glycoprotein precursor antigenic peptides across ten common allotypes of endoplasmic reticulum aminopeptidase 1 (ERAP1), a polymorphic intracellular enzyme that can regulate cytotoxic T-lymphocyte responses by generating or destroying antigenic peptides. We utilized a systematic proteomic approach that allows the concurrent analysis of hundreds of trimming reactions in parallel, thus better emulating antigen processing in the cell. While all ERAP1 allotypes were capable of producing optimal ligands for major histocompatibility complex class I molecules, including known severe acute respiratory syndrome coronavirus 2 epitopes, they presented significant differences in peptide sequences produced, suggesting allotype-dependent sequence biases. Allotype 10, previously suggested to be enzymatically deficient, was rather found to be functionally distinct from other allotypes. Our findings suggest that common ERAP1 allotypes can be a major source of heterogeneity in antigen processing and through this mechanism contribute to variable immune responses in coronavirus disease 2019.

Conformational dynamics linked to domain closure and substrate binding explain the ERAP1 allosteric regulation mechanism.

The endoplasmic-reticulum aminopeptidase ERAP1 processes antigenic peptides for loading on MHC-I proteins and recognition by CD8 T cells as they survey the body for infection and malignancy. Crystal structures have revealed ERAP1 in either open or closed conformations, but whether these occur in solution and are involved in catalysis is not clear. Here, we assess ERAP1 conformational states in solution in the presence of substrates, allosteric activators, and inhibitors by small-angle X-ray scattering. We also characterize changes in protein conformation by X-ray crystallography, and we localize alternate C-terminal binding sites by chemical crosslinking. Structural and enzymatic data suggest that the structural reconfigurations of ERAP1 active site are physically linked to domain closure and are promoted by binding of long peptide substrates. These results clarify steps required for ERAP1 catalysis, demonstrate the importance of conformational dynamics within the catalytic cycle, and provide a mechanism for the observed allosteric regulation and Lys/Arg528 polymorphism disease association.

ASCT1 and ASCT2: Brother and Sister?

The SLC1 family includes seven members divided into two groups, namely, EAATs and ASCTs, that share similar 3D architecture; the first one includes high-affinity glutamate transporters, and the second one includes SLC1A4 and SLC1A5, known as ASCT1 and ASCT2, respectively, responsible for the traffic of neutral amino acids across the cell plasma membrane. The physiological role of ASCT1 and ASCT2 has been investigated over the years, revealing different properties in terms of substrate specificities, affinities, and regulation by physiological effectors and posttranslational modifications. Furthermore, ASCT1 and ASCT2 are involved in pathological conditions, such as neurodegenerative disorders and cancer. This has driven research in the pharmaceutical field aimed to find drugs able to target the two proteins.This review focuses on structural, functional, and regulatory aspects of ASCT1 and ASCT2, highlighting similarities and differences.

ERAP1 binds peptide C-termini of different sequences and/or lengths by a common recognition mechanism.

Endoplasmic reticulum aminopeptidase 1 (ERAP1) plays a key role in controlling the immunopeptidomes available for presentation by MHC (major histocompatibility complex) molecules, thus influences immunodominance and cell-mediated immunity. It carries out this critical function by a unique molecular ruler mechanism that trims antigenic precursors in a peptide-length and sequence dependent manner. Acting as a molecular ruler, ERAP1 is capable of concurrently binding antigen peptide N- and C-termini by its N-terminal catalytic and C-terminal regulatory domains, respectively. As such ERAP1 can not only monitor substrate's lengths, but also exhibit a degree of sequence specificity at substrates' N- and C-termini. On the other hand, it also allows certain sequence and length flexibility in the middle part of peptide substrates that is critical for shaping MHC restricted immunopeptidomes. Here we report structural and biochemical studies to understand the molecular details on how ERAP1 can accommodate side chains of different anchoring residues at the substrate's C-terminus. We also examine how ERAP1 can accommodate antigen peptide precursors with length flexibility. Based on two newly determined complex structures, we find that ERAP1 binds the C-termini of peptides similarly even with different substrate sequences and/or lengths, by utilizing the same hydrophobic specificity pocket to accommodate peptides with either a Phe or Leu as the C-terminal anchor residue. In addition, SPR (surface plasmon resonance) binding analyses in solution further confirm the biological significance of these peptide-ERAP1 interactions. Similar to the binding mode of MHC-I molecules, ERAP1 accommodates for antigenic peptide length difference by allowing the peptide middle part to kink or bulge at the middle of its substrate binding cleft. This explains how SNP coded variants located at the middle of ERAP1 substrate binding cleft would influence the antigen pool and an individual's susceptibility to diseases.

Elucidation of the Complicated Scenario of Primate APOBEC3 Gene Evolution.

APOBEC3 proteins play pivotal roles in defenses against retroviruses, including HIV-1, as well as retrotransposons. Presumably due to the evolutionary arms race between the hosts and retroelements, APOBEC3 genes have rapidly evolved in primate lineages through sequence diversification, gene amplification and loss, and gene fusion. Consequently, modern primates possess a unique set or "repertoire" of APOBEC3 genes. The APOBEC3 gene repertoire of humans has been well investigated. There are three types of catalytic domains (Z domain; A3Z1, A3Z2, and A3Z3), 11 Z domains, and 7 independent genes, including 4 genes encoding double Z domains. However, the APOBEC3 gene repertoires of nonhuman primates remain largely unclear. Here, we characterize APOBEC3 gene repertoires among primates and investigated the evolutionary scenario of primate APOBEC3 genes using phylogenetic and comparative genomics approaches. In the 21 primate species investigated, we identified 145 APOBEC3 genes, including 69 double-domain type APOBEC3 genes. We further estimated the ages of the respective APOBEC3 genes and revealed that APOBEC3B, APOBEC3D, and APOBEC3F are the youngest in humans and were generated in the common ancestor of Catarrhini. Notably, invasion of the LINE1 retrotransposon peaked during the same period as the generation of these youngest APOBEC3 genes, implying that LINE1 invasion was one of the driving forces of the generation of these genes. Moreover, we found evidence suggesting that sequence diversification by gene conversions among APOBEC3 paralogs occurred in multiple primate lineages. Together, our analyses reveal the hidden diversity and the complicated evolutionary scenario of APOBEC3 genes in primates.IMPORTANCE In terms of virus-host interactions and coevolution, the APOBEC3 gene family is one of the most important subjects in the field of retrovirology. APOBEC3 genes are composed of a repertoire of subclasses based on sequence similarity, and a paper by LaRue et al. provides the standard guideline for the nomenclature and genomic architecture of APOBEC3 genes. However, it has been more than 10 years since this publication, and new information, including RefSeq, which we used in this study, is accumulating. Based on accumulating knowledge, APOBEC3 genes, particularly those of primates, should be refined and reannotated. This study updates knowledge of primate APOBEC3 genes and their genomic architectures. We further inferred the evolutionary scenario of primate APOBEC3 genes and the potential driving forces of APOBEC3 gene evolution. This study will be a landmark for the elucidation of the multiple aspects of APOBEC3 family genes in the future.

Design, Synthesis, and Structural Characterization of Lysine Covalent BH3 Peptides Targeting Mcl-1.

Modulating disease-relevant protein-protein interactions (PPIs) using pharmacological tools is a critical step toward the design of novel therapeutic strategies. Over the years, however, targeting PPIs has proven a very challenging task owing to the large interfacial areas. Our recent efforts identified possible novel routes for the design of potent and selective inhibitors of PPIs using a structure-based design of covalent inhibitors targeting Lys residues. In this present study, we report on the design, synthesis, and characterizations of the first Lys-covalent BH3 peptide that has a remarkable affinity and selectivity for hMcl-1 over the closely related hBfl-1 protein. Our structural studies, aided by X-ray crystallography, provide atomic-level details of the inhibitor interactions that can be used to further translate these discoveries into novel generation, Lys-covalent pro-apoptotic agents.

Small-Angle X-ray Scattering Models of APOBEC3B Catalytic Domain in a Complex with a Single-Stranded DNA Inhibitor.

In normal cells APOBEC3 (A3A-A3H) enzymes as part of the innate immune system deaminate cytosine to uracil on single-stranded DNA (ssDNA) to scramble DNA in order to give protection against a range of exogenous retroviruses, DNA-based parasites, and endogenous retroelements. However, some viruses and cancer cells use these enzymes, especially A3A and A3B, to escape the adaptive immune response and thereby lead to the evolution of drug resistance. We have synthesized first-in-class inhibitors featuring modified ssDNA. We present models based on small-angle X-ray scattering (SAXS) data that (1) confirm that the mode of binding of inhibitor to an active A3B C-terminal domain construct in the solution state is the same as the mode of binding substrate to inactive mutants of A3A and A3B revealed in X-ray crystal structures and (2) give insight into the disulfide-linked inactive dimer formed under the oxidizing conditions of purification.

Selective Covalent Targeting of Anti-apoptotic BFL-1 by a Sulfonium-Tethered Peptide.

Anti-apoptotic B cell lymphoma 2 (BCL-2) family proteins are proven targets for human cancers. Targeting the BH3-binding pockets of these anti-apoptotic proteins could reactivate apoptosis in BCL-2-depedent cancers. BFL-1 is a BCL-2 family protein overexpressed in various chemoresistant cancers. A unique cysteine at the binding interface of the BH3 and BFL-1 was previously proven to be an intriguing targeting site to irreversibly inhibit BFL-1 functions with stabilized cyclic peptide bearing a covalent warhead. Recently, we developed a sulfonium-tethered peptide cyclization strategy to construct peptide ligands that could selectively and efficiently react with the cysteine(s) of target proteins near the interacting interface. Using this method, we constructed a BFL-1 peptide inhibitor, B4-MC, that could selectively conjugate with BFL-1 both in vitro and in cell. B4-MC showed good cellular uptake, colocalized with BFL-1 on mitochondria, and showed obvious growth inhibition of BFL-1 over-expressed cancer cell lines.

The cryo-EM structure of the endocytic receptor DEC-205.

DEC-205 (CD205), a member of the macrophage mannose receptor protein family, is the prototypic endocytic receptor of dendritic cells, whose ligands include phosphorothioated cytosine-guanosine oligonucleotides, a motif often seen in bacterial or viral DNA. However, despite growing biological and clinical significance, little is known about the structural arrangement of this receptor or any of its family members. Here, we describe the 3.2 Å cryo-EM structure of human DEC-205, thereby illuminating the structure of the mannose receptor protein family. The DEC-205 monomer forms a compact structure comprising two intercalated rings of C-type lectin-like domains, where the N-terminal cysteine-rich and fibronectin domains reside at the central intersection. We establish a pH-dependent oligomerization pathway forming tetrameric DEC-205 using solution-based techniques and ultimately solved the 4.9 Å cryo-EM structure of the DEC-205 tetramer to identify the unfurling of the second lectin ring which enables tetramer formation. Furthermore, we suggest the relevance of this oligomerization pathway within a cellular setting, whereby cytosine-guanosine binding appeared to disrupt this cell-surface oligomer. Accordingly, we provide insight into the structure and oligomeric assembly of the DEC-205 receptor.

Antigen specificities and functional properties of MR1-restricted T cells.

MR1 is an MHC class I-like molecule with unique structural and biological features that make it an important member among the molecules involved in antigen presentation to T cells. Distinctive features include ubiquitous expression of the MR1 gene and its monomorphism. Another relevant property is that the MR1 protein appears at very low levels on the plasma membrane and its surface expression is regulated by antigen binding. Finally, the nature of presented antigens differs from those that bind other presenting molecules and includes small metabolites of microbial and self-origin, small drugs and tumor-associated antigens. This opinion paper describes in detail some of those features and discusses recent literature in the field.

Impact of Natural Occurring ERAP1 Single Nucleotide Polymorphisms within miRNA-Binding Sites on HCMV Infection.

Human cytomegalovirus (HCMV) is a ß-herpesvirus that causes serious problems in people with a compromised immune system, whereas it coexists asymptomatically within the host with a healthy immune system. Like other viruses, HCMV has adopted multiples strategies to manipulate the host's immune responses. Among them, expression of viral microRNAs (miRNAs) is one of the most intriguing. HCMV miR-UL112-5p and miR-US4-1 have been found to contribute to immune evasion by targeting the endoplasmic reticulum aminopeptidase 1 (ERAP1), a highly polymorphic key component of antigen processing. The current incomplete picture on the interplay between viral miRNAs and host immunity implies the need to better characterize the host genetic determinants. Naturally occurring single nucleotide polymorphisms (SNPs) within the miRNA binding sites of target genes may affect miRNA-target interactions. In this review, we focus on the relevance of 3' untranslated region (3'UTR) ERAP1 SNPs within miRNA binding sites in modulating miRNA-mRNA interactions and the possible consequent individual susceptibility to HCMV infection. Moreover, we performed an in silico analysis using different bioinformatic algorithms to predict ERAP1 variants with a putative powerful biological function. This evidence provides a basis to deepen the knowledge on how 3'UTR ERAP1 variants may alter the mechanism of action of HCMV miRNAs, in order to develop targeted antiviral therapies.

Atypical TRAV1-2- T cell receptor recognition of the antigen-presenting molecule MR1.

MR1 presents vitamin B-related metabolites to mucosal associated invariant T (MAIT) cells, which are characterized, in part, by the TRAV1-2+ αß T cell receptor (TCR). In addition, a more diverse TRAV1-2- MR1-restricted T cell repertoire exists that can possess altered specificity for MR1 antigens. However, the molecular basis of how such TRAV1-2- TCRs interact with MR1-antigen complexes remains unclear. Here, we describe how a TRAV12-2+ TCR (termed D462-E4) recognizes an MR1-antigen complex. We report the crystal structures of the unliganded D462-E4 TCR and its complex with MR1 presenting the riboflavin-based antigen 5-OP-RU. Here, the TRBV29-1 ß-chain of the D462-E4 TCR binds over the F'-pocket of MR1, whereby the complementarity-determining region (CDR) 3ß loop surrounded and projected into the F'-pocket. Nevertheless, the CDR3ß loop anchored proximal to the MR1 A'-pocket and mediated direct contact with the 5-OP-RU antigen. The D462-E4 TCR footprint on MR1 contrasted that of the TRAV1-2+ and TRAV36+ TCRs' docking topologies on MR1. Accordingly, diverse MR1-restricted T cell repertoire reveals differential docking modalities on MR1, thus providing greater scope for differing antigen specificities.

A redox switch regulates the structure and function of anti-apoptotic BFL-1.

Apoptosis is regulated by BCL-2 family proteins. Anti-apoptotic members suppress cell death by deploying a surface groove to capture the critical BH3 α-helix of pro-apoptotic members. Cancer cells hijack this mechanism by overexpressing anti-apoptotic BCL-2 family proteins to enforce cellular immortality. We previously identified and harnessed a unique cysteine (C55) in the groove of anti-apoptotic BFL-1 to selectively neutralize its oncogenic activity using a covalent stapled-peptide inhibitor. Here, we find that disulfide bonding between a native cysteine pair at the groove (C55) and C-terminal α9 helix (C175) of BFL-1 operates as a redox switch to control the accessibility of the anti-apoptotic pocket. Reducing the C55-C175 disulfide triggers α9 release, which promotes mitochondrial translocation, groove exposure for BH3 interaction and inhibition of mitochondrial permeabilization by pro-apoptotic BAX. C55-C175 disulfide formation in an oxidative cellular environment abrogates the ability of BFL-1 to bind BH3 domains. Thus, we identify a mechanism of conformational control of BFL-1 by an intramolecular redox switch.

Target the human Alanine/Serine/Cysteine Transporter 2(ASCT2): Achievement and Future for Novel Cancer Therapy.

Glutamine metabolism, described as major energy and building blocks supply to cell growth, has gained great attention. Alanine-Serine-Cysteine Transporter (ASCT2), which belongs to solute carried (SLC) family transporters and is encoded by the SLC1A5 gene serves as a significant role for glutamine transport. Indeed, ASCT2 is often overexpressed in highly proliferative cancer cells to fulfill enhanced glutamine demand. So far, ASCT2 has been proved to be a significant target during the carcinogenesis process, and emerging evidence reveals that ASCT2 inhibitors can provide a benefit strategy for cancer therapy. Herein, we describe the structure of ASCT2, and summarize its related regulatory factors which are associated with antitumor activity. Moreover, this review article highlights the remarkable reform of discovery and development for ASCT2 inhibitors. On the basis of case studies, our perspectives for targeting ASCT2 and development of ASCT2 antagonist are discussed in the final part.

Agonistic or antagonistic mucosal-associated invariant T (MAIT) cell activity is determined by the 6-alkylamino substituent on uracil MR1 ligands.

Mucosal-associated invariant T (MAIT) are a subset of innate-like T cells that are activated by uracil ligands presented by MR1. For the first time, we demonstrate that changes to the 6-aminoalkyl chain on uracil agonist 5-OP-RU can determine agonistic or antagonistic MAIT cell activity. Insomuch, a simplified agonist with a functional profile similar to 5-OP-RU, and a new structural class of antagonist that exhibits similar activity to known MAIT cell antagonist Ac-6-FP, were identified.