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Post-translational modification (PTM) of antigens provides an additional source of specificities targeted by immune responses to tumors or pathogens, but identifying antigen PTMs and assessing their role in shaping the immunopeptidome is challenging. Here we describe the Protein Modification Integrated Search Engine (PROMISE), an antigen discovery pipeline that enables the analysis of 29 different PTM combinations from multiple clinical cohorts and cell lines. We expanded the antigen landscape, uncovering human leukocyte antigen class I binding motifs defined by specific PTMs with haplotype-specific binding preferences and revealing disease-specific modified targets, including thousands of new cancer-specific antigens that can be shared between patients and across cancer types. Furthermore, we uncovered a subset of modified peptides that are specific to cancer tissue and driven by post-translational changes that occurred in the tumor proteome. Our findings highlight principles of PTM-driven antigenicity, which may have broad implications for T cell-mediated therapies in cancer and beyond.

The ERM (ezrin, radixin,and moesin) family of proteins and therelated protein merlin participate in scaffolding and signaling eventsat the cell cortex. The proteins share an N-terminal FERM [band four-point-one(4.1) ERM] domain composed of three subdomains (F1, F2, and F3) withbinding sites for short linear peptide motifs. By screening the FERMdomains of the ERMs and merlin against a phage library that displayspeptides representing the intrinsically disordered regions of thehuman proteome, we identified a large number of novel ligands. Wedetermined the affinities for the ERM and merlin FERM domains interactingwith 18 peptides and validated interactions with full-length proteinsthrough pull-down experiments. The majority of the peptides containedan apparent Yx-[FILV] motif; others show alternative motifs. We defineddistinct binding sites for two types of similar but distinct bindingmotifs (YxV and FYDF) using a combination of Rosetta FlexPepDock computationalpeptide docking protocols and mutational analysis. We provide a detailedmolecular understanding of how the two types of peptides with distinctmotifs bind to different sites on the moesin FERM phosphotyrosinebinding-like subdomain and uncover interdependencies between the differenttypes of ligands. The study expands the motif-based interactomes ofthe ERMs and merlin and suggests that the FERM domain acts as a switchableinteraction hub.

Item does not contain fulltext PURPOSE: This study aimed to investigate the clinical and genetic aspects of solute carrier (SLC) genes in inherited retinal diseases (IRDs). METHODS: Exome sequencing data were filtered to identify pathogenic variants in SLC genes. Analysis of transcript and protein expression was performed on fibroblast cell lines and retinal sections. RESULTS: Comprehensive analysis of 433 SLC genes in 913 exome sequencing IRD samples revealed homozygous pathogenic variants in 6 SLC genes, including 2 candidate novel genes, which were 2 variants in SLC66A1, causing autosomal recessive retinitis pigmentosa (ARRP), and a variant in SLC39A12, causing autosomal recessive mild widespread retinal degeneration with marked macular involvement. In addition, we present 4 families with ARRP and homozygous null variants in SLC37A3 that were previously suggested to cause retinitis pigmentosa, 2 of which cause exon skipping. The recently reported SLC4A7- c.2007dup variant was found in 2 patients with ARRP resulting in the absence of protein. Finally, variants in SLC24A1 were found in 4 individuals with either ARRP or congenital stationary night blindness. CONCLUSION: We report on SLC66A1 and SLC39A12 as candidate novel IRD genes, establish SLC37A3 pathogenicity, and provide further evidence of SLC4A7 as IRD genes. We extend the phenotypic spectrum of SLC24A1 and suggest that its ARRP phenotype may be more common than previously reported.

Reliably scoring and ranking candidate models of protein complexes and assigning their oligomeric state from the structure of the crystal lattice represent outstanding challenges. A community-wide effort was launched to tackle these challenges. The latest resources on protein complexes and interfaces were exploited to derive a benchmark dataset consisting of 1677 homodimer protein crystal structures, including a balanced mix of physiological and non-physiological complexes. The non-physiological complexes in the benchmark were selected to bury a similar or larger interface area than their physiological counterparts, making it more difficult for scoring functions to differentiate between them. Next, 252 functions for scoring protein-protein interfaces previously developed by 13 groups were collected and evaluated for their ability to discriminate between physiological and non-physiological complexes. A simple consensus score generated using the best performing score of each of the 13 groups, and a cross-validated Random Forest (RF) classifier were created. Both approaches showed excellent performance, with an area under the Receiver Operating Characteristic (ROC) curve of 0.93 and 0.94 respectively, outperforming individual scores developed by different groups. Additionally, AlphaFold2 engines were shown to recall the physiological dimers with significantly higher accuracy than the non-physiological set, lending support for the pertinence of our benchmark dataset. Optimizing the combined power of interface scoring functions and evaluating it on challenging benchmark datasets appears to be a promising strategy.

A major effort in cancer research is to organize the complexities of the disease into fundamental traits. Despite conceptual progress in the last decades and the synthesis of hallmark features, no organizing principles governing cancer beyond cellular features exist. We analyzed experimentally determined structures harboring the most significant and prevalent driver missense mutations in human cancer, covering 73% (n = 168178) of the Catalog of Somatic Mutation in Cancer tumor samples (COSMIC). The results reveal that a single structural element—κ-helix (polyproline II helix)—lies at the core of driver point mutations, with significant enrichment in all major anatomical sites, suggesting that a small number of molecular traits are shared by most and perhaps all types of cancer. Thus, we uncovered the lowest possible level of organization at which carcinogenesis takes place at the protein level. This framework provides an initial scheme for a mechanistic understanding underlying the development of tumors and pinpoints key vulnerabilities.

Ufmylation, a protein modification by Ubiquitin-like (UBL) protein UFM1, plays a crucial role in several cellular processes including DNA damage response, protein translation and ER homeostasis. To date, little is known how the enzymes responsible for this modification coordinate their action. Here we have studied the details of UFL1 (E3) activity, its binding to UFC1 (E2), and its relation to UBA5 (E1), using a combination of structural modeling with Alphafold2, X-ray crystallography, NMR, andin vitrobiochemical activity assays. Guided by an Alphafold2 model, we generated an active UFL1 fusion construct that includes its cofactor DDRGK1, and solved the first crystal structure of this critical interaction. This fusion construct also unveiled the importance of the N-terminal helix of UFL1 for its binding to UFC1, which was validated by ITC and NMR experiments. Importantly, the binding site suggested by our structural model of the UFL1-UFC1 interaction reveals a conserved interface, and suggests a competition for binding to UFC1 between UFL1 and UBA5, which we reconfirmed by NMR. Altogether, our study reveals a novel, terminal helix-mediated regulatory mechanism which coordinates the cascade of E1-E2-E3 mediated transfer of UFM1 to its substrate, and provides new leads to target this important modification.Significance statementUfmylation is an important post-translational modification, but little is known about the mechanistic details of its machinery, and in particular how the UFM1 E3 ligase (UFL1) binds and functions together with the E2 conjugating enzyme (UFC1). We combined AlphaFold2 modeling, X-ray crystallography, NMR and biochemical experiments to reveal crucial elements that govern UFL1 activity and ufmylation. We discover a crucial role for the UFL1 N-terminal helix in binding to UFC1 and productive ufmylation. This helix competes with the E1 (UBA5) C-terminal helix for binding to UFC1. Altogether, our findings uncover a new, helix-mediated regulatory mechanism in ufmylation.

Visualization of inhibitory synapses requires protocol tailoring for different sample types and imaging techniques, and usually relies on genetic manipulation or the use of antibodies that underperform in tissue immunofluorescence. Starting from an endogenous ligand of gephyrin, a universal marker of the inhibitory synapse, we developed a short peptidic binder and dimerized it, significantly increasing affinity and selectivity. We further tailored fluorophores to the binder, yielding "Sylite"-a probe with outstanding signal-to-background ratio that outperforms antibodies in tissue staining with rapid and efficient penetration, mitigation of staining artifacts, and simplified handling. In super-resolution microscopy Sylite precisely localizes the inhibitory synapse and enables nanoscale measurements. Sylite profiles inhibitory inputs and synapse sizes of excitatory and inhibitory neurons in the midbrain and combined with complimentary tracing techniques reveals the synaptic connectivity.

Histone deacetylases play important biological roles well beyond the deacetylation of histone tails. In particular, HDAC6 is involved in multiple cellular processes such as apoptosis, cytoskeleton reorganization, and protein folding, affecting substrates such as ɑ-tubulin, Hsp90 and cortactin proteins. We have applied a biochemical enzymatic assay to measure the activity of HDAC6 on a set of candidate unlabeled peptides. These served for the calibration of a structure-based substrate prediction protocol, Rosetta FlexPepBind, previously used for the successful substrate prediction of HDAC8 and other enzymes. A proteome-wide screen of reported acetylation sites using our calibrated protocol together with the enzymatic assay provide new peptide substrates and avenues to novel potential functional regulatory roles of this promiscuous, multi-faceted enzyme. In particular, we propose novel regulatory roles of HDAC6 in tumorigenesis and cancer cell survival via the regulation of EGFR/Akt pathway activation. The calibration process and comparison of the results between HDAC6 and HDAC8 highlight structural differences that explain the established promiscuity of HDAC6.