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Singly protonated, doubly protonated, and sodiated pentaglucosamide (GIcNAc)[SUB5], oligoglucosamines (GlcN)[SUBm], and (GIcN)[SUB3]GlcN(3OH14:0) were analyzed in an YTICR mass spectrometer by electron-ion dissociation reactions and compared to collision activation. The general fragmentation mode was found as the asymmetrical sequence fragments (B[SUBn] and minor C[SUBn] ion series) with full sequence coverage. Molecular mass information of each glucosamide or glucosamine residue can be readily obtained from the ion series. Fragmentation by electron capture dissociation revealed additional fragmentation of the N-acetyl moiety compared to sustained off-resonance irradiation collision-activated dissociation (SORI-CAD) and electroninduced dissociation (ELD). Sodiated GlcNAc[SUB5] molecular adduct ions were analyzed by ElD and compared to CAD. Both techniques provided full sequence coverage. ElD was more effective, but CAD resulted in the cross-ring ion products [SUP0,2]A[SUBn] and [SUP2,4]A[SUBn] for all relevant glucosamide residues. [ABSTRACT FROM AUTHOR]

The Human Proteome Project (HPP), the flagship initiative of the Human Proteome Organization (HUPO), has pursued two goals: (1) to credibly identify at least one isoform of every protein-coding gene and (2) to make proteomics an integral part of multiomics studies of human health and disease. The past year has seen major transitions for the HPP. neXtProt was retired as the official HPP knowledge base, UniProtKB became the reference proteome knowledge base, and Ensembl-GENCODE provides the reference protein target list. A function evidence FE1-5 scoring system has been developed for functional annotation of proteins, parallel to the PE1-5 UniProtKB/neXtProt scheme for evidence of protein expression. This report includes updates from neXtProt (version 2023-09) and UniProtKB release 2024_04, with protein expression detected (PE1) for 18138 of the 19411 GENCODE protein-coding genes (93%). The number of non-PE1 proteins ("missing proteins") is now 1273. The transition to GENCODE is a net reduction of 367 proteins (19,411 PE1-5 instead of 19,778 PE1-4 last year in neXtProt). We include reports from the Biology and Disease-driven HPP, the Human Protein Atlas, and the HPP Grand Challenge Project. We expect the new Functional Evidence FE1-5 scheme to energize the Grand Challenge Project for functional annotation of human proteins throughout the global proteomics community, including π-HuB in China.

Engineering the genetic code of an organism provides the basis for (i) making any organism safely resistant to natural viruses and (ii) preventing genetic information flow into and out of genetically modified organisms while (iii) allowing the biosynthesis of genetically encoded unnatural polymers 1-4 . Achieving these three goals requires the reassignment of multiple of the 64 codons nature uses to encode proteins. However, synonymous codon replacement-recoding-is frequently lethal, and how recoding impacts fitness remains poorly explored. Here, we explore these effects using whole-genome synthesis, multiplexed directed evolution, and genome-transcriptome-translatome-proteome co-profiling on multiple recoded genomes. Using this information, we assemble a synthetic Escherichia coli genome in seven sections using only 57 codons to encode proteins. By discovering the rules responsible for the lethality of synonymous recoding and developing a data-driven multi-omics-based genome construction workflow that troubleshoots synthetic genomes, we overcome the lethal effects of 62,007 synonymous codon swaps and 11,108 additional genomic edits. We show that synonymous recoding induces transcriptional noise including new antisense RNAs, leading to drastic transcriptome and proteome perturbation. As the elimination of select codons from an organism's genetic code results in the widespread appearance of cryptic promoters, we show that synonymous codon choice may naturally evolve to minimize transcriptional noise. Our work provides the first genome-scale description of how synonymous codon changes influence organismal fitness and paves the way for the construction of functional genomes that provide genetic firewalls from natural ecosystems and safely produce biopolymers, drugs, and enzymes with an expanded chemistry., Competing Interests: Conflict of interest statement The authors declare competing financial interests. A.N. is an inventor on a patent related to directed evolution with random genomic mutations (DIvERGE) (US10669537B2: Mutagenizing Intracellular Nucleic Acids) that has been outlicensed. Harvard Medical School has filed provisional patent applications related to this work on which A.N. and G.M.C. are listed as inventors. Q.Z., M.W., M.L., A.J., K.C., Z.L., and F.H. are employed by GenScript USA Inc., but the company had no role in designing or executing experiments. G.M.C. is a founder of GRO Biosciences and EnEvolv (now part of Ginkgo Bioworks), in which he has related financial interests. Other potentially relevant financial interests of G.M.C. are listed at http://arep.med.harvard.edu/gmc/tech.html.

Modulation of the cervix by steroid hormones and commensal microbiome play a central role in the health of the female reproductive tract. Here we describe organ-on-a-chip (Organ Chip) models that recreate the human cervical epithelial-stromal interface with a functional epithelial barrier and production of mucus with biochemical and hormone-responsive properties similar to living cervix. When Cervix Chips are populated with optimal healthy versus dysbiotic microbial communities (dominated by Lactobacillus crispatus and Gardnerella vaginalis, respectively), significant differences in tissue innate immune responses, barrier function, cell viability, proteome, and mucus composition are observed that are similar to those seen in vivo. Thus, human Cervix Organ Chips represent physiologically relevant in vitro models to study cervix physiology and host-microbiome interactions, and hence may be used as a preclinical testbed for development of therapeutic interventions to enhance women's health., (© 2024. The Author(s).)

Mucolipidosis IV (MLIV) is a rare, autosomal recessive, lysosomal disease characterized by intellectual disability, motor deficits, and progressive vision loss. Using adeno-associated vector 9 (AAV9) and AAV-PHP.B as delivery vectors, we previously demonstrated the feasibility of modifying disease course in a mouse model of MLIV by the human MCOLN1 gene transfer. Here, using a primate-enabling capsid AAV.CPP.16 (CPP16), we constructed a new, clinic-oriented MCOLN1 gene expression vector and demonstrated its efficacy in the preclinical model of MLIV. Systemic administration of CPP16-MCOLN1 in adult symptomatic Mcoln1 -/- mice at a dose of 1e12 vg per mouse resulted in MCOLN1 expression in the brain and peripheral tissues, alleviated brain pathology, rescued neuromotor function, and completely prevented paralysis. Notable expression of MCOLN1 transcripts was also detected in the retina of the mouse, which had exhibited significant degeneration at the time of the treatment. However, no increase in retinal thickness was observed after gene therapy treatment. Our results suggest a new AAV-based systemic gene replacement therapy for the treatment of MLIV that could be translated into clinical studies., Competing Interests: Y.G. and F.B. are co-inventors on a provisional IP filing “Targeted Gene Therapy Approaches to Mucolipidosis IV (MLIV)”, No. 29539–0720P02. F.B. is a co-founder of and scientific advisor to Brave Bio Inc. F.B. is a paid consultant for XinGene Therapeutics Inc. Y.G.’s immediate family member is a former CSO and a paid consultant for Brave Bio Inc., (© 2024 The Authors.)

Single-cell analysis has clearly established itself in biology and biomedical fields as an invaluable tool that allows one to comprehensively understand the relationship between cells, including their types, states, transitions, trajectories, and spatial position. Scientific methods such as fluorescence labeling, nanoscale super-resolution microscopy, advances in single cell RNAseq and proteomics technologies, provide more detailed information about biological processes which were not evident with the analysis of bulk material. This new era of single-cell biology provides a better understanding of such complex biological systems as cancer, inflammation, immunity mechanism and aging processes, and opens the door into the field of drug response heterogeneity. The latest discoveries of cellular heterogeneity gives us a unique understanding of complex biological processes, such as disease mechanism, and will lead to new strategies for better and personalized treatment strategies. Recently, single-cell proteomics techniques that allow quantification of thousands of proteins from single mammalian cells have been introduced. Here we present an improved single-cell mass spectrometry-based proteomics platform called SCREEN ( S ingle C ell p R ot E om E a N alysis) for deep and high-throughput single-cell proteome coverage with high efficiency, less turnaround time and with an improved ability for protein quantitation across more cells than previously achieved. We applied this new platform to analyze the single-cell proteomic landscape under different drug treatment over time to uncover heterogeneity in cancer cell response, which for the first time, to our knowledge, has been achieved by mass spectrometry based analytical methods. We discuss challenges in single-cell proteomics, future improvements and general trends with the goal to encourage forthcoming technical developments.

With advances in sample preparation, small-volume liquid dispensing technologies, high-resolution MS/MS instrumentation, and data acquisition methodologies, it has become increasingly possible to confidently investigate the heterogeneous proteome found within individual cells. In this chapter, we present an automated high-throughput sample preparation workflow based on the Tecan Uno instrument for quantitative single-cell mass spectrometry-based proteomics. Cells are analyzed by the Single-Cell Proteome Analysis platform (SCREEN), which was introduced earlier and provides deeper proteome coverage across single cells., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Single-cell methodologies and technologies have started a revolution in biology which until recently has primarily been limited to deep sequencing and imaging modalities. With the advent and subsequent torrid development of single-cell proteomics over the last 5 years, despite the fact that proteins cannot be amplified like transcripts, it has now become abundantly clear that it is a worthy complement to single-cell transcriptomics. In this review, we engage in an assessment of the current state of the art of single-cell proteomics including workflow, sample preparation techniques, instrumentation, and biological applications. We investigate the challenges associated with working with very small sample volumes and the acute need for robust statistical methods for data interpretation. We delve into what we believe is a promising future for biological research at single-cell resolution and highlight some of the exciting discoveries that already have been made using single-cell proteomics, including the identification of rare cell types, characterization of cellular heterogeneity, and investigation of signaling pathways and disease mechanisms. Finally, we acknowledge that there are a number of outstanding and pressing problems that the scientific community vested in advancing this technology needs to resolve. Of prime importance is the need to set standards so that this technology becomes widely accessible allowing novel discoveries to be easily verifiable. We conclude with a plea to solve these problems rapidly so that single-cell proteomics can be part of a robust, high-throughput, and scalable single-cell multi-omics platform that can be ubiquitously applied to elucidating deep biological insights into the diagnosis and treatment of all diseases that afflict us., (© 2023. The Author(s).)

Mucolipidosis IV (MLIV) is an ultra-rare, recessively inherited lysosomal disorder resulting from inactivating mutations in MCOLN1 , the gene encoding the lysosomal cation channel TRPML1. The disease primarily affects the central nervous system (CNS) and manifests in the first year with cognitive and motor developmental delay, followed by a gradual decline in neurological function across the second decade of life, blindness, and premature death in third or fourth decades. Brain pathology manifestations in MLIV are consistent with hypomyelinating leukodystrophy with brain iron accumulation. Presently, there are no approved or investigational therapies for MLIV, and pathogenic mechanisms remain largely unknown. The MLIV mouse model, Mcoln1 -/- mice, recapitulates all major manifestations of the human disease. Here, to better understand the pathological mechanisms in the MLIV brain, we performed cell type specific LC-MS/MS proteomics analysis in the MLIV mouse model and reconstituted molecular signatures of the disease in either freshly isolated populations of neurons, astrocytes, oligodendrocytes, and neural stem cells, or whole tissue cortical homogenates from young adult symptomatic Mcoln1 -/- mice. Our analysis confirmed on the molecular level major histopathological hallmarks of MLIV universally present in Mcoln1 -/- tissue and brain cells, such as hypomyelination, lysosomal dysregulation, and impaired metabolism of lipids and polysaccharides. Importantly, pathway analysis in brain cells revealed mitochondria-related alterations in all Mcoln1 -/- brain cells, except oligodendrocytes, that was not possible to resolve in whole tissue. We also report unique proteome signatures and dysregulated pathways for each brain cell population used in this study. These data shed new light on cell-intrinsic mechanisms of MLIV and provide new insights for biomarker discovery and validation to advance translational studies for this disease., Competing Interests: YG is an inventor on US patent application PCT/US2020/057839 titled GENE THERAPY APPROACHES TO MUCOLIPIDOSIS IV (MLIV) assigned to Massachusetts General Brigham Corporation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Sangster, Shahriar, Niziolek, Carisi, Lewandowski, Budnik and Grishchuk.)

Non-native conformations drive protein-misfolding diseases, complicate bioengineering efforts, and fuel molecular evolution. No current experimental technique is well suited for elucidating them and their phenotypic effects. Especially intractable are the transient conformations populated by intrinsically disordered proteins. We describe an approach to systematically discover, stabilize, and purify native and non-native conformations, generated in vitro or in vivo, and directly link conformations to molecular, organismal, or evolutionary phenotypes. This approach involves high-throughput disulfide scanning (HTDS) of the entire protein. To reveal which disulfides trap which chromatographically resolvable conformers, we devised a deep-sequencing method for double-Cys variant libraries of proteins that precisely and simultaneously locates both Cys residues within each polypeptide. HTDS of the abundant E. coli periplasmic chaperone HdeA revealed distinct classes of disordered hydrophobic conformers with variable cytotoxicity depending on where the backbone was cross-linked. HTDS can bridge conformational and phenotypic landscapes for many proteins that function in disulfide-permissive environments., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Extracellular vesicles (EVs) are released by all cells into biofluids such as plasma. The separation of EVs from highly abundant free proteins and similarly sized lipoproteins remains technically challenging. We developed a digital ELISA assay based on Single Molecule Array (Simoa) technology for ApoB-100, the protein component of several lipoproteins. Combining this ApoB-100 assay with previously developed Simoa assays for albumin and three tetraspanin proteins found on EVs (Ter-Ovanesyan, Norman et al., 2021), we were able to measure the separation of EVs from both lipoproteins and free proteins. We used these five assays to compare EV separation from lipoproteins using size exclusion chromatography with resins containing different pore sizes. We also developed improved methods for EV isolation based on combining several types of chromatography resins in the same column. We present a simple approach to quantitatively measure the main impurities of EV isolation in plasma and apply this approach to develop novel methods for enriching EVs from human plasma. These methods will enable applications where high-purity EVs are required to both understand EV biology and profile EVs for biomarker discovery., Competing Interests: DT DT has filed IP on methods for EV isolation and analysis, TG TG has filed IP on methods for EV isolation and analysis, BB BB has filed IP on methods for EV isolation and analysis, AN, SW, RL, WT No competing interests declared, DK DK has filed IP on methods for EV isolation and analysis, GC GMC has filed IP on methods for EV isolation and analysis, DW DRW has filed IP on methods for EV isolation and analysis, (© 2023, Ter-Ovanesyan, Gilboa et al.)

Stem cell differentiation is a highly dynamic process involving pervasive changes in gene expression. The large majority of existing studies has characterized differentiation at the level of individual molecular profiles, such as the transcriptome or the proteome. To obtain a more comprehensive view, we measured protein, mRNA and microRNA abundance during retinoic acid-driven differentiation of mouse embryonic stem cells. We found that mRNA and protein abundance are typically only weakly correlated across time. To understand this finding, we developed a hierarchical dynamical model that allowed us to integrate all data sets. This model was able to explain mRNA-protein discordance for most genes and identified instances of potential microRNA-mediated regulation. Overexpression or depletion of microRNAs identified by the model, followed by RNA sequencing and protein quantification, were used to follow up on the predictions of the model. Overall, our study shows how multi-omics integration by a dynamical model could be used to nominate candidate regulators., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 van den Berg et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Engineering the genetic code of an organism has been proposed to provide a firewall from natural ecosystems by preventing viral infections and gene transfer 1-6 . However, numerous viruses and mobile genetic elements encode parts of the translational apparatus 7-9 , potentially rendering a genetic-code-based firewall ineffective. Here we show that such mobile transfer RNAs (tRNAs) enable gene transfer and allow viral replication in Escherichia coli despite the genome-wide removal of 3 of the 64 codons and the previously essential cognate tRNA and release factor genes. We then establish a genetic firewall by discovering viral tRNAs that provide exceptionally efficient codon reassignment allowing us to develop cells bearing an amino acid-swapped genetic code that reassigns two of the six serine codons to leucine during translation. This amino acid-swapped genetic code renders cells resistant to viral infections by mistranslating viral proteomes and prevents the escape of synthetic genetic information by engineered reliance on serine codons to produce leucine-requiring proteins. As these cells may have a selective advantage over wild organisms due to virus resistance, we also repurpose a third codon to biocontain this virus-resistant host through dependence on an amino acid not found in nature 10 . Our results may provide the basis for a general strategy to make any organism safely resistant to all natural viruses and prevent genetic information flow into and out of genetically modified organisms., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

As ultimate parasites, viruses depend on host factors for every step of their life cycle. On the other hand, cells evolved multiple mechanisms of detecting and interfering with viral replication. Yet, our understanding of the complex ensembles of pro- and anti-viral factors is very limited in virtually every virus-cell system. Here we investigated the proteins recruited to the replication organelles of poliovirus, a representative of the genus Enterovirus of the Picornaviridae family. We took advantage of a strict dependence of enterovirus replication on a host protein GBF1, and established a stable cell line expressing a truncated GBF1 fused to APEX2 peroxidase that effectively supported viral replication upon inhibition of the endogenous GBF1. This construct biotinylated multiple host and viral proteins on the replication organelles. Among the viral proteins, the polyprotein cleavage intermediates were overrepresented, suggesting that the GBF1 environment is linked to viral polyprotein processing. The proteomics characterization of biotinylated host proteins identified multiple proteins previously associated with enterovirus replication, as well as more than 200 new factors recruited to the replication organelles. RNA metabolism proteins, many of which normally localize in the nucleus, constituted the largest group, underscoring the massive release of nuclear factors into the cytoplasm of infected cells and their involvement in viral replication. Functional analysis of several newly identified proteins revealed both pro- and anti-viral factors, including a novel component of infection-induced stress granules. Depletion of these proteins similarly affected the replication of diverse enteroviruses indicating broad conservation of the replication mechanisms. Thus, our data significantly expand the knowledge of the composition of enterovirus replication organelles, provide new insights into viral replication, and offer a novel resource for identifying targets for anti-viral interventions., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Moghimi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Tissue-specific stem cells persist for a lifetime and can differentiate to maintain homeostasis or transform to initiate cancer. Despite their importance, there are no described quality assurance mechanisms for newly formed stem cells. We observed intimate and specific interactions between macrophages and nascent blood stem cells in zebrafish embryos. Macrophage interactions frequently led to either removal of cytoplasmic material and stem cell division or complete engulfment and stem cell death. Stressed stem cells were marked by surface Calreticulin, which stimulated macrophage interactions. Using cellular barcoding, we found that Calreticulin knock-down or embryonic macrophage depletion reduced the number of stem cell clones that established adult hematopoiesis. Our work supports a model in which embryonic macrophages determine hematopoietic clonality by monitoring stem cell quality.

Pluripotent stem cells (PSC) endocrine differentiation at a large scale allows sampling of transcriptome and proteome with phosphoproteome (proteoform) at specific time points. We describe the dynamic time course of changes in cells undergoing directed beta-cell differentiation and show target proteins or previously unknown phosphorylation of critical proteins in pancreas development, NKX6-1, and Chromogranin A (CHGA). We describe fluctuations in the correlation between gene expression, protein abundance, and phosphorylation, following differentiation protocol perturbations at all stages to identify proteoform profiles. Our modeling recognizes outliers on a phenomic landscape of endocrine differentiation, and we describe new biological pathways involved. We have validated our proteomic data by analyzing independent single-cell RNAseq datasets for in-vitro pancreatic islet production and corroborated our findings for several proteins suggestive as targets for future research. The single-cell analysis combined with proteoform data places new protein targets within the specific time point and at the specific pancreatic lineage of differentiating stem cells. We suggest that non-correlating proteins abundances or new phosphorylation motifs of NKX6.1 and CHGA point to new signaling pathways that may play an essential role in beta-cell development. We present our findings for the research community's use to improve endocrine differentiation protocols and developmental studies., (© 2022 Wiley-VCH GmbH.)

Ratiometric delivery of combination chemotherapy can achieve therapeutic efficacy based on synergistic interactions between drugs. It is critical to design such combinations with drugs that complement each other and reduce cancer growth through multiple mechanisms. Using hyaluronic acid (HA) as a carrier, two chemotherapeutic agents-doxorubicin (DOX) and camptothecin (CPT)-were incorporated and tested for their synergistic potency against a broad panel of blood-cancer cell lines. The pair also demonstrated the ability to achieve immunogenic cell death by increasing the surface exposure levels of Calreticulin, thereby highlighting its ability to induce apoptosis via an alternate pathway. Global proteomic profiling of cancer cells treated with HA-DOX-CPT identified pathways that could potentially predict patient sensitivity to HA-DOX-CPT. This lays the foundation for further exploration of integrating drug delivery and proteomics in personalized immunogenic chemotherapy.

The thalidomide analogue lenalidomide (Len) is a clinical therapeutic that alters the substrate engagement of cereblon (CRBN), a substrate receptor for the CRL4 E3 ubiquitin ligase. Here, we report the development of photolenalidomide (pLen), a Len probe with a photoaffinity label and enrichment handle, designed for target identification by chemical proteomics. pLen preserves the substrate degradation profile, phenotypic antiproliferative and immunomodulatory properties of Len, and enhances interactions with the thalidomide-binding domain of CRBN, as revealed by binding site mapping and molecular modeling. Using pLen, we captured the known targets IKZF1 and CRBN from multiple myeloma MM.1S cells and further identified a new target, eukaryotic translation initiation factor 3 subunit i (eIF3i), from HEK293T cells. eIF3i is directly labeled by pLen and forms a ternary complex with CRBN in the presence of Len across several epithelial cell lines but is itself not ubiquitylated or degraded. These data point to the existence of a broader array of targets induced by ligands to CRBN that may or may not be degraded, which can be identified by the highly translatable application of pLen to additional biological systems.

Iron is essential for erythropoiesis and other biological processes, but is toxic in excess. Dietary absorption of iron is a highly regulated process and is a major determinant of body iron levels. Iron excretion, however, is considered a passive, unregulated process, and the underlying pathways are unknown. Here we investigated the role of metal transporters SLC39A14 and SLC30A10 in biliary iron excretion. While SLC39A14 imports manganese into the liver and other organs under physiological conditions, it imports iron under conditions of iron excess. SLC30A10 exports manganese from hepatocytes into the bile. We hypothesized that biliary excretion of excess iron would be impaired by SLC39A14 and SLC30A10 deficiency. We therefore analyzed biliary iron excretion in Slc39a14-and Slc30a10-deficient mice raised on iron-sufficient and -rich diets. Bile was collected surgically from the mice, then analyzed with nonheme iron assays, mass spectrometry, ELISAs, and an electrophoretic assay for iron-loaded ferritin. Our results support a model in which biliary excretion of excess iron requires iron import into hepatocytes by SLC39A14, followed by iron export into the bile predominantly as ferritin, with iron export occurring independently of SLC30A10. To our knowledge, this is the first report of a molecular determinant of mammalian iron excretion and can serve as basis for future investigations into mechanisms of iron excretion and relevance to iron homeostasis., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Chromosome 13q deletion [del(13q)], harboring the miR-15a/16-1 cluster, is one of the most common genetic alterations in mature B-cell malignancies, which originate from germinal center (GC) and post-GC B cells. Moreover, miR-15a/16 expression is frequently reduced in lymphoma and multiple myeloma (MM) cells without del(13q), suggesting important tumor-suppressor activity. However, the role of miR-15a/16-1 in B-cell activation and initiation of mature B-cell neoplasms remains to be determined. We show that conditional deletion of the miR-15a/16-1 cluster in murine GC B cells induces moderate but widespread molecular and functional changes including an increased number of GC B cells, percentage of dark zone B cells, and maturation into plasma cells. With time, this leads to development of mature B-cell neoplasms resembling human extramedullary plasmacytoma (EP) as well as follicular and diffuse large B-cell lymphomas. The indolent nature and lack of bone marrow involvement of EP in our murine model resembles human primary EP rather than MM that has progressed to extramedullary disease. We corroborate human primary EP having low levels of miR-15a/16 expression, with del(13q) being the most common genetic loss. Additionally, we show that, although the mutational profile of human EP is similar to MM, there are some exceptions such as the low frequency of hyperdiploidy in EP, which could account for different disease presentation. Taken together, our studies highlight the significant role of the miR-15a/16-1 cluster in the regulation of the GC reaction and its fundamental context-dependent tumor-suppression function in plasma cell and B-cell malignancies., (© 2021 by The American Society of Hematology.)

The molecular characterization of extracellular vesicles (EVs) has revealed a great heterogeneity in their composition at a cellular and tissue level. Current isolation methods fail to efficiently separate EV subtypes for proteomic and functional analysis. The aim of this study was to develop a reproducible and scalable isolation workflow to increase the yield and purity of EV preparations. Through a combination of polymer-based precipitation and size exclusion chromatography (Pre-SEC), we analyzed two subsets of EVs based on their CD9, CD63 and CD81 content and elution time. EVs were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blot assays. To evaluate differences in protein composition between the early- and late-eluting EV fractions, we performed a quantitative proteomic analysis of MDA-MB-468-derived EVs. We identified 286 exclusive proteins in early-eluting fractions and 148 proteins with a differential concentration between early- and late-eluting fractions. A density gradient analysis further revealed EV heterogeneity within each analyzed subgroup. Through a systems biology approach, we found significant interactions among proteins contained in the EVs which suggest the existence of functional clusters related to specific biological processes. The workflow presented here allows the study of EV subtypes within a single cell type and contributes to standardizing the EV isolation for functional studies., (© 2021 The Authors. Journal of Extracellular Vesicles published by Wiley Periodicals, LLC on behalf of the International Society for Extracellular Vesicles.)

Cohesin is a multiprotein ring complex that regulates 3D genome organization, sister chromatid cohesion, gene expression, and DNA repair. Cohesin is known to be ubiquitinated, although the mechanism, regulation, and effects of cohesin ubiquitination remain poorly defined. We previously used gene editing to introduce a dual epitope tag into the endogenous allele of each of 11 known components of cohesin in human HCT116 cells. Here we report that mass spectrometry analysis of dual-affinity purifications identified the USP13 deubiquitinase as a novel cohesin-interacting protein. Subsequent immunoprecipitation/Western blots confirmed the endogenous interaction in HCT116, 293T, HeLa, and RPE-hTERT cells; demonstrated that the interaction occurs specifically in the soluble nuclear fraction (not in the chromatin); requires the ubiquitin-binding domains (UBA1/2) of USP13; and occurs preferentially during DNA replication. Reciprocal dual-affinity purification of endogenous USP13 followed by mass spectrometry demonstrated that cohesin is its primary interactor in the nucleus. Ectopic expression and CRISPR knockout of USP13 showed that USP13 is paradoxically required for both deubiquitination and ubiquitination of cohesin subunits in human cells. USP13 was dispensable for sister chromatid cohesion in HCT116 and HeLa cells, whereas it was required for the dissociation of cohesin from chromatin as cells transit through mitosis. Together these results identify USP13 as a new cohesin-interacting protein that regulates the ubiquitination of cohesin and its cell cycle regulated interaction with chromatin., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The protozoan parasite Trichomonas vaginalis (TV), exclusively adapted to the human genital tract, is one of the most common sexually transmitted pathogens. Adding to the complexity of the host-pathogen interactions, the parasite harbors TV-specific endosymbiont viruses ( Trichomonasvirus , TVV). It was reported that small extracellular vesicles (sEVs) released by TV play a role in host immunity; however, the role of the viral endosymbiosis in this process remained unknown. We hypothesized that the virus may offer evolutionary benefit to its protozoan host at least in part by altering the immunomodulatory properties of sEVs spreading from the site of infection to non-infected immune effector cells. We infected human vaginal epithelial cells, the natural host of the parasite, with TV natively harboring TVV and an isogenic derivative of the parasite cured from the viral infection. sEVs were isolated from vaginal cell culture 24 h post TV infection and from medium where the isogenic TV strains were cultured in the absence of the human host. sEVs from TVV-negative but not TVV-positive parasites cultured alone caused NF-κB activation and increase of IL-8 and RANTES expression by uterine endocervical cells, which provide innate immune defense at the gate to the upper reproductive tract. Similarly, mononuclear leukocytes increased their IL-8, IL-6 and TNF-α output in response to sEVs from virus-negative, but not isogenic virus-positive parasites, the latter exosomes being immunosuppressive in comparison to TV medium control. The same phenomenon of suppressed immunity induced by the TVV-positive compared to TVV-negative phenotype was seen when stimulating the leukocytes with sEVs originating from infected vaginal cultures. In addition, the sEVs from the TVV-positive infection phenotype suppressed immune signaling of a toll-like receptor ligand derived from mycoplasma, another frequent TV symbiont. Quantitative comparative proteome analysis of the secreted sEVs from virus-positive versus virus-negative TV revealed differential expression of two functionally uncharacterized proteins and five proteins involved in Zn binding, protein binding, electron transfer, transferase and catalytic activities. These data support the concept that symbiosis with viruses may provide benefit to the protozoan parasite by exploiting sEVs as a vehicle for inter-cellular communications and modifying their protein cargo to suppress host immune activation., (Copyright © 2020 Govender, Chan, Yamamoto, Budnik and Fichorova.)

Extracellular vesicles (EVs) derived from various stem cell sources induce cardioprotective effects during ischemia-reperfusion injury (IRI). These have been attributed mainly to the antiapoptotic, proangiogenic, microRNA (miRNA) cargo within the stem cell-derived EVs. However, the mechanisms of EV-mediated endothelial signaling to cardiomyocytes, as well as their therapeutic potential toward ischemic myocardial injury, are not clear. EV content beyond miRNA that may contribute to cardioprotection has not been fully illuminated. This study characterized the protein cargo of human vascular endothelial EVs (EEVs) to identify lead cardioactive proteins and assessed the effect of EEVs on human laminar cardiac tissues (hlCTs) exposed to IRI. We mapped the protein content of human vascular EEVs and identified proteins that were previously associated with cellular metabolism, redox state, and calcium handling, among other processes. Analysis of the protein landscape of human cardiomyocytes revealed corresponding modifications induced by EEV treatment. To assess their human-specific cardioprotection in vitro, we developed a human heart-on-a-chip IRI assay using human stem cell-derived, engineered cardiac tissues. We found that EEVs alleviated cardiac cell death as well as the loss in contractile capacity during and after simulated IRI in an uptake- and dose-dependent manner. Moreover, we found that EEVs increased the respiratory capacity of normoxic cardiomyocytes. These results suggest that vascular EEVs rescue hlCTs exposed to IRI possibly by supplementing injured myocytes with cargo that supports multiple metabolic and salvage pathways and therefore may serve as a multitargeted therapy for IRI., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The functional state of the neurovascular unit (NVU), composed of the blood-brain barrier and the perivasculature that forms a dynamic interface between the blood and the central nervous system (CNS), plays a central role in the control of brain homeostasis and is strongly affected by CNS drugs. Human primary brain microvascular endothelium, astrocyte, pericyte, and neural cell cultures are often used to study NVU barrier functions as well as drug transport and efficacy; however, the proteomic and metabolomic responses of these different cell types are not well characterized. Culturing each cell type separately, using deep coverage proteomic analysis and characterization of the secreted metabolome, as well as measurements of mitochondrial activity, the responses of these cells under baseline conditions and when exposed to the NVU-impairing stimulant methamphetamine (Meth) are analyzed. These studies define the previously unknown metabolic and proteomic profiles of human brain pericytes and lead to improved characterization of the phenotype of each of the NVU cell types as well as cell-specific metabolic and proteomic responses to Meth., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

An emerging principle of cell biology is the regulated conversion of macromolecules between soluble and condensed states. To screen for such regulation of the cyanobacterial proteome, we use quantitative mass spectrometry to identify proteins that change solubility during the day-night cycle. We find a set of night-insoluble proteins that includes many enzymes in essential metabolic pathways. Using time-lapse microscopy and isotope labeling, we show that these proteins reversibly transition between punctate structures at night and a soluble state during the day without substantial degradation. We find that the cyanobacterial circadian clock regulates the kinetics of puncta formation during the night and that the appearance of puncta indicates the metabolic status of the cell. Reversible condensation of specific enzymes is thus a regulated response to the day-night cycle and may reflect a general bacterial strategy used in fluctuating growth conditions., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

The neural substrates and pathophysiological mechanisms underlying the onset of cognitive and motor deficits in autism spectrum disorders (ASDs) remain unclear. Mutations in ASD-associated SHANK3 in mice (Shank3B -/- ) result in the accelerated maturation of corticostriatal circuits during the second and third postnatal weeks. Here, we show that during this period, there is extensive remodeling of the striatal synaptic proteome and a developmental switch in glutamatergic synaptic plasticity induced by cortical hyperactivity in striatal spiny projection neurons (SPNs). Behavioral abnormalities in Shank3B -/- mice emerge during this stage and are ameliorated by normalizing excitatory synapse connectivity in medial striatal regions by the downregulation of PKA activity. These results suggest that the abnormal postnatal development of striatal circuits is implicated in the onset of behavioral deficits in Shank3B -/- mice and that modulation of postsynaptic PKA activity can be used to regulate corticostriatal drive in developing SPNs of mouse models of ASDs and other neurodevelopmental disorders., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

The proteinaceous extracellular matrix (ECM) is vital for the survival, proliferation, migration, and differentiation of many types of cancer. However, little is known regarding metabolic pathways required for ECM secretion. By using an unbiased computational approach, we searched for enzymes whose suppression may lead to disruptions in protein secretion. Here, we show that 6-phosphogluconate dehydrogenase (PGD), a cytosolic enzyme involved in carbohydrate metabolism, is required for ER structural integrity and protein secretion. Chemical inhibition or genetic suppression of PGD activity led to cell stress accompanied by significantly expanded ER volume and was rescued by compensating endogenous glutathione supplies. Our results also suggest that this characteristic ER-dilation phenotype may be a general marker indicating increased ECM protein congestion inside cells and decreased secretion. Thus, PGD serves as a link between cytosolic carbohydrate metabolism and protein secretion., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Poison frogs sequester small molecule lipophilic alkaloids from their diet of leaf litter arthropods for use as chemical defenses against predation. Although the dietary acquisition of chemical defenses in poison frogs is well documented, the physiological mechanisms of alkaloid sequestration has not been investigated. Here, we used RNA sequencing and proteomics to determine how alkaloids impact mRNA or protein abundance in the little devil frog ( Oophaga sylvatica ), and compared wild-caught chemically defended frogs with laboratory frogs raised on an alkaloid-free diet. To understand how poison frogs move alkaloids from their diet to their skin granular glands, we focused on measuring gene expression in the intestines, skin and liver. Across these tissues, we found many differentially expressed transcripts involved in small molecule transport and metabolism, as well as sodium channels and other ion pumps. We then used proteomic approaches to quantify plasma proteins, where we found several protein abundance differences between wild and laboratory frogs, including the amphibian neurotoxin binding protein saxiphilin. Finally, because many blood proteins are synthesized in the liver, we used thermal proteome profiling as an untargeted screen for soluble proteins that bind the alkaloid decahydroquinoline. Using this approach, we identified several candidate proteins that interact with this alkaloid, including saxiphilin. These transcript and protein abundance patterns suggest that the presence of alkaloids influences frog physiology and that small molecule transport proteins may be involved in toxin bioaccumulation in dendrobatid poison frogs., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The cohesin complex regulates sister chromatid cohesion, chromosome organization, gene expression, and DNA repair. Cohesin is a ring complex composed of four core subunits and seven regulatory subunits. In an effort to comprehensively identify additional cohesin-interacting proteins, we used gene editing to introduce a dual epitope tag into the endogenous allele of each of 11 known components of cohesin in cultured human cells, and we performed MS analyses on dual-affinity purifications. In addition to reciprocally identifying all known components of cohesin, we found that cohesin interacts with a panoply of splicing factors and RNA-binding proteins (RBPs). These included diverse components of the U4/U6.U5 tri-small nuclear ribonucleoprotein complex and several splicing factors that are commonly mutated in cancer. The interaction between cohesin and splicing factors/RBPs was RNA- and DNA-independent, occurred in chromatin, was enhanced during mitosis, and required RAD21. Furthermore, cohesin-interacting splicing factors and RBPs followed the cohesin cycle and prophase pathway of cell cycle-regulated interactions with chromatin. Depletion of cohesin-interacting splicing factors and RBPs resulted in aberrant mitotic progression. These results provide a comprehensive view of the endogenous human cohesin interactome and identify splicing factors and RBPs as functionally significant cohesin-interacting proteins., (© 2019 Kim et al.)

Many proteoforms-arising from alternative splicing, post-translational modifications (PTM), or paralogous genes-have distinct biological functions, such as histone PTM proteoforms. However, their quantification by existing bottom-up mass-spectrometry (MS) methods is undermined by peptide-specific biases. To avoid these biases, we developed and implemented a first-principles model (HI quant ) for quantifying proteoform stoichiometries. We characterized when MS data allow inferring proteoform stoichiometries by HI quant and derived an algorithm for optimal inference. We applied this algorithm to infer proteoform stoichiometries in two experimental systems that supported rigorous bench-marking: alkylated proteoforms spiked-in at known ratios and endogenous histone 3 PTM proteoforms quantified relative to internal heavy standards. When compared with the benchmarks, the proteoform stoichiometries interfered by HI quant without using external standards had relative error of 5-15% for simple proteoforms and 20-30% for complex proteoforms. A HI quant server is implemented at: https://web.northeastern.edu/slavov/2014HIquant/., (© 2019 Malioutov et al.)