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Abstract The presence of autoantibodies is a defining feature of many autoimmune diseases. The number of unique autoantibody clones is conceivably limited by immune tolerance mechanisms, but unknown due to limitations of the currently applied technologies. Here, we introduce an autoantigen-specific liquid chromatography-mass spectrometry-based IgG1 Fab profiling approach using the anti-citrullinated protein antibody (ACPA) repertoire in rheumatoid arthritis (RA) as an example. We show that each patient harbors a unique and diverse ACPA IgG1 repertoire dominated by only a few antibody clones. In contrast to the total plasma IgG1 antibody repertoire, the ACPA IgG1 sub-repertoire is characterised by an expansion of antibodies that harbor one, two or even more Fab glycans, and different glycovariants of the same clone can be detected. Together, our data indicate that the autoantibody response in a prominent human autoimmune disease is complex, unique to each patient and dominated by a relatively low number of clones.

Abstract In obesity, sustained adipose tissue (AT) inflammation constitutes a cellular memory that limits the effectiveness of weight loss interventions. Yet, the impact of fasting regimens on the regulation of AT immune infiltration is still elusive. Here we show that intermittent fasting (IF) exacerbates the lipid-associated macrophage (LAM) inflammatory phenotype of visceral AT in obese mice. Importantly, this increase in LAM abundance is strongly p53 dependent and partly mediated by p53-driven adipocyte apoptosis. Adipocyte-specific deletion of p53 prevents LAM accumulation during IF, increases the catabolic state of adipocytes, and enhances systemic metabolic flexibility and insulin sensitivity. Finally, in cohorts of obese/diabetic patients, we describe a p53 polymorphism that links to efficacy of a fasting-mimicking diet and that the expression of p53 and TREM2 in AT negatively correlates with maintaining weight loss after bariatric surgery. Overall, our results demonstrate that p53 signalling in adipocytes dictates LAM accumulation in AT under IF and modulates fasting effectiveness in mice and humans.

Abstract Antibodies play a key role in the immune defence against Gram-negative bacteria. After binding to bacterial surface antigens, IgG and IgM can activate the complement system and trigger formation of lytic membrane attack complex (MAC) pores. Molecular studies to compare functional activity of antibodies on bacteria are hampered by the limited availability of well-defined antibodies against bacterial surface antigens. Therefore, we genetically engineered E. coli by expressing the StrepTagII antigen into outer membrane protein X (OmpX) and validated that these engineered bacteria were recognised by anti-StrepTagII antibodies. We then combined this antigen–antibody system with a purified complement assay to avoid interference of serum components and directly compare MAC-mediated bacterial killing via IgG1 and pentameric IgM. While both IgG1 and IgM could induce MAC-mediated killing, we show that IgM has an increased capacity to induce complement-mediated killing of E. coli compared to IgG1. While Fc mutations that enhance IgG clustering after target binding could not improve MAC formation, mutations that cause formation of pre-assembled IgG hexamers enhanced the complement activating capacity of IgG1. Altogether, we here present a system to study antibody-dependent complement activation on E. coli and show IgM’s enhanced capacity over IgG to induce complement-mediated lysis of E. coli.

Abstract Cell cycle transitions result from global changes in protein phosphorylation states triggered by cyclin-dependent kinases (CDKs). To understand how this complexity produces an ordered and rapid cellular reorganisation, we generated a high-resolution map of changing phosphosites throughout unperturbed early cell cycles in single Xenopus embryos, derived the emergent principles through systems biology analysis, and tested them by biophysical modelling and biochemical experiments. We found that most dynamic phosphosites share two key characteristics: they occur on highly disordered proteins that localise to membraneless organelles, and are CDK targets. Furthermore, CDK-mediated multisite phosphorylation can switch homotypic interactions of such proteins between favourable and inhibitory modes for biomolecular condensate formation. These results provide insight into the molecular mechanisms and kinetics of mitotic cellular reorganisation.

IntroductionUpon vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) humans will start to produce antibodies targeting virus specific antigens that will end up in circulation. In lactating women such antibodies will also end up in breastmilk, primarily in the form of secretory immunoglobulin A1 (SIgA1), the most abundant immunoglobulin (Ig) in human milk. Here we set out to investigate the SIgA1 clonal repertoire response to repeated SARS-CoV-2 vaccination, using a LC–MS fragment antigen-binding (Fab) clonal profiling approach.MethodsWe analyzed the breastmilk of six donors from a larger cohort of 109 lactating mothers who received one of three commonly used SARS-CoV-2 vaccines. We quantitatively monitored the SIgA1 Fab clonal profile over 16 timepoints, from just prior to the first vaccination until 15 days after the second vaccination.ResultsIn all donors, we detected a population of 89–191 vaccine induced clones. These populations were unique to each donor and heterogeneous with respect to individual clonal concentrations, total clonal titer, and population size. The vaccine induced clones were dominated by persistent clones (68%) which came up after the first vaccination and were retained or reoccurred after the second vaccination. However, we also observe transient SIgA1 clones (16%) which dissipated before the second vaccination, and vaccine induced clones which uniquely emerged only after the second vaccination (16%). These distinct populations were observed in all analyzed donors, regardless of the administered vaccine.DiscussionOur findings suggest that while individual donors have highly unique human milk SIgA1 clonal profiles and a highly personalized SIgA1 response to SARS-CoV-2 vaccination, there are also commonalities in vaccine induced responses.

ABSTRACTThe development of increasingly complex antibody formats, such as bispecifics, can lead to the formation of increasingly complex high- and low-molecular-weight by-products. Here, we focus on the characterization of high molecular weight species (HMWs) representing the highest complexity of size variants. Standard methods used for product release, such as size exclusion chromatography (SEC), can separate HMW by-products from the main product, but cannot distinguish smaller changes in mass. Here, for the identification of the diverse and complex HMW variants of a trivalent bispecific CrossMAb antibody, offline fractionation, as well as production of HMW by-products combined with comprehensive analytical testing, was applied. Furthermore, HMW variants were analyzed regarding their chemical binding nature and tested in functional assays regarding changes in potency of the variants. Changes in potency were explained by detailed characterization using mass photometry, SDS-PAGE analysis, native mass spectrometry (MS) coupled to SEC and bottom-up proteomics. We identified a major portion of the HMW by-products to be non-covalently linked, leading to dissociation and changes in activity. We also identified and localized high heterogeneity of a by-product of concern and applied a CD3 affinity column coupled to native MS to annotate unexpected by-products. We present here a multi-method approach for the characterization of complex HMW by-products. A better understanding of these by-products is beneficial to guide analytical method development and proper specification setting for therapeutic bispecific antibodies to ensure constant efficacy and patient safety of the product through the assessment of by-products.

In this work the authors show how a combination of glycosylations, rigid and flexible parts within neurofascin 155 and contactin 1 define a 7.4 nm distance at the myelin-axon interface and allow bridging of three-fold larger distances in the synapse.

Profiling antibody repertoires using proteomic approaches may provide a way of screening for antibody cross-reactivity against SARS-CoV-2 variants. Here the authors use a IgG1 specific cleavage method to analyse the IgG1 repertoire within recovered patients and relate this to antibody binding and neutralisation.

Formation of the mitotic checkpoint complex (MCC) is catalysed by a phosphorylation-dependent scaffold. This work provides structural details of how a tripartite Mad1:Bub1:Cdc20 complex presents Cdc20 to Mad2, triggering open-to-closed conversion of Mad2 to assemble the MCC.

The tricarboxylic acid cycle is the central pathway of energy production in eukaryotic cells and plays a key part in aerobic respiration throughout all kingdoms of life. One of the pivotal enzymes in this cycle is 2-oxoglutarate dehydrogenase complex (OGDHC), which generates NADH by oxidative decarboxylation of 2-oxoglutarate to succinyl-CoA. OGDHC is a megadalton protein complex originally thought to be assembled from three catalytically active subunits (E1o, E2o, E3). In fungi and animals, however, the protein MRPS36 has more recently been proposed as a putative additional component. Based on extensive cross-linking mass spectrometry data supported by phylogenetic analyses, we provide evidence that MRPS36 is an important member of the eukaryotic OGDHC, with no prokaryotic orthologues. Comparative sequence analysis and computational structure predictions reveal that, in contrast with bacteria and archaea, eukaryotic E2o does not contain the peripheral subunit-binding domain (PSBD), for which we propose that MRPS36 evolved as an E3 adaptor protein, functionally replacing the PSBD. We further provide a refined structural model of the complete eukaryotic OGDHC of approximately 3.45 MDa with novel mechanistic insights.

Currently available enzyme replacement therapies for lysosomal storage diseases are limited in their effectiveness due in part to short circulation times and suboptimal biodistribution of the therapeutic enzymes. We previously engineered Chinese hamster ovary (CHO) cells to produce α-galactosidase A (GLA) with various N-glycan structures and demonstrated that elimination of mannose-6-phosphate (M6P) and conversion to homogeneous sialylated N-glycans prolonged circulation time and improved biodistribution of the enzyme following a single-dose infusion into Fabry mice. Here, we confirmed these findings using repeated infusions of the glycoengineered GLA into Fabry mice and further tested whether this glycoengineering approach, Long-Acting-GlycoDesign (LAGD), could be implemented on other lysosomal enzymes. LAGD-engineered CHO cells stably expressing a panel of lysosomal enzymes [aspartylglucosamine (AGA), beta-glucuronidase (GUSB), cathepsin D (CTSD), tripeptidyl peptidase (TPP1), alpha-glucosidase (GAA) or iduronate 2-sulfatase (IDS)] successfully converted all M6P-containing N-glycans to complex sialylated N-glycans. The resulting homogenous glycodesigns enabled glycoprotein profiling by native mass spectrometry. Notably, LAGD extended the plasma half-life of all three enzymes tested (GLA, GUSB, AGA) in wildtype mice. LAGD may be widely applicable to lysosomal replacement enzymes to improve their circulatory stability and therapeutic efficacy.

Harding et al. present a biophysical and structural characterization of the complex between huntingtin (HTT) and HAP40 proteins. They show that the abundance of HAP40 is coupled with that of HTT and that there is greater conformational variety in the exon 1 of the mutant HTT than WT, important for the future drug discovery studies targeting Huntington’s disease.

A key step in therapeutic and endogenous humoral antibody characterization is identifying the amino acid sequence. So far, this task has been mainly tackled through sequencing of B-cell receptor (BCR) repertoires at the nucleotide level. Mass spectrometry (MS) has emerged as an alternative tool for obtaining sequence information directly at the – most relevant – protein level. Although several MS methods are now well established, analysis of recombinant and endogenous antibodies comes with a specific set of challenges, requiring approaches beyond the conventional proteomics workflows. Here, we review the challenges in MS-based sequencing of both recombinant as well as endogenous humoral antibodies and outline state-of-the-art methods attempting to overcome these obstacles. We highlight recent examples and discuss remaining challenges. We foresee a great future for these approaches making de novo antibody sequencing and discovery by MS-based techniques feasible, even for complex clinical samples from endogenous sources such as serum and other liquid biopsies.

To prevent unregulated complement activation, extracellular chaperones capture soluble precursors to the membrane attack complex (sMAC). Here, structural analysis of sMAC reveals how clusterin recognizes heterogeneous sMAC complexes and inhibits polymerization of complement protein C9.

Reiding et al. performed conventional proteomics as well as glycoproteomics on protein extracts from lysates of pooled neutrophils obtained from ten healthy donors. They analyse and compare the glycan occupancy on membrane and secreted glycoproteins, which is helpful to reveal the relationship between neutrophil glycoproteins and their function.

Bauzá-Martinez et al. analyzed the HLA-I peptide ligandome in extracellular vesicles (EVs) from an EBV-immortalized B cell line, JY, and compare HLA-I-binding peptides recovered from EVs to those recovered from whole-cells. The authors report an enrichment for HLA-B-binding peptides and for post-translationally modified cysteinylated peptides in EVs as compared to whole-cells.

Adeno-associated viruses (AAVs) have emerged as promising gene therapy vectors.The AAV capsid consists of 60 subunits made up from three distinct viral proteins (VPs). Here authors record high-resolution native mass spectra of intact AAV capsids to assess the VP stoichiometries in a panel of serotypes and reveals an extremely heterogeneous population of capsids of variable composition.

Recent advances in computational methods have enabled the predictive design of self-assembling protein nanomaterials with atomic-level accuracy. Here authors investigate the assembly of two computationally designed, 120-subunit icosahedral complexes and find that assembly of each material from its two constituent protein building blocks was highly cooperative.

Immunotherapy may exploit alternative vulnerabilities of drug resistant cells. Here, the authors show that the HLA peptide presentation landscape is heterogeneous even within one individual, hinting that a multi-peptide vaccination approach against highly conserved tumor suppressors may be needed.

Little is known about how cardiac metabolism remodels following cardiac injury. Here, the authors show that mitochondrial CaMKII plays an important role in remodeling cardiac metabolism after injury and that replacement of mitochondrial creatine kinase improves energetics and protects against adverse remodeling.

Here, Giansanti et al. perform a system-wide and time-resolved characterization of the changes in the host cell proteome and phosphoproteome of cells infected with the enterovirus coxsackievirus B3 during a full round of replication and identify mTORC1 signalling as a major regulation network during virus infection.

Here the authors describe PhosID, an enrichment strategy using phosphonate-handles, that combines click chemistry and IMAC-based phospho-enrichment for quantitative proteomics analysis of newly synthesized proteins.

The perinuclear theca (PT) is a highly condensed, largely insoluble protein structure that surrounds the nucleus of eutherian spermatozoa. Recent reports have indicated that the PT unexpectedly houses several somatic proteins, such as core histones, which may be important post-fertilization during re-modelling of the male pronucleus, yet little is known regarding the overall proteomic composition of the PT. Here, we report the first in depth, label-free proteomic characterization of the PT of boar spermatozoa following the implementation of a long-established subcellular fractionation protocol designed to increase the detection of low abundance proteins. A total of 1,802 proteins were identified, a result that represents unparalleled depth of coverage for the boar sperm proteome and exceeds the entire annotated proteome of the Sus scrofa species so far. In the PT structure itself, we identified 813 proteins and confirmed the presence of previously characterized PT proteins including the core histones H2A, H2B, H3 and H4, as well as Ras-related protein Rab-2A (RAB2A) and Rab-2B (RAB2B) amongst other RAB proteins. In addition to these previously characterized PT proteins, our data revealed that the PT is replete in proteins critical for sperm-egg fusion and egg activation, including: Izumo family members 1–4 (IZUMO1-4) and phosphoinositide specific phospholipase ζ (PLCZ1). Through Ingenuity Pathway Analysis, we found surprising enrichment of endoplasmic reticulum (ER) proteins and the ER-stress response in the PT. This is particularly intriguing as it is currently held that the ER structure is lost during testicular sperm maturation. Using the String and Cytoscape tools to visualize protein-protein interactions revealed an intricate network of PT protein complexes, including numerous proteasome subunits. Collectively, these data suggest that the PT may be a unique site of cellular homeostasis that houses an abundance of protein degradation machinery. This fits with previous observations that the PT structure dissociates first within the oocyte post-fertilization. It remains to be explored whether proteasome subunits within the PT actively assist in the protein degradation of paternal cell structures post-fertilization and how aberrations in PT protein content may delay embryonic development.

The Alpha-1-Antitrypsin (A1AT) protein is an important protease inhibitor highly abundant in human serum and other body fluids. Additional to functioning as a protease inhibitor, A1AT is an important acute phase protein. Here, we set out to compare the proteoform profiles of A1AT purified from the human serum and milk of eight healthy donors to determine the origin of human milk A1AT. Following affinity purification, size-exclusion chromatography coupled to native mass spectrometry was used to monitor individual proteoform profiles comparing inter- and intra-donor profiles. The A1AT intra-donor proteoform profiles were found to be highly identical between serum and milk, while they were highly distinct between donors, even when comparing only serum or milk samples. The observed inter-donor proteoform variability was due to differences in the abundances of different N-glycoforms, mainly due to branching, fucosylation, and the relative abundance of N-terminally processed A1AT fragments. From our data we conclude that nearly all A1AT in serum and milk is synthesized by a common source, i.e. the liver, and then secreted into the circulation and enters the mammary gland via diffusion or transport. Thereby, proteoform profile changes, as seen upon infection and/or inflammation in the blood will be reflected in the milk, which may then be transferred to the breastfed infant.

Mass-spectrometry based immunopeptidomics has provided unprecedented insights into antigen presentation, not only charting an enormous ligandome of self-antigens, but also cancer neoantigens and peptide antigens harbouring post-translational modifications. Here we concentrate on the latter, focusing on the small subset of HLA Class I peptides (less than 1%) that has been observed to be post-translationally modified (PTM) by a O-linked N-acetylglucosamine (GlcNAc). Just like neoantigens these modified antigens may have specific immunomodulatory functions. Here we compiled from literature, and a new dataset originating from the JY B cell lymphoblastoid cell line, a concise albeit comprehensive list of O-GlcNAcylated HLA class I peptides. This cumulative list of O-GlcNAcylated HLA peptides were derived from normal and cancerous origin, as well as tissue specimen. Remarkably, the overlap in detected O-GlcNAcylated HLA peptides as well as their source proteins is strikingly high. Most of the O-GlcNAcylated HLA peptides originate from nuclear proteins, notably transcription factors. From this list, we extract that O-GlcNAcylated HLA Class I peptides are preferentially presented by the HLA-B*07:02 allele. This allele loads peptides with a Proline residue anchor at position 2, and features a binding groove that can accommodate well the recently proposed consensus sequence for O-GlcNAcylation, P(V/A/T/S)g(S/T), essentially explaining why HLA-B*07:02 is a favoured binding allele. The observations drawn from the compiled list, may assist in the prediction of novel O-GlcNAcylated HLA antigens, which will be best presented by patients harbouring HLA-B*07:02 or related alleles that use Proline as anchoring residue.

Recently, a mass spectrometry-based approach was introduced to directly assess the IgG1 immunoglobulin clonal repertoires in plasma. Here we expanded upon this approach by describing a mass spectrometry-based technique to assess specifically the clonal repertoire of another important class of immunoglobulin molecules, IgA1, and show it is efficiently and robustly applicable to either milk or plasma samples. Focusing on two individual healthy donors, whose milk was sampled longitudinally during the first 16 weeks of lactation, we demonstrate that the total repertoire of milk sIgA1 is dominated by only 50-500 clones, even though the human body theoretically can generate several orders of magnitude more clones. We show that in each donor the sIgA1 repertoire only changes marginally and quite gradually over the monitored 16-week period of lactation. Furthermore, the observed overlap in clonal repertoires between the two individual donors is close to non-existent. Mothers provide protection to their newborn infants directly by the transfer of antibodies via breastfeeding. The approach introduced here, can be used to visualize the clonal repertoire transferred from mother to infant and to detect changes in-time in that repertoire adapting to changes in maternal physiology.

RIPK1 kinase activity is known to transduce a death signal, but the molecular mechanisms that normally prevent RIPK1 activation are unclear. Here, the authors report that IKK-mediated phosphorylation on RIPK1 Ser25 directly represses its enzymatic activity and thus RIPK1-dependent cell death.

Sepsis and septic shock remain the leading causes of death in intensive care units (ICUs), yet the pathogenesis originating from the inflammatory response during sepsis remains ambiguous. Acute-phase proteins are typically highly glycosylated, and the nature of the glycans have been linked to the incidence and severity of such inflammatory responses. To further build upon these findings we here monitored, the longitudinal changes in the plasma proteome and, in molecular detail, glycoproteoform profiles of alpha-1-antichymotrypsin (AACT) extracted from plasma of ten individual septic patients. For each patient we included four different time-points, including post-operative (before sepsis) and following discharge from the ICU. We isolated AACT from plasma depleted for albumin, IgG and serotransferrin and used high-resolution native mass spectrometry to qualitatively and quantitatively monitor the multifaceted glycan microheterogeneity of desialylated AACT, which allowed us to monitor how changes in the glycoproteoform profiles reflected the patient’s physiological state. Although we observed a general trend in the remodeling of the AACT glycoproteoform profiles, e.g. increased fucosylation and branching/LacNAc elongation, each patient exhibited unique features and responses, providing a resilient proof-of-concept for the importance of personalized longitudinal glycoproteoform profiling. Importantly, we observed that the AACT glycoproteoform changes induced by sepsis did not readily subside after discharge from ICU.

Gene therapy approaches now allow for the production of therapeutic antibodies by healthy or cancerous human tissues directly in vivo, and, with an increasing number of gene delivery methods available, the cell type for expression can be chosen. Yet, little is known about the biophysical changes introduced by expressing antibodies from producer cells or tissues targeted by gene therapy approaches, nor about the consequences for the type of glycosylation. The effects of different glycosylation on therapeutic antibodies have been well studied by controlling their glycan compositions in non-human mammalian production cells, i.e., Chinese hamster ovary cells. Therefore, we investigated the glycosylation state of clinically approved antibodies secreted from cancer tissues frequently targeted by in vivo gene therapy, using native mass spectrometry and glycoproteomics. We found that antibody sialylation and fucosylation depended on the producer tissue and the antibody isotype, allowing us to identify optimal producer cell types according to the desired mode of action of the antibody. Furthermore, we discovered that high amounts (>20%) of non-glycosylated antibodies were produced in cells sensitive to the action of the produced antibodies. Different glycosylation in different producer cells can translate into an altered potency of in-vivo produced antibodies, depending on the desired mode of action, and can affect their serum half-lives. These results increase our knowledge about antibodies produced from cells targeted by gene therapy, enabling development of improved cancer gene therapy vectors that can include in vivo glycoengineering of expressed antibodies to optimize their efficacies, depending on the desired mode of action.

Fucosidases are associated with several pathological conditions and play an important role in the health of the human gut. For example, fucosidases have been shown to be indicators and/or involved in hepatocellular carcinoma, breast cancer, and helicobacter pylori infections. A prerequisite for the detection and profiling of fucosidases is the formation of a specific covalent linkage between the enzyme of interest and the activity-based probe (ABP). The most commonly used fucosidase ABPs are limited to only one of the classes of fucosidases, the retaining fucosidases. New approaches are needed that allow for the detection of the second class of fucosidases, the inverting type. Here, we report an ortho-quinone methide-based probe with an azide mini-tag that selectively labels both retaining and inverting bacterial α-l-fucosidases. Mass spectrometry-based intact protein and sequence analysis of a probe-labeled bacterial fucosidase revealed almost exclusive single labeling at two specific tryptophan residues outside of the active site. Furthermore, the probe could detect and image extracellular fucosidase activity on the surface of live bacteria.

Triple-negative breast cancer (TNBC) lacks prognostic and predictive markers. Here, the authors use phosphoproteomics to define kinases with distinct activity profiles in TNBC, demonstrating their prognostic value as well as their utility for simplifying TNBC classification and designing drug regimens.

Several therapeutics are glycosylated proteins, yet the analysis of their specific glycosylation patterns remains challenging. Here the authors demonstrate an approach for the detailed characterization of glycosylated biopharmaceuticals applied to the determination of the glycoproteoform profile of glycoengineered variants of erythropoietin.

The authors demonstrate a 3-tier mass spectrometry approach, including bottom-up and top-down proteomics, as well as native mass spectrometry to provide a detailed description of proteoforms, protein processing and post-translational modifications present within ribosomes from bacteria, plant, and human.

Artificial compartments have been expressed in prokaryotes and yeast, but similar capabilities have been missing for mammalian cell engineering. Here the authors use bacterial encapsulins to engineer genetically controlled multifunctional orthogonal compartments in mammalian cells.

Human milk is a vital biofluid containing a myriad of molecular components to ensure an infant’s best start at a healthy life. One key component of human milk is β-casein, a protein which is not only a structural constituent of casein micelles but also a source of bioactive, often antimicrobial, peptides contributing to milk’s endogenous peptidome. Importantly, post-translational modifications (PTMs) like phosphorylation and glycosylation typically affect the function of proteins and peptides; however, here our understanding of β-casein is critically limited. To uncover the scope of proteoforms and endogenous peptidoforms we utilized mass spectrometry (LC-MS/MS) to achieve in-depth longitudinal profiling of β-casein from human milk, studying two donors across 16 weeks of lactation. We not only observed changes in β-casein’s known protein and endogenous peptide phosphorylation, but also in previously unexplored O-glycosylation. This newly discovered PTM of β-casein may be important as it resides on known β-casein-derived antimicrobial peptide sequences.

Hyperphosphorylation of the calcium release channel/ryanodine receptor type 2 (RyR2) at serine 2814 (S2814) is associated with multiple cardiac diseases including atrial fibrillation and heart failure. Despite recent advances, the molecular mechanisms driving pathological changes associated with RyR2 S2814 phosphorylation are still not well understood. Methods: Using affinity-purification coupled to mass spectrometry (AP-MS), we investigated the RyR2 interactome in ventricles from wild-type (WT) mice and two S2814 knock-in mutants: the unphosphorylated alanine mutant (S2814A) and hyperphosphorylated mimic aspartic acid mutant (S2814D). Western blots were used for validation. Results: In WT mouse ventricular lysates, we identified 22 proteins which were enriched with RyR2 pull-down relative to both IgG control and no antibody (beads-only) pull-downs. Parallel AP-MS using WT, S2814A, and S2814D mouse ventricles identified 72 proteins, with 20 being high confidence RyR2 interactors. Of these, 14 had an increase in their binding to RyR2 S2814A but a decrease in their binding to RyR2 S2814D. We independently validated three protein hits, Idh3b, Aifm1, and Cpt1b, as RyR2 interactors by western blots and showed that Aifm1 and Idh3b had significantly decreased binding to RyR2 S2814D compared to WT and S2814A, consistent with MS findings. Conclusion: By applying state-of-the-art proteomic approaches, we discovered a number of novel RyR2 interactors in the mouse heart. In addition, we found and defined specific alterations in the RyR2 interactome that were dependent on the phosphorylation status of RyR2 at S2814. These findings yield mechanistic insights into RyR2 regulation which may guide future drug designs.

Cysteine palmitoylation affects the localization and function of membrane proteins, but its stoichiometry and specificity are not well understood. Here, the authors show that palmitoylation is a stochastic process that depends on the accessibility of cysteines rather than a defined substrate motif.

Sortilin is an endocytosis receptor with a luminal β-propeller domain. Here the authors present the structures of the β-propeller domain at neutral and acidic pH, which reveal that sortilin dimerises and undergoes conformational changes at low pH and further propose a model for low pH-induced ligand release by endocytosis receptors.

Chemical cross-linking combined with mass spectrometry (XL-MS) can provide information on protein conformations and interactions in highly complex samples. Here the authors describe an improved XL-MS workflow to increase the depth and fidelity of cross-link identification using whole proteome databases.

Self-assembling proteins that form capsid-like structures act as molecular containers for diverse cargoes. Here, the authors solve the cryo-EM structures of lumazine synthase shells, and show that supercharged mutants form expanded assemblies, indicating that electrostatics can be exploited to engineer cage architecture.

Myelin-associated glycoprotein (MAG) maintains myelin-axon spacing. Here, the authors report the crystal structures of the MAG full ectodomain in complex with oligosaccharide, and use additional assays to provide insights into the mechanism of MAG-mediated signalling.

O-GlcNAcylation is an abundant post-translational modification in the nervous system, linked to both neurodevelopmental and neurodegenerative disease. However, the mechanistic links between these phenotypes and site-specific O-GlcNAcylation remain largely unexplored. Here, we show that Ser517 O-GlcNAcylation of the microtubule-binding protein Collapsin Response Mediator Protein-2 (CRMP2) increases with age. By generating and characterizing a Crmp2S517A knock-in mouse model, we demonstrate that loss of O-GlcNAcylation leads to a small decrease in body weight and mild memory impairment, suggesting that Ser517 O-GlcNAcylation has a small but detectable impact on mouse physiology and cognitive function.

Background: Since the outbreak of coronavirus disease 2019 (COVID-19), many put their hopes in the rapid availability of effective immunizations. Human milk, containing antibodies against syndrome coronavirus 2 (SARS-CoV-2), may serve as means of protection through passive immunization. We aimed to determine the presence and pseudovirus neutralization capacity of SARS-CoV-2 specific IgA in human milk of mothers who recovered from COVID-19, and the effect of pasteurization on these antibodies. Methods: This prospective case control study included lactating mothers, recovered from (suspected) COVID-19 and healthy controls. Human milk and serum samples were collected. To assess the presence of SARS-CoV-2 antibodies we used multiple complementary assays, namely ELISA with the SARS-CoV-2 spike protein (specific for IgA and IgG), receptor binding domain (RBD) and nucleocapsid (N) protein for IgG in serum, and bridging ELISA with the SARS-CoV-2 RBD and N protein for specific Ig (IgG, IgM and IgA in human milk and serum). To assess the effect of pasteurization, human milk was exposed to Holder (HoP) and High Pressure Pasteurization (HPP). Results: Human milk contained abundant SARS-CoV-2 antibodies in 83% of the proven cases and in 67% of the suspected cases. Unpasteurized milk with and without these antibodies was found to be capable of neutralizing a pseudovirus of SARS-CoV-2 in (97% and 85% of the samples respectively). After pasteurization, total IgA antibody levels were affected by HoP, while SARS-CoV-2 specific antibody levels were affected by HPP. Pseudovirus neutralizing capacity of the human milk samples was only retained with the HPP approach. No correlation was observed between milk antibody levels and neutralization capacity. Conclusions: Human milk from recovered COVID-19-infected mothers contains SARS-CoV-2 specific antibodies which maintained neutralization capacity after HPP. All together this may represent a safe and effective immunization strategy after HPP.

Many biopharmaceuticals exhibit mixed heterogeneity in their post-translational modifications (PTMs) that are essential for their function. Here the authors use a combination of mass spectrometry techniques to analyse human erythropoietin (EPO) and properdin to discover new PTMs on properdin and derive a biosimilarity score for various sources of EPO.

Regulatory T cells (Tregs) control key events of immune tolerance, primarily by suppression of effector T cells. We previously revealed that Tregs rapidly suppress T cell receptor (TCR)-induced calcium store depletion in conventional CD4+CD25− T cells (Tcons) independently of IP3 levels, consequently inhibiting NFAT signaling and effector cytokine expression. Here, we study Treg suppression mechanisms through unbiased phosphoproteomics of primary human Tcons upon TCR stimulation and Treg-mediated suppression, respectively. Tregs induced a state of overall decreased phosphorylation as opposed to TCR stimulation. We discovered novel phosphosites (T595_S597) in the DEF6 (SLAT) protein that were phosphorylated upon TCR stimulation and conversely dephosphorylated upon coculture with Tregs. Mutation of these DEF6 phosphosites abrogated interaction of DEF6 with the IP3 receptor and affected NFAT activation and cytokine transcription in primary Tcons. This novel mechanism and phosphoproteomics data resource may aid in modifying sensitivity of Tcons to Treg-mediated suppression in autoimmune disease or cancer.

3-Hydroxybenzoate 6-hydroxylase (3HB6H, EC 1.13.14.26) is a FAD-dependent monooxygenase involved in the catabolism of aromatic compounds in soil microorganisms. 3HB6H is unique among flavoprotein hydroxylases in that it harbors a phospholipid ligand. The purified protein obtained from expressing the gene encoding 3HB6H from Rhodococcus jostii RHA1 in the host Escherichia coli contains a mixture of phosphatidylglycerol and phosphatidylethanolamine, which are the major constituents of E. coli’s cytoplasmic membrane. Here, we purified 3HB6H (RjHB6H) produced in the host R. jostii RHA#2 by employing a newly developed actinomycete expression system. Biochemical and biophysical analysis revealed that Rj3HB6H possesses similar catalytic and structural features as 3HB6H, but now contains phosphatidylinositol, which is a specific constituent of actinomycete membranes. Native mass spectrometry suggests that the lipid cofactor stabilizes monomer-monomer contact. Lipid analysis of 3HB6H from Pseudomonas alcaligenes NCIMB 9867 (Pa3HB6H) produced in E. coli supports the conclusion that 3HB6H enzymes have an intrinsic ability to bind phospholipids with different specificity, reflecting the membrane composition of their bacterial host.

Proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme that catalyzes the oxidation of proline to Δ1-pyrroline-5-carboxylate. Thermus thermophilus ProDH (TtProDH) contains in addition to its flavin-binding domain an N-terminal arm, consisting of helices αA, αB, and αC. Here, we report the biochemical properties of the helical arm truncated TtProDH variants ΔA, ΔAB, and ΔABC, produced with maltose-binding protein as solubility tag. All three truncated variants show similar spectral properties as TtProDH, indicative of a conserved flavin-binding pocket. ΔA and ΔAB are highly active tetramers that rapidly react with the suicide inhibitor N-propargylglycine. Removal of the entire N-terminal arm (ΔABC) results in barely active dimers that are incapable of forming a flavin adduct with N-propargylglycine. Characterization of V32D, Y35F, and V36D variants of ΔAB established that a hydrophobic patch between helix αC and helix α8 is critical for TtProDH catalysis and tetramer stabilization.