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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.

Posttranslational spliced peptides (PTSPs) are a unique class of peptides that have been found to be presented by HLA class-I molecules in cancer. Thus far, no consensus has been reached on the proportion of PTSPs in the immunopeptidome, with estimates ranging from 2% to as high as 45% and stirring significant debate. Furthermore, the role of the HLA class-II pathway in PTSP presentation has been studied only in diabetes. Here, we exploit our large-scale cancer peptidomics database and our newly devised pipeline for filtering spliced peptide predictions to identify recurring spliced peptides, both for HLA class-I and class-II complexes. Our results indicate that HLA class-I–spliced peptides account for a low percentage of the immunopeptidome (less than 3.1%) yet are larger in number relative to other types of identified aberrant peptides. Therefore, spliced peptides significantly contribute to the repertoire of presented peptides in cancer cells. In addition, we identified HLA class-II–bound spliced peptides, but to a lower extent (less than 0.5%). The identified spliced peptides include cancer- and immune-associated genes, such as the MITF oncogene, DAPK1 tumor suppressor, and HLA-E, which were validated using synthetic peptides. The potential immunogenicity of the DAPK1- and HLA-E–derived PTSPs was also confirmed. In addition, a reanalysis of our published mouse single-cell clone immunopeptidome dataset showed that most of the spliced peptides were found repeatedly in a large number of the single-cell clones. Establishing a novel search-scheme for the discovery and evaluation of recurring PTSPs among cancer patients may assist in identifying potential novel targets for immunotherapy.

Inflammation in ulcerative colitis is typically restricted to the mucosal layer of distal gut. Disrupted mucus barrier coupled with microbial dysbiosis has been reported to occur prior to the onset of inflammation. Here, we show the involvement of vesicular trafficking protein Rab7 in regulating the colonic mucus system. We identified a lowered Rab7 expression in goblet cells of colon during human and murine colitis. In vivo Rab7 knocked down mice (Rab7KD) displayed a compromised mucus layer, increased microbial permeability and depleted gut microbiota with enhanced susceptibility to dextran sodium-sulfate induced colitis. These abnormalities emerged owing to altered mucus composition, as revealed by mucus proteomics, with increased expression of mucin protease Chloride channel accessory 1 (CLCA1). Mechanistically, Rab7 maintained optimal CLCA1 levels by controlling its lysosomal degradation, a process that was dysregulated during colitis. Overall, our work establishes a role for Rab7 dependent control of CLCA1 secretion required for maintaining mucosal homeostasis.

SummaryProtein degradation by proteasomes is important for the immune response against tumors. Antigens generated by the proteasome promote immune cell infiltration into tumors and improve tumors’ responses to immunotherapy. For example, immunoproteasomes – a subset of proteasomes induced by inflammatory signals – may improve the response of melanomas to immune checkpoint inhibitors (ICI) by eliciting tumor inflammation. Yet, it is unclear whether and how protein degradation by proteasomes impacts cancer progression and contributes to immune evasion and resistance. Here, we profile the proteasome-cleaved peptides in lung cancers and find that PSME4 serves as a novel inhibitory regulator of the immunoproteasome, playing an anti-inflammatory role in cancer. Biochemical assays combined with scRNA-seq, immunopeptidomics and in vivo analyses demonstrate that PSME4 promotes an immunosuppressive environment around the tumor and abrogates anti-tumor immunity by inhibiting antigen presentation and attenuating tumor inflammation. Furthermore, we find that PSME4 expression is correlated with responsiveness to ICI across several cancer types. Our findings suggest that PSME4-mediated regulation of proteasome activity is a novel mechanism of immune evasion in non-small-cell lung carcinoma and may be targeted therapeutically for restoring anti-tumor immunity.Graphical AbstractHighlightsMapping the degradation landscape in Non-Small Cell Lung Cancer (NSCLC) uncovers altered proteasome activity and compositionProteasome regulator PSME4 plays an anti-inflammatory role in NSCLC by attenuating immunoproteasome activityPSME4 restricts tumor antigen presentation and cytokine secretion, defining a ‘cold’ tumor environmentPSME4 drives tumor immune evasion and is associated with resistance to immunotherapy

Antigen processing and presentation are critical for modulating tumor-host interactions. While post-translational modifications (PTMs) can alter the binding and recognition of antigens, their identification remains challenging. Here we uncover the role PTMs may play in antigen presentation and recognition in human cancers by profiling 29 different PTM combinations in immunopeptidomics data from multiple clinical samples and cell lines. We established and validated an antigen discovery pipeline and showed that newly identified modified antigens from a murine cancer model are cancer-specific and can elicit T cell killing. Systematic analysis of PTMs across multiple cohorts defined new haplotype preferences and binding motifs in association with specific PTM types. By expanding the antigenic landscape with modifications, we uncover disease-specific targets, including thousands of novel cancer-specific antigens and reveal insight into PTM-driven antigenicity. Collectively, our findings highlight an immunomodulatory role for modified peptides presented on HLA I, which may have broad implications for T-cell mediated therapies in cancer and beyond.SignificanceMajor efforts are underway to identify cancer-specific antigens for personalized immunotherapy. Here, we enrich the immunopeptidome landscape by uncovering thousands of novel putative antigens that are post-translationally modified. We define unique preferences for PTM-driven alterations affecting HLA binding and TCR recognition, which in turn alter tumor-immune interactions.Conflict of interest statementAuthors declare no conflicts of interest.

Histones constitute the primary protein building blocks of the chromatin and play key roles in the dynamic control of chromatin compaction and epigenetic regulation. Histones are regulated by intricate mechanisms that alter their functionality and stability, thereby expanding the regulation of chromatin-transacting processes. As such, histone degradation is tightly regulated to provide spatiotemporal control of cellular histone abundance. While several mechanisms have been implicated in controlling histone stability, here, we discuss proteasome-dependent degradation of histones and the protein modifications that are associated with it. We then highlight specific cellular and physiological states that are associated with altered histone degradation by cellular proteasomes.

The fidelity of the early embryonic program is underlined by tight regulation of the chromatin. Yet, how the chromatin is organized to prohibit the reversal of the developmental program remains unclear. Specifically, the totipotency-to-pluripotency transition marks one of the most dramatic events to the chromatin, and yet, the nature of histone alterations underlying this process is incompletely characterized. Here, we show that linker histone H1 is post-translationally modulated by SUMO2/3, which facilitates its fixation onto ultra-condensed heterochromatin in embryonic stem cells (ESCs). Upon SUMOylation depletion, the chromatin becomes de-compacted and H1 is evicted, leading to totipotency reactivation. Furthermore, we show that H1 and SUMO2/3 jointly mediate the repression of totipotent elements. Lastly, we demonstrate that preventing SUMOylation on H1 abrogates its ability to repress the totipotency program in ESCs. Collectively, our findings unravel a critical role for SUMOylation of H1 in facilitating chromatin repression and desolation of the totipotent identity.

The Golgi is a dynamic organelle whose correct assembly is crucial for cellular homeostasis. Perturbations in Golgi structure are associated with numerous disorders from neurodegeneration to cancer. However, whether and how dispersal of the Golgi apparatus is actively regulated under stress, and the consequences of Golgi dispersal, remain unknown. Here we demonstrate that 26S proteasomes are associated with the cytosolic surface of Golgi membranes to facilitate Golgi Apparatus-Related Degradation (GARD) and degradation of GM130 in response to Golgi stress. The degradation of GM130 is dependent on p97/VCP and 26S proteasomes, and required for Golgi dispersal. Finally, we show that perturbation of Golgi homeostasis induces cell death of multiple myeloma in vitro and in vivo, offering a therapeutic strategy for this malignancy. Taken together, this work reveals a mechanism of Golgi-localized proteasomal degradation, providing a functional link between proteostasis control and Golgi architecture, which may be critical in various secretion-related pathologies., Correct Golgi assembly is important to cellular homeostasis but regulation of its structure under stress remains unclear. Here, the authors identify stress-induced degradation of GM130 by Golgi-localized 26S proteasomes, leading to Golgi dispersal.

Introduction: Degradation of proteins by cellular proteasomes is critical for the fidelity of protein homeostasis and proper cell function. Indeed, perturbations in proteasome function, as well as the degradation of specific substrates, are associated with a variety of human diseases. Yet, monitoring and analyzing protein degradation in a high throughput manner in physiology and pathology remains limited. Areas covered: Here we discuss several of the recently developed mass spectrometry-based methods for studying proteasome-mediated cellular degradation and discuss their advantages and limitations. We highlight Mass Spectrometry Analysis of Proteolytic Peptides (MAPP), a method designed to purify and identify proteasome-cleaved cellular proteins as a novel approach in molecular and clinical profiling of human disease. Expert opinion: The recent improvement of proteomics technologies now offers an unprecedented ability to study disease in clinical settings. Expanding clinical studies to include the degradation landscape will provide a new resolution to complement the cellular proteome. In turn, this holds promise to provide both new disease targets and novel peptide biomarkers which will further enhance personalized proteomics.

Antigen presentation on HLA molecules is a major mechanism by which the immune system monitors self and non-self-recognition. Importantly, HLA-I presentation has gained much attention through its role in eliciting anti-tumor immunity. Several determinants controlling the peptides presented on HLA have been uncovered, mainly through the study of model substrates and large-scale immunopeptidome analyses. These determinants include the relative abundances of proteins in the cell, the stability or turnover rate of these proteins and the binding affinities of a given peptide to the HLA haplotypes found in a cell. However, the regulatory principles involved in selection and regulation of specific antigens in response to tumor pro-inflammatory signals remain largely unknown. Here, we chose to examine the effect that TNFα and IFNγ stimulation may exert on the immunopeptidome landscape of lung cancer cells. We show that the expression of many of the proteins involved in the class I antigen presentation pathway are changed by pro-inflammatory cytokines. Further, we could show that increased expression of the HLA-B allomorph drives a significant change in HLA-bound antigens, independently of the significant changes observed in the cellular proteome. Finally, we observed increased HLA-B levels in correlation with tumor infiltration across the TCGA lung cancer cohorts. Taken together, our results suggest that the immunopeptidome landscape should be examined in the context of anti-tumor immunity whereby signals in the microenvironment may be critical in shaping and modulating this important aspect of host-tumor interactions.

Introduction Proteasome inhibition has emerged as an important therapeutic strategy in multiple myeloma (MM) and its first-in-class agent, bortezomib, has become a major player in MM treatment. Bortezomib reversibly inhibits the proteasome, inducing an accumulation of ubiquitinated proteins in MM cells, which results in endoplasmic reticulum stress-related apoptosis. However, up to 30% of patients (pts) have been shown to fail bortezomib-based induction and the underlying mechanisms remain poorly understood. As protein degradation by the proteasome, is reliant on protein ubiquitination, uncovering the ubiquitination profile of bone marrow mononuclear cells (BM-MNC) of MM pts, may offer putative biomarkers for predicting the response to treatment and in the future shed new light on disease pathophysiology. In collaboration with PTM Biosciences, we investigated ubiquitination patterns in BM-MNC samples obtained from MM pts treated with bortezomib-based induction. We hypothesized that ubiquitination patterns at diagnosis would differ between responders vs non-responders and would serve as a platform for identifying potential new therapeutic targets involved in induction of primary resistance. Methods BM-MNC samples, collected prior to therapy from 31 NDMM pts, treated with Velcade (bortezomib)-Cyclophosphamide-Dexamethasone (VCD) or VD, were analyzed. Response to treatment was determined after 4-6 cycles of treatment (employing the IMWG criteria), unless progressed earlier. Functional total cells extract that preserve the cellular enzymatic activity were prepared from each sample and applied to protein microarrays containing ~9000 known proteins (ProtoArray®, Thermo Fisher). Following the exposure of the protein array surface to the sample, enzymes from the sample modified the thousands of proteins spotted on the array, to generate a patient-specific signatures of ubiquitination (Experimental Biology and Medicine 2016; 0: 1-8). The ubiquitination level of every protein was quantified, data was normalized using the quantile normalization method and sorted by descending order for each sample. The ranking position of a protein at each sample within one group was compared to its ranking positions on the other group samples. The proteins differing in their ranking level (p-value ≤ 0.005) between groups were selected. Results Thirty-one BM-MNC samples obtained from MM pts prior VCD (n=27) or VD (n=4) induction, were tested. The cohort included 16 responding patient that achieved at least PR and 15 non-responding pts that obtained SD or less (Table 1). PTM analysis initially revealed 241 proteins that were considered as differentially modified between the 2 groups, mostly in the non-responders (153 vs 88). Ubiquitination of a specific protein was determined as "highly discriminating", if shown to be discriminating in at least 50% of the rounds. We identified 44 classifier proteins; 11 that were highly ubiquitinated in the responders and 33 in the non-responders. These 44 proteins were defined as "highly discriminating", providing a unique PTM signature for responders vs non-responders (Figure 1). Among the 44 proteins, 14 (31.8%) were previously suggested to serve as biomarkers in other cancers and 6 other proteins (13.6 %) were found to be related to the ubiquitin/ubiquitin-like or proteasome machinery. At least 3 of these proteins (USP28, SQSTM1 & PSME1) are known to be related to MM and responsiveness to bortezomib treatment. Further validation of 6 of these targets, confirmed their expression in BM-MNC samples in an independent assay. Prospective clinical validation is required to examine the specificity and sensitivity of the identified biomarkers. Conclusions Here we provide a proof-of-concept for the ability of ubiquitination profiling of BM-MNC samples to identify putative biomarkers for predicting response to bortezomib-based therapy. Among the proteins identified some were associated with response to bortezomib treatment and other novel candidates require further prospective validation. Algorithm training, validation cohort and investigation of the predictive value of this approach in MM pts treated with other therapeutic regimens are underway. Our study provides the first example in clinical samples, highlighting the potential significance of this approach in the clinic and offers a novel strategy for proteomics-based personalized medicine. Disclosures Tsubery: PTM Biosciences: Employment. Gali:PTM Biosciences: Employment. Gali:PTM Biosciences: Employment. Sela:PTM Biosciences: Consultancy. Merbl:PTM Biosciences: Consultancy, Equity Ownership. Cohen:VBL Therapeutics Â: Employment; Madison Â: Consultancy; Takeda Â: Consultancy; Neopharm Â: Consultancy; Amgen Â: Consultancy, Research Funding; Janssen Â: Consultancy.

Summary Mbd3, a member of nucleosome remodeling and deacetylase (NuRD) co-repressor complex, was previously identified as an inhibitor for deterministic induced pluripotent stem cell (iPSC) reprogramming, where up to 100% of donor cells successfully complete the process. NuRD can assume multiple mutually exclusive conformations, and it remains unclear whether this deterministic phenotype can be attributed to a specific Mbd3/NuRD subcomplex. Moreover, since complete ablation of Mbd3 blocks somatic cell proliferation, we aimed to explore functionally relevant alternative ways to neutralize Mbd3-dependent NuRD activity. We identify Gatad2a, a NuRD-specific subunit, whose complete deletion specifically disrupts Mbd3/NuRD repressive activity on the pluripotency circuitry during iPSC differentiation and reprogramming without ablating somatic cell proliferation. Inhibition of Gatad2a facilitates deterministic murine iPSC reprogramming within 8 days. We validate a distinct molecular axis, Gatad2a-Chd4-Mbd3, within Mbd3/NuRD as being critical for blocking reestablishment of naive pluripotency and further highlight signaling-dependent and post-translational modifications of Mbd3/NuRD that influence its interactions and assembly.

The Nucleosome Remodeling and Deacytelase (NuRD) complex is a co-repressive complex involved in many pathological and physiological processes in the cell. Previous studies have identified one of its components, Mbd3, as a potent inhibitor for reprogramming of somatic cells to pluripotency. Following OSKM induction, early and partial depletion of Mbd3 protein followed by applying naïve ground-state pluripotency conditions, results in a highly efficient and near-deterministic generation of mouse iPS cells. Increasing evidence indicates that the NuRD complex assumes multiple mutually exclusive protein complexes, and it remains unclear whether the deterministic iPSC phenotype is the result of a specific NuRD sub complex. Since complete ablation of Mbd3 blocks somatic cell proliferation, here we aimed to identify alternative ways to block Mbd3-dependent NuRD activity by identifying additional functionally relevant components of the Mbd3/NuRD complex during early stages of reprogramming. We identified Gatad2a (also known as P66α), a relatively uncharacterized NuRD-specific subunit, whose complete deletion does not impact somatic cell proliferation, yet specifically disrupts Mbd3/NuRD repressive activity on the pluripotency circuit during both stem cell differentiation and reprogramming to pluripotency. Complete ablation of Gatad2a in somatic cells, but not Gatad2b, results in a deterministic naïve iPSC reprogramming where up to 100% of donor somatic cells successfully complete the process within 8 days. Genetic and biochemical analysis established a distinct sub-complex within the NuRD complex (Gatad2a-Chd4-Mbd3) as the functional and biochemical axis blocking reestablishment of murine naïve pluripotency. Disassembly of this axis by depletion of Gatad2a, results in resistance to conditions promoting exit of naïve pluripotency and delays differentiation. We further highlight context- and posttranslational dependent modifications of the NuRD complex affecting its interactions and assembly in different cell states. Collectively, our work unveils the distinct functionality, composition and interactions of Gatad2a-Chd4-Mbd3/NuRD subcomplex during the resolution and establishment of mouse naïve pluripotency.

Posttranslational modifications (PTMs) of proteins are an integral part of major cellular regulatory mechanisms dictating protein function, localization, and stability. The capacity to screen PTMs using protein microarrays has advanced our ability to identify their targets and regulatory role. This chapter discusses a unique procedure that combines functional extract-based activity assay with large-scale screening utilities of protein microarrays. This "PTM-profiling" system offers advantages in quantitatively identifying modifications in an unbiased manner in the context of specific cellular conditions. While the possibilities of studying PTMs in different settings are enormous, the immune system presents an attractive model for studying the effects of perturbations in PTMs, and specifically the ubiquitin system, as these were already implicated in both immune function and dysfunction. This chapter discusses the significance of PTM profiling in addressing basic questions in immunology. We describe detailed protocols for the preparation of functional cell extracts from immune cell cultures, following differentiation or induced signals, and screening PTMs on protein arrays, as well as basic guidelines for data analysis and interpretation.

O-GlcNAc transferase (OGT) is a serine/threonine glycosyltransferase that is essential for development and continues to be critically important throughout life. Understanding OGT’s complex biology requires identifying its substrates. Here we demonstrate the utility of a microarray approach for discovering novel OGT substrates. We also report a rapid method to validate OGT substrates that combines in vitro transcription-translation with O-GlcNAc mass tagging. Among the validated new OGT targets is Orthodenticle homeobox 2 (OTX2), a transcription factor critical for brain development, which is primarily expressed only during early embryogenesis and in medulloblastomas, where it functions as an oncogene. We show that endogenous OTX2 from a medulloblastoma cell line is O-GlcNAcylated at several sites. Our results demonstrate that protein microarray technology, combined with the target validation strategy we report, is useful for identifying biologically important OGT substrates, including substrates not present in most tissue types or cell lines.

SummaryUbiquitin and ubiquitin-like (Ubl) protein modifications affect protein stability, activity, and localization, but we still lack broad understanding of the functions of Ubl modifications. We have profiled the protein targets of ubiquitin and six additional Ubls in mitosis using a functional assay that utilizes active mammalian cell extracts and protein microarrays and identified 1,500 potential substrates; 80–200 protein targets were exclusive to each Ubl. The network structure is nonrandom, with most targets mapping to a single Ubl. There are distinct molecular functions for each Ubl, suggesting divergent biological roles. Analysis of differential profiles between mitosis and G1 highlighted a previously underappreciated role for the Ubl, FAT10, in mitotic regulation. In addition to its role as a resource for Ubl modifications, our study provides a systematic approach to analyze changes in posttranslational modifications at various cellular states.

The ubiquitin system plays an important role in essentially every cellular process, regulating numerous pathways ranging from development, transcription, DNA damage response, cell cycle, and signal transduction. Its best studied role involves removal of faulty proteins or those that are not necessary anymore. Aberrations in the ubiquitin system have been implicated in various pathologies including cancer, where specific mutations in E3 ligases such as Mdm2, pVHL, and BRCA1 have been linked to disease progression, prognosis, and resistance to drugs. Yet, there are hundreds of E3 ligases in the human genome and our knowledge of their target proteins and their dynamic regulation in the cellular environment is largely limited. In addition, fundamental questions related to recognition and specificity in ubiquitin conjugation remain unanswered. It is thus of major importance to characterize the ubiquitin landscape under various cellular conditions, and study how the regulatory network is altered in health and disease. To do so, analytical tools that allow identification of ubiquitin substrates, the conjugation and removal of ubiquitin, and the nature of specific ubiquitin linkages that are formed are needed. In this mini-review, we discuss common proteomic methodologies applied to studying the ubiquitome, and specifically focus on our recently developed post-translational modification (PTM) profiling approach. PTM profiling is a functional assay, amenable to biochemical manipulation, which allows the detection of protein modifications in a high-throughput manner. We discuss in detail the advantages and limitations of this system, focusing primarily on examples for analyzing the ubiquitin system in cancer. Uncovering the intricate signaling dynamics governed by and regulating ubiquitin modifications should clearly evolve into a new paradigm in understanding the molecular basis of malignant transformation and the development of novel therapeutic modalities.

To investigate the role of genes encoding regulators of G protein signaling in early therapeutic response to antipsychotic drugs and in susceptibility to drug-induced extrapyramidal symptoms. As regulators of G protein signaling and regulators of G protein signaling-like proteins play a pivotal role in dopamine receptor signaling, genetically based, functional variation could contribute to interindividual variability in therapeutic and adverse effects.Consecutively hospitalized, psychotic patients with Diagnostic and Statistical Manual of Mental Disorder-IV schizophrenia (n=121) were included in the study if they received treatment with typical antipsychotic medication (n=72) or typical antipsychotic drugs and risperidone (n=49) for at least 2 weeks. Clinical state and adverse effects were rated at baseline and after 2 weeks. Twenty-four single nucleotide polymorphisms were genotyped in five regulators of G protein signaling genes.None of the single nucleotide polymorphisms were related to clinical response to antipsychotic treatment at 2 weeks. Five out of six single nucleotide polymorphisms within or flanking the RGS2 gene were nominally associated with development or worsening of parkinsonian symptoms (PARK+) as measured by the Simpson Angus Scale, one of them after correction for multiple testing (rs4606, P=0.002). A GCCTG haplotype encompassing tagging single nucleotide polymorphisms within and flanking RGS2 was significantly overrepresented among PARK+ compared with PARK--patients (0.23 vs. 0.08, P=0.003). A second, 'protective', GTGCA haplotype was significantly overrepresented in PARK--patients (0.13 vs. 0.30, P=0.009). Both haplotype associations survive correction for multiple testing.Subject to replication, these findings suggest that genetic variation in the RGS2 gene is associated with susceptibility to extrapyramidal symptoms induced by antipsychotic drugs.

Autoimmune diseases are often marked by autoantibodies binding to self antigens. However, many healthy persons also manifest autoantibodies that bind to self antigens, known as natural autoantibodies. In order to characterize natural autoantibodies present at birth, we used an antigen microarray (antigen chip) to analyze informatically (with clustering algorithms and correlation mapping) the natural IgM, IgA, and IgG autoantibody repertoires present in 10 pairs of sera from healthy mothers and the cords of their newborn babies. These autoantibodies were found to bind to 305 different, mostly self, molecules. We report that in utero, humans develop IgM and IgA autoantibodies to relatively uniform sets of self molecules. The global patterns of maternal IgM autoantibodies significantly diverged from those at birth, although certain reactivities remained common to both maternal and cord samples. Because maternal IgG antibodies (unlike IgM and IgA) cross the placenta, maternal and cord IgG autoantibodies showed essentially identical reactivities. We found that some self antigens that bind cord autoantibodies were among the target self antigens associated with autoimmune diseases later in life. Thus, the obviously benign autoimmunity prevalent at birth may provide the basis for the emergence of some autoimmune diseases relatively prevalent later in life.

One's present repertoire of antibodies encodes the history of one's past immunological experience. Can the present autoantibody repertoire be consulted to predict resistance or susceptibility to the future development of an autoimmune disease? Here, we developed an antigen microarray chip and used bioinformatic analysis to study a model of type 1 diabetes developing in nonobese diabetic male mice in which the disease was accelerated and synchronized by exposing the mice to cyclophosphamide at 4 weeks of age. We obtained sera from 19 individual mice, treated the mice to induce cyclophosphamide-accelerated diabetes (CAD), and found, as expected, that 9 mice became severely diabetic, whereas 10 mice permanently resisted diabetes. We again obtained serum from each mouse after CAD induction. We then analyzed, by using rank-order and superparamagnetic clustering, the patterns of antibodies in individual mice to 266 different antigens spotted on the chip. A selected panel of 27 different antigens (10% of the array) revealed a pattern of IgG antibody reactivity in the pre-CAD sera that discriminated between the mice resistant or susceptible to CAD with 100% sensitivity and 82% specificity (P= 0.017). Surprisingly, the set of IgG antibodies that was informative before CAD induction did not separate the resistant and susceptible groups after the onset of CAD; new antigens became critical for post-CAD repertoire discrimination. Thus, at least for a model disease, present antibody repertoires can predict future disease, predictive and diagnostic repertoires can differ, and decisive information about immune system behavior can be mined by bioinformatic technology. Repertoires matter.

Protein microarrays are extremely useful for detecting substrates of protein kinases, substrates of ubiquitin ligases and other post translational modifications. The ability to screen binding interactions as well as post-translational modifications of thousands of proteins at once has improved our ability to identify their targets. Utilizing such systems in combination with functional mammalian cell extracts that preserve enzymatic activity offers advantages in identifying semi-quantitative changes of these interactions in the context of specific cellular conditions. This article provides a detailed procedure for setting up an extract-based activity assay for high content detection of protein post-translation modifications. It also provides basic guidelines for data analysis.

Traditionally, immunologic diagnosis has been based on an attempt to correlate each disease with a specific immune reactivity, such as an antibody or a T-cell response to a single antigen specific for the disease entity. The state of the body, however, appears to be encoded by the immune system in collectives of reactivities and not by single reactivities. Here we describe our use of microarray technology and informatics to develop an antigen chip capable of detecting global patterns of antibodies binding to hundreds of antigens simultaneously. The patterns fashion diagnostic signatures.

Networks can be found everywhere—in technology, in nature and in our bodies. In this paper we present how antigen networks can be used as a model to study network interaction and architecture. Utilizing antigen microarray data of the reactivity of hundreds of antibodies of sera of ten mothers and their newborns, we reconstruct networks, either isotype specific (IgM or IgG) or person specific—mothers or newborns—and investigate the network properties. Such an approach makes it possible to decipher fundamental information regarding the personal immune network state and its unique characteristics. In the current paper we demonstrate how we are successful in studying the interaction between two dependent networks, the maternal IgG repertoire and the one of the offspring, using the concept of meta-network provides essential information regarding the biological phenomenon of cross placental transfer. Such an approach is useful in the study of coupled networks in variety of scientific fields. S Online supplementary data available from stacks.iop.org/PhysBio/10/025003/mmedia

Protein microarray technology has emerged as a powerful tool for comparing binding interactions, expression level, substrate specificities, and posttranslational modifications (PTMs) of different proteins in a parallel and high-throughput manner. The ability to immobilize proteins to a solid surface and register the specific address of each protein has bridged major limitations for investigating the proteome in biological samples, namely, the wide dynamic range of protein concentrations and the perturbation of the physical and chemical properties of proteins by their modification. Recent advances introduced the use of functional mammalian cell extracts to assay PTMs under different cellular conditions. This assay offers a new approach for performing large-scale complex biochemical analysis of protein modifications. Here, we review studies of PTM profiling using protein microarrays and discuss the limitations and potential applications of the system. We believe that the information generated from such proteomic studies may be of significant value in our elucidation of the molecular mechanisms that govern human physiology.

Protein microarray technology has emerged as a powerful tool for comparing binding interactions, expression level, substrate specificities, and posttranslational modifications (PTMs) of different proteins in a parallel and high-throughput manner. The ability to immobilize proteins to a solid surface and register the specific address of each protein has bridged major limitations for investigating the proteome in biological samples, namely, the wide dynamic range of protein concentrations and the perturbation of the physical and chemical properties of proteins by their modification. Recent advances introduced the use of functional mammalian cell extracts to assay PTMs under different cellular conditions. This assay offers a new approach for performing large-scale complex biochemical analysis of protein modifications. Here, we review studies of PTM profiling using protein microarrays and discuss the limitations and potential applications of the system. We believe that the information generated from such proteomic studies may be of significant value in our elucidation of the molecular mechanisms that govern human physiology.

MotivationNew antigen microarray technology enables parallel recording of antibody reactivities with hundreds of antigens. Such data affords system level analysis of the immune system's organization using methods and approaches from network theory. Here we measured the reactivity of 290 antigens (for both the IgG and IgM isotypes) of 10 healthy mothers and their term newborns. We constructed antigen correlation networks (or immune networks) whose nodes are the antigens and the edges are the antigen-antigen reactivity correlations, and we also computed their corresponding minimum spanning trees (MST)--maximal information reduced sub-graphs. We quantify the network organization (topology) in terms of the network theory divergence rate measure and rank the antigen importance in the full antigen correlation networks by the eigen-value centrality measure. This analysis makes possible the characterization and comparison of the IgG and IgM immune networks at birth (newborns) and adulthood (mothers) in terms of topology and node importance.ResultsComparison of the immune network topology at birth and adulthood revealed partial conservation of the IgG immune network topology, and significant reorganization of the IgM immune networks. Inspection of the antigen importance revealed some dominant (in terms of high centrality) antigens in the IgG and IgM networks at birth, which retain their importance at adulthood.

The Anaphase-Promoting Complex (APC) is an E3 ubiquitin ligase that regulates mitosis and G1 by sequentially targeting cell-cycle regulators for ubiquitination and proteasomal degradation. The mechanism of ubiquitin chain formation by APC and the resultant chain topology remains controversial. By using a single-lysine APC substrate to dissect the topology of ubiquitinated substrates, we find that APC-catalyzed ubiquitination has an intrinsic preference for the K11 linkage of ubiquitin that is essential for substrate degradation. K11 specificity is determined by an E2 enzyme, UBE2S/E2-EPF, that elongates ubiquitin chains after the substrates are pre-ubiquitinated by UbcH10 or UbcH5. UBE2S copurifies with APC; dominant-negative Ube2S slows down APC substrate degradation in functional cell-cycle extracts. We propose that Ube2S is a critical, unique component of the APC ubiquitination pathway.

The immune system is essential to body defense and maintenance. Specific antibodies to foreign invaders function in body defense, and it has been suggested that autoantibodies binding to self molecules are important in body maintenance. Recently, the autoantibody repertoires in the bloods of healthy mothers and their newborns were studied using an antigen microarray containing hundreds of self molecules. It was found that the mothers expressed diverse repertoires for both IgG and IgM autoantibodies. Each newborn shares with its mother a similar repertoire of IgG antibodies, which cross the placental but its IgM repertoire is more similar to those of other newborns. Here, we took a system-level approach and analyzed the correlations between autoantibody reactivities of the previous data and extended the study to new data from newborns at birth and a week later, and from healthy young women. For the young women, we found modular organization of both IgG and IgM isotypes into antigen cliques—subgroups of highly correlated antigen reactivities. In contrast, the newborns were found to share a universal congenital IgM profile with no modular organization. Moreover, the IgG autoantibodies of the newborns manifested buds of the mothers' antigen cliques, but they were noticeably less structured. These findings suggest that the natural autoantibody repertoire of humans shows relatively little organization at birth, but, by young adulthood, it becomes sorted out into a modular organization of subgroups (cliques) of correlated antigens. These features revealed by antigen microarrays can be used to define personal states of autoantibody organizational motifs.

Identification of protein targets of post-translational modification is an important analytical problem in biology. Protein microarrays exposed to cellular extracts could offer a rapid and convenient means of identifying modified proteins, but this kind of biochemical assay, unlike DNA microarrays, depends on a faithful reconstruction of in vivo conditions. Over several years, concentrated cellular extracts have been developed, principally for cell cycle studies that reproduce very complex cellular states. We have used extracts that replicate the mitotic checkpoint and anaphase release to identify differentially regulated poyubiquitination. Protein microarrays were exposed to these complex extracts, and the polyubiquitinated products were detected by specific antibodies. We expected that among the substrates revealed by the microarray should be substrates of the anaphase promoting complex (APC). Among 8,000 proteins on the chip, 10% were polyubiquitinated. Among those, we found 11 known APC substrates (out of 16 present on the chip) to be polyubiquitinated. Interestingly, only 1.5% of the proteins were differentially ubiquitinated on exit from the checkpoint. When we arbitrarily chose 6 proteins thought to be involved in mitosis from the group of differentially modified proteins, all registered as putative substrates of the APC, and among 4 arbitrarily chosen non-mitotic proteins picked from the same list, 2 were ubiquitinated in an APC-dependent manner. The striking yield of potential APC substrates from a simple assay with concentrated cell extracts suggests that combining microarray analysis of the products of post-translational modifications with extracts that preserve the physiological state of the cell can yield information on protein modification under various in vivo conditions.

Multiple sclerosis (MS) is a chronic relapsing disease of the central nervous system (CNS) in which immune processes are believed to play a major role. To date, there is no reliable method by which to characterize the immune processes and their changes associated with different forms of MS and disease progression. We performed antigen microarray analysis to characterize patterns of antibody reactivity in MS serum against a panel of CNS protein and lipid autoantigens and heat shock proteins. Informatic analysis consisted of a training set that was validated on a blinded test set. The results were further validated on an independent cohort of relapsing–remitting (RRMS) samples. We found unique autoantibody patterns that distinguished RRMS, secondary progressive (SPMS), and primary progressive (PPMS) MS from both healthy controls and other neurologic or autoimmune driven diseases including Alzheimer's disease, adrenoleukodystropy, and lupus erythematosus. RRMS was characterized by autoantibodies to heat shock proteins that were not observed in PPMS or SPMS. In addition, RRMS, SPMS, and PPMS were characterized by unique patterns of reactivity to CNS antigens. Furthermore, we examined sera from patients with different immunopathologic patterns of MS as determined by brain biopsy, and we identified unique antibody patterns to lipids and CNS-derived peptides that were linked to each type of pathology. The demonstration of unique serum immune signatures linked to different stages and pathologic processes in MS provides an avenue to monitor MS and to characterize immunopathogenic mechanisms and therapeutic targets in the disease.

Much effort has been devoted to assess the importance of nodes in complex biological networks (such as gene transcriptional regulatory networks, protein interaction networks, and neural networks). Examples of commonly used measures of node importance include node degree, node centrality, and node vulnerability score (the effect of the node deletion on the network efficiency). Here, we present a new approach to compute and investigate the mutual dependencies between network nodes from the matrices of node-node correlations. To this end, we first define the dependency of node i on node j (or the influence of node j on node i), D(i, j) as the average over all nodes k of the difference between the i - k correlation and the partial correlations between these nodes with respect to node j. Note that the dependencies, D(i, j) define a directed weighted matrix, since, in general, D(i, j) differs from D( j, i). For this reason, many of the commonly used measures of node importance, such as node centrality, cannot be used. Hence, to assess the node importance of the dependency networks, we define the system level influence (SLI) of antigen j, SLI( j) as the sum of the influence of j on all other antigens i. Next, we define the system level influence or the influence score of antigen j, SLI( j) as the sum of D(i, j) over all nodes i. We introduce the new approach and demonstrate that it can unveil important biological information in the context of the immune system. More specifically, we investigated antigen dependency networks computed from antigen microarray data of autoantibody reactivity of IgM and IgG isotypes present in the sera of ten mothers and their newborns. We found that the analysis was able to unveil that there is only a subset of antigens that have high influence scores (SLI) common both to the mothers and newborns. Networks comparison in terms of modularity (using the Newman's algorithm) and of topology (measured by the divergence rate) revealed that, at birth, the IgG networks exhibit a more profound global reorganization while the IgM networks exhibit a more profound local reorganization. During immune system development, the modularity of the IgG network increases and becomes comparable to that of the IgM networks at adulthood. We also found the existence of several conserved IgG and IgM network motifs between the maternal and newborns networks, which might retain network information as our immune system develops. If correct, these findings provide a convincing demonstration of the effectiveness of the new approach to unveil most significant biological information. Whereas we have introduced the new approach within the context of the immune system, it is expected to be effective in the studies of other complex biological social, financial, and manmade networks.

Genetic variation in antipsychotic drug targets could underlie variability among patients in the time required for antipsychotic effects to be elicited. In a clinical, pharmacogenetic study we focused on the dopamine receptor interacting protein (DRIP) gene family. DRIPs are pivotally involved in regulating dopamine receptor signal transduction. Consecutively hospitalized, acutely psychotic patients with DSM-IV schizophrenia ( n =121) were included in the study if they received treatment with typical antipsychotic medication (TYP, n =72) or TYP plus risperidone (TYP-R, n =49) for at least 2 wk. Clinical state and adverse effects were rated at baseline and after 2 wk. Patients improved significantly on both TYP and TYP-R with no significant difference between them. Early responders were defined as patients whose PANSS change scores were greater than the median. Twenty-two single nucleotide polymorphisms (SNPs) were analysed in five DRIP-encoding genes. Two SNPs in NEF3 , which encodes the DRIP, neurofilament-medium (NF-M), were associated with early response (rs1457266, p =0.01; rs1379357, p =0.006). A 5 SNP haplotype spanning NEF3 was over-represented in early responders ( p =0.015), in the combined patient group and in the TYP group alone. These findings suggest that variation in NEF3 , most likely functional variants that are in linkage disequilibrium with the SNPs that we studied, influences rate of response to TYP. Since NEF3 is primarily associated with dopamine D1 receptor function, the evidence for a complementary role of dopamine D1 receptors in antipsychotic effects is considered. The findings reported here open an interesting research avenue in the pharmacogenetics of antipsychotic effects but require replication in larger samples treated in a controlled context.

Cellular function is critically regulated through degradation of substrates by the proteasome. To enable direct analysis of naturally cleaved proteasomal peptides under physiological conditions, we developed mass spectrometry analysis of proteolytic peptides (MAPP), a method for proteasomal footprinting that allows capture, isolation and analysis of proteasome-cleaved peptides. Application of MAPP to cancer cell lines as well as primary immune cells reveals dynamic modulation of the cellular degradome in response to various stimuli, such as pro-inflammatory signals. Further, we demonstrate analysis of minute amounts of clinical samples by studying cells from peripheral blood of patients with systemic lupus erythematosus (SLE). We find increased degradation of histones in patient immune cells, which suggests a role for aberrant proteasomal degradation in the pathophysiology of SLE. Taken together, MAPP offers a broadly applicable method to facilitate the study of the cellular degradation landscape in various cellular conditions and diseases involving changes in proteasomal degradation, including protein aggregation diseases, autoimmunity and cancer.

FAT10 is a ubiquitin-like protein suggested to target proteins for proteasomal degradation. It is highly upregulated upon pro-inflammatory cytokines, namely, TNFα, IFNγ, and IL6, and was found to be highly expressed in various epithelial cancers. Evidence suggests that FAT10 is involved in cancer development and may have a pro-tumorigenic role. However, its biological role is still unclear, as well as its biochemical and cellular regulation. To identify pathways underlying FAT10 expression in the context of pro-inflammatory stimulation, which characterizes the cancerous environment, we implemented a phenotypic transcriptional reporter screen with a library of annotated compounds. We identified AZ960, a potent JAK2 inhibitor, which significantly downregulates FAT10 under pro-inflammatory cytokines induction, in an NFκB-independent manner. We validated JAK2 as a major regulator of FAT10 expression via knockdown, and we suggest that the transcriptional effects are mediated through pSTAT1/3/5. Overall, we have elucidated a pathway regulating FAT10 transcription and discovered a tool compound to chemically downregulate FAT10 expression, and to further study its biology.

Posttranslational modifications (PTMs) of proteins are an integral part of major cellular regulatory mechanisms dictating protein function, localization, and stability. The capacity to screen PTMs using protein microarrays has advanced our ability to identify their targets and regulatory role. This chapter discusses a unique procedure that combines functional extract-based activity assay with large-scale screening utilities of protein microarrays. This "PTM-profiling" system offers advantages in quantitatively identifying modifications in an unbiased manner in the context of specific cellular conditions. While the possibilities of studying PTMs in different settings are enormous, the immune system presents an attractive model for studying the effects of perturbations in PTMs, and specifically the ubiquitin system, as these were already implicated in both immune function and dysfunction. This chapter discusses the significance of PTM profiling in addressing basic questions in immunology. We describe detailed protocols for the preparation of functional cell extracts from immune cell cultures, following differentiation or induced signals, and screening PTMs on protein arrays, as well as basic guidelines for data analysis and interpretation.