To identify the molecular signaling pathways underlying sudden unexpected death in epilepsy (SUDEP) and high-risk SUDEP compared to control patients with epilepsy.

Sudden unexplained death in childhood (SUDC) is death of a child over 1 year of age that is unexplained after review of clinical history, circumstances of death, and complete autopsy with ancillary testing. Multiple etiologies may cause SUDC. SUDC and sudden unexpected death in epilepsy (SUDEP) share clinical and pathological features, suggesting some similarities in mechanism of death and possible abnormalities in hippocampus and cortex. To identify molecular signaling pathways, we performed label-free quantitative mass spectrometry on microdissected frontal cortex, hippocampal dentate gyrus (DG), and cornu ammonis (CA1-3) in SUDC (n = 19) and pediatric control cases (n = 19) with an explained cause of death. At a 5% false discovery rate (FDR), we found differential expression of 660 proteins in frontal cortex, 170 in DG, and 57 in CA1-3. Pathway analysis of altered proteins identified top signaling pathways associated with activated oxidative phosphorylation (p = 6.3 × 10-15, z = 4.08) and inhibited EIF2 signaling (p = 2.0 × 10-21, z = - 2.56) in frontal cortex, and activated acute phase response in DG (p = 8.5 × 10-6, z = 2.65) and CA1-3 (p = 4.7 × 10-6, z = 2.00). Weighted gene correlation network analysis (WGCNA) of clinical history indicated that SUDC-positive post-mortem virology (n = 4/17) had the most significant module in each brain region, with the top most significant associated with decreased mRNA metabolic processes (p = 2.8 × 10-5) in frontal cortex. Additional modules were associated with clinical history, including fever within 24 h of death (top: increased mitochondrial fission in DG, p = 1.8 × 10-3) and febrile seizure history (top: decreased small molecule metabolic processes in frontal cortex, p = 8.8 × 10-5) in all brain regions, neuropathological hippocampal findings in the DG (top: decreased focal adhesion, p = 1.9 × 10-3). Overall, cortical and hippocampal protein changes were present in SUDC cases and some correlated with clinical features. Our studies support that proteomic studies of SUDC cohorts can advance our understanding of the pathogenesis of these tragedies and may inform the development of preventive strategies.

Despite minimal disparity at the sequence level, mammalian H3 variants bind to distinct sets of polypeptides. Although histone H3.1 predominates in cycling cells, our knowledge of the soluble complexes that it forms en route to deposition or following eviction from chromatin remains limited. Here, we provide a comprehensive analysis of the H3.1-binding proteome, with emphasis on its interactions with histone chaperones and components of the replication fork. Quantitative mass spectrometry revealed 170 protein interactions, whereas a large-scale biochemical fractionation of H3.1 and associated enzymatic activities uncovered over twenty stable protein complexes in dividing human cells. The sNASP and ASF1 chaperones play pivotal roles in the processing of soluble histones but do not associate with the active CDC45/MCM2-7/GINS (CMG) replicative helicase. We also find TONSL-MMS22L to function as a H3-H4 histone chaperone. It associates with the regulatory MCM5 subunit of the replicative helicase.

The vast majority of human tissue specimens are formalin-fixed, paraffin embedded (FFPE) archival samples, making this type of tissue a potential gold mine for medical research. It is now accepted that proteomics can be done using FFPE tissue and can generate similar results as snap-frozen tissue. However, the current methodology requires a large amount of starting protein, limiting the questions that can be answered in these types of proteomics studies and making cell-type specific proteomics studies difficult. Cell-type specific proteomics has the potential to greatly enhance understanding of cell functioning in both normal and disease states. Therefore, here we describe a new method that allows localized proteomics on individual cell populations isolated from FFPE tissue sections using laser capture microdissection. To demonstrate this technique we microdissected neurons from archived tissue blocks of the temporal cortex from patients with Alzheimer's disease. Using this method we identified over 400 proteins in microdissected neurons; on average 78% that were neuronal and 50% that were associated with Alzheimer's disease. Therefore, this technique is able to provide accurate and meaningful data and has great potential for any future study that wishes to perform localized proteomics using very small amounts of archived FFPE tissue.

Genotypic differences greatly influence susceptibility and resistance to disease. Understanding genotype-phenotype relationships requires that phenotypes be viewed as manifestations of network properties, rather than simply as the result of individual genomic variations. Genome sequencing efforts have identified numerous germline mutations, and large numbers of somatic genomic alterations, associated with a predisposition to cancer. However, it remains difficult to distinguish background, or 'passenger', cancer mutations from causal, or 'driver', mutations in these data sets. Human viruses intrinsically depend on their host cell during the course of infection and can elicit pathological phenotypes similar to those arising from mutations. Here we test the hypothesis that genomic variations and tumour viruses may cause cancer through related mechanisms, by systematically examining host interactome and transcriptome network perturbations caused by DNA tumour virus proteins. The resulting integrated viral perturbation data reflects rewiring of the host cell networks, and highlights pathways, such as Notch signalling and apoptosis, that go awry in cancer. We show that systematic analyses of host targets of viral proteins can identify cancer genes with a success rate on a par with their identification through functional genomics and large-scale cataloguing of tumour mutations. Together, these complementary approaches increase the specificity of cancer gene identification. Combining systems-level studies of pathogen-encoded gene products with genomic approaches will facilitate the prioritization of cancer-causing driver genes to advance the understanding of the genetic basis of human cancer.

PIM1 is a serine/threonine kinase that promotes and maintains prostate tumorigenesis. While PIM1 protein levels are elevated in prostate cancer relative to local disease, the mechanisms by which PIM1 contributes to oncogenesis have not been fully elucidated. Here, we performed a direct, unbiased chemical genetic screen to identify PIM1 substrates in prostate cancer cells. The PIM1 substrates we identified were involved in a variety of oncogenic processes, and included N-Myc Downstream-Regulated Gene 1 (NDRG1), which has reported roles in suppressing cancer cell invasion and metastasis. NDRG1 is phosphorylated by PIM1 at serine 330 (pS330), and the level of NDRG1 pS330 is associated higher grade prostate tumors. We have shown that PIM1 phosphorylation of NDRG1 at S330 reduced its stability, nuclear localization, and interaction with AR, resulting in enhanced cell migration and invasion.

Generation of virus-host protein-protein interactions (PPIs) maps may provide clues to uncover SARS-CoV-2-hijacked cellular processes. However, these PPIs maps were created by expressing each viral protein singularly, which does not reflect the life situation in which certain viral proteins synergistically interact with host proteins. Our results reveal the host-viral protein-protein interactome of SARS-CoV-2 NSP3, NSP4, and NSP6 expressed individually or in combination. Furthermore, REEP5/TRAM1 complex interacts with NSP3 at ROs and promotes viral replication. The significance of our research is identifying virus-host interactions that may be targeted for therapeutic intervention.

Animal toxins operate by binding to receptors and ion channels. These proteins are short and vary in sequence, structure and function. Sporadic discoveries have also revealed endogenous toxin-like proteins in non-venomous organisms. Viral proteins are the largest group of quickly evolving proteomes. We tested the hypothesis that toxin-like proteins exist in viruses and that they act to modulate functions of their hosts.

Association of aberrant glycosylation with melanoma progression is based mainly on analyses of cell lines. Here we present a systems-based study of glycomic changes and corresponding enzymes associated with melanoma metastasis in patient samples. Upregulation of core fucosylation (FUT8) and downregulation of α-1,2 fucosylation (FUT1, FUT2) were identified as features of metastatic melanoma. Using both in vitro and in vivo studies, we demonstrate FUT8 is a driver of melanoma metastasis which, when silenced, suppresses invasion and tumor dissemination. Glycoprotein targets of FUT8 were enriched in cell migration proteins including the adhesion molecule L1CAM. Core fucosylation impacted L1CAM cleavage and the ability of L1CAM to support melanoma invasion. FUT8 and its targets represent therapeutic targets in melanoma metastasis.

Programmed cell death protein 1 (PD-1) is a critical inhibitory receptor that limits excessive T cell responses. Cancer cells have evolved to evade these immunoregulatory mechanisms by upregulating PD-1 ligands and preventing T cell-mediated anti-tumor responses. Consequently, therapeutic blockade of PD-1 enhances T cell-mediated anti-tumor immunity, but many patients do not respond and a significant proportion develop inflammatory toxicities. To improve anti-cancer therapy, it is critical to reveal the mechanisms by which PD-1 regulates T cell responses. We performed global quantitative phosphoproteomic interrogation of PD-1 signaling in T cells. By complementing our analysis with functional validation assays, we show that PD-1 targets tyrosine phosphosites that mediate proximal T cell receptor signaling, cytoskeletal organization, and immune synapse formation. PD-1 ligation also led to differential phosphorylation of serine and threonine sites within proteins regulating T cell activation, gene expression, and protein translation. In silico predictions revealed that kinase/substrate relationships engaged downstream of PD-1 ligation. These insights uncover the phosphoproteomic landscape of PD-1-triggered pathways and reveal novel PD-1 substrates that modulate diverse T cell functions and may serve as future therapeutic targets. These data are a useful resource in the design of future PD-1-targeting therapeutic approaches.

Approximately 30% of human lung cancers acquire mutations in either Keap1 or Nfe2l2, resulting in the stabilization of Nrf2, the Nfe2l2 gene product, which controls oxidative homeostasis. Here, we show that heme triggers the degradation of Bach1, a pro-metastatic transcription factor, by promoting its interaction with the ubiquitin ligase Fbxo22. Nrf2 accumulation in lung cancers causes the stabilization of Bach1 by inducing Ho1, the enzyme catabolizing heme. In mouse models of lung cancers, loss of Keap1 or Fbxo22 induces metastasis in a Bach1-dependent manner. Pharmacological inhibition of Ho1 suppresses metastasis in a Fbxo22-dependent manner. Human metastatic lung cancer display high levels of Ho1 and Bach1. Bach1 transcriptional signature is associated with poor survival and metastasis in lung cancer patients. We propose that Nrf2 activates a metastatic program by inhibiting the heme- and Fbxo22-mediated degradation of Bach1, and that Ho1 inhibitors represent an effective therapeutic strategy to prevent lung cancer metastasis.

Intracellular degradation of proteins and organelles by the autophagy-lysosome system is essential for cellular quality control and energy homeostasis. Besides degradation, endolysosomal organelles can fuse with the plasma membrane and contribute to unconventional secretion. Here, we identify a function for mammalian SKP1 in endolysosomes that is independent of its established role as an essential component of the family of SCF/CRL1 ubiquitin ligases. We found that, under nutrient-poor conditions, SKP1 is phosphorylated on Thr131, allowing its interaction with V1 subunits of the vacuolar ATPase (V-ATPase). This event, in turn, promotes V-ATPase assembly to acidify late endosomes and enhance endolysosomal degradation. Under nutrient-rich conditions, SUMOylation of phosphorylated SKP1 allows its binding to and dephosphorylation by the PPM1B phosphatase. Dephosphorylated SKP1 interacts with SEC22B to promote unconventional secretion of the content of less acidified hybrid endosomal/autophagic compartments. Collectively, our study implicates SKP1 phosphorylation as a switch between autophagy and unconventional secretion in a manner dependent on cellular nutrient status.

The cell wall of mycobacteria plays a key role in interactions with the environment. Its ability to act as a selective filter is crucial to bacterial survival. Proteins in the cell wall enable this function by mediating the import and export of diverse metabolites, from ions to lipids to proteins. Identifying cell wall proteins is an important step in assigning function, especially as many mycobacterial proteins lack functionally characterized homologues. Current methods for protein localization have inherent limitations that reduce accuracy. Here we showed that although chemical labeling of live cells did not exclusively label surface proteins, protein tagging by the engineered peroxidase APEX2 within live Mycobacterium tuberculosis accurately identified the cytosolic and cell wall proteomes. Our data indicate that substrates of the virulence-associated Type VII ESX secretion system are exposed to the periplasm, providing insight into the currently unknown mechanism by which these proteins cross the mycobacterial cell envelope.

Although mismatch repair (MMR) is essential for correcting DNA replication errors, it can also recognize other lesions, such as oxidized bases. In G0 and G1, MMR is kept in check through unknown mechanisms as it is error-prone during these cell cycle phases. We show that in mammalian cells, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins inhibit the proteasomal degradation of p21, which competes with MMR proteins for binding to PCNA, thereby inhibiting MMR. The ability of D-type cyclins to limit MMR is CDK4- and CDK6-independent and is conserved in G0 and G1. At the G1/S transition, the timely, cullin-RING ubiquitin ligase (CRL)-dependent degradation of D-type cyclins and p21 enables MMR activity to efficiently repair DNA replication errors. Persistent expression of D-type cyclins during S-phase inhibits the binding of MMR proteins to PCNA, increases the mutational burden, and promotes microsatellite instability.

The large majority of oxidative DNA lesions occurring in the G1 phase of the cell cycle are repaired by base excision repair (BER) rather than mismatch repair (MMR) to avoid long resections that can lead to genomic instability and cell death. However, the molecular mechanisms dictating pathway choice between MMR and BER have remained unknown. Here, we show that, during G1, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins shield p21 from its two ubiquitin ligases CRL1SKP2 and CRL4CDT2 in a CDK4/6-independent manner. In turn, p21 competes through its PCNA-interacting protein degron with MMR components for their binding to PCNA. This inhibits MMR while not affecting BER. At the G1/S transition, the CRL4AMBRA1-dependent degradation of D-type cyclins renders p21 susceptible to proteolysis. These timely degradation events allow the proper binding of MMR proteins to PCNA, enabling the repair of DNA replication errors. Persistent expression of cyclin D1 during S-phase increases the mutational burden and promotes microsatellite instability. Thus, the expression of D-type cyclins inhibits MMR in G1, whereas their degradation is necessary for proper MMR function in S.

The progression of pancreatic oncogenesis requires immune-suppressive inflammation in cooperation with oncogenic mutations. However, the drivers of intratumoral immune tolerance are uncertain. Dectin 1 is an innate immune receptor crucial for anti-fungal immunity, but its role in sterile inflammation and oncogenesis has not been well defined. Furthermore, non-pathogen-derived ligands for dectin 1 have not been characterized. We found that dectin 1 is highly expressed on macrophages in pancreatic ductal adenocarcinoma (PDA). Dectin 1 ligation accelerated the progression of PDA in mice, whereas deletion of Clec7a-the gene encoding dectin 1-or blockade of dectin 1 downstream signaling was protective. We found that dectin 1 can ligate the lectin galectin 9 in mouse and human PDA, which results in tolerogenic macrophage programming and adaptive immune suppression. Upon disruption of the dectin 1-galectin 9 axis, CD4+ and CD8+ T cells, which are dispensable for PDA progression in hosts with an intact signaling axis, become reprogrammed into indispensable mediators of anti-tumor immunity. These data suggest that targeting dectin 1 signaling is an attractive strategy for developing an immunotherapy for PDA.

Programmed cell death-1 (PD-1) is an essential inhibitory receptor in T cells. Antibodies targeting PD-1 elicit durable clinical responses in patients with multiple tumor indications. Nevertheless, a significant proportion of patients do not respond to anti-PD-1 treatment, and a better understanding of the signaling pathways downstream of PD-1 could provide biomarkers for those whose tumors respond and new therapeutic approaches for those whose tumors do not. We used affinity purification mass spectrometry to uncover multiple proteins associated with PD-1. Among these proteins, signaling lymphocytic activation molecule-associated protein (SAP) was functionally and mechanistically analyzed for its contribution to PD-1 inhibitory responses. Silencing of SAP augmented and overexpression blocked PD-1 function. T cells from patients with X-linked lymphoproliferative disease (XLP), who lack functional SAP, were hyperresponsive to PD-1 signaling, confirming its inhibitory role downstream of PD-1. Strikingly, signaling downstream of PD-1 in purified T cell subsets did not correlate with PD-1 surface expression but was inversely correlated with intracellular SAP levels. Mechanistically, SAP opposed PD-1 function by acting as a molecular shield of key tyrosine residues that are targets for the tyrosine phosphatase SHP2, which mediates PD-1 inhibitory properties. Our results identify SAP as an inhibitor of PD-1 function and SHP2 as a potential therapeutic target in patients with XLP.

LINE-1/L1 retrotransposon sequences comprise 17% of the human genome. Among the many classes of mobile genetic elements, L1 is the only autonomous retrotransposon that still drives human genomic plasticity today. Through its co-evolution with the human genome, L1 has intertwined itself with host cell biology. However, a clear understanding of L1's lifecycle and the processes involved in restricting its insertion and intragenomic spread remains elusive. Here we identify modes of L1 proteins' entrance into the nucleus, a necessary step for L1 proliferation. Using functional, biochemical, and imaging approaches, we also show a clear cell cycle bias for L1 retrotransposition that peaks during the S phase. Our observations provide a basis for novel interpretations about the nature of nuclear and cytoplasmic L1 ribonucleoproteins (RNPs) and the potential role of DNA replication in L1 retrotransposition.

The pathogen Staphylococcus aureus colonizes and infects a variety of different sites within the human body. To adapt to these different environments, S. aureus relies on a complex and finely tuned regulatory network. While some of these networks have been well-elucidated, the functions of more than 50% of the transcriptional regulators in S. aureus remain unexplored. Here, we assess the contribution of the LacI family of metabolic regulators to staphylococcal virulence. We found that inactivating the purine biosynthesis regulator purR resulted in a strain that was acutely virulent in bloodstream infection models in mice and in ex vivo models using primary human neutrophils. Remarkably, these enhanced pathogenic traits are independent of purine biosynthesis, as the purR mutant was still highly virulent in the presence of mutations that disrupt PurR's canonical role. Through the use of transcriptomics coupled with proteomics, we revealed that a number of virulence factors are differentially regulated in the absence of purR Indeed, we demonstrate that PurR directly binds to the promoters of genes encoding virulence factors and to master regulators of virulence. These results guided us into further ex vivo and in vivo studies, where we discovered that S. aureus toxins drive the death of human phagocytes and mice, whereas the surface adhesin FnbA contributes to the increased bacterial burden observed in the purR mutant. Thus, S. aureus repurposes a metabolic regulator to directly control the expression of virulence factors, and by doing so, tempers its pathogenesis.

Proteomic studies of human Alzheimer's disease brain tissue have potential to identify protein changes that drive disease, and to identify new drug targets. Here, we analyse 38 published Alzheimer's disease proteomic studies, generating a map of protein changes in human brain tissue across thirteen brain regions, three disease stages (preclinical Alzheimer's disease, mild cognitive impairment, advanced Alzheimer's disease), and proteins enriched in amyloid plaques, neurofibrillary tangles, and cerebral amyloid angiopathy. Our dataset is compiled into a searchable database (NeuroPro). We found 848 proteins were consistently altered in 5 or more studies. Comparison of protein changes in early-stage and advanced Alzheimer's disease revealed proteins associated with synapse, vesicle, and lysosomal pathways show change early in disease, but widespread changes in mitochondrial associated protein expression change are only seen in advanced Alzheimer's disease. Protein changes were similar for brain regions considered vulnerable and regions considered resistant. This resource provides insight into Alzheimer's disease brain protein changes and highlights proteins of interest for further study.