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Necroptosis is a form of regulated necrotic cell death that promotes inflammation. In cells undergoing necroptosis, activated RIPK1 kinase mediates the formation of RIPK1/RIPK3/MLKL complex to promote MLKL oligomerization and execution of necroptosis. RIPK1 kinase activity also promotes cell-autonomous activation of proinflammatory cytokine production in necroptosis. However, the signaling pathways downstream of RIPK1 kinase in necroptosis and how RIPK1 kinase activation controls inflammatory response induced by necroptosis are still largely unknown. Here, we quantitatively measured the temporal dynamics of over 7000 confident phosphorylation-sites during necroptosis using mass spectrometry. Our study defined a RIPK1-dependent phosphorylation pattern in late necroptosis that is associated with a proinflammatory component marked by p-S473 TRIM28. We show that the activation of p38 MAPK mediated by oligomerized MLKL promotes the phosphorylation of S473 TRIM28, which in turn mediates inflammation during late necroptosis. Taken together, our study illustrates a mechanism by which p38 MAPK may be activated by oligomerized MLKL to promote inflammation in necroptosis.

Amyloid aggregation of phosphorylated Tau (pTau) into neurofibrillary tangles is closely associated with Alzheimer’s disease (AD). Several molecular chaperones have been reported to bind Tau and impede its pathological aggregation. Recent findings of elevated levels of Hsp27 in the brains of patients with AD suggested its important role in pTau pathology. However, the molecular mechanism of Hsp27 in pTau aggregation remains poorly understood. Here, we show that Hsp27 partially co-localizes with pTau tangles in the brains of patients with AD. Notably, phosphorylation of Tau by microtubule affinity regulating kinase 2 (MARK2), dramatically enhances the binding affinity of Hsp27 to Tau. Moreover, Hsp27 efficiently prevents pTau fibrillation in vitro and mitigates neuropathology of pTau aggregation in a Drosophila tauopathy model. Further mechanistic study reveals that Hsp27 employs its N-terminal domain to directly interact with multiple phosphorylation sites of pTau for specific binding. Our work provides the structural basis for the specific recognition of Hsp27 to pathogenic pTau, and highlights the important role of Hsp27 in preventing abnormal aggregation and pathology of pTau in AD.

Summary Mammalian cyclic dinucleotide 2′3′-cGAMP functions as a second messenger in innate immune response. Here, we report a protocol to utilize 2′3′-cGAMP photoaffinity probes to capture 2′3′-cGAMP-binding or 2′3′-cGAMP-interacting proteins from HeLa cell lysate for in-gel visualization by fluorescent imaging or identification by SILAC–based quantitative MS. Further validation is also executed using photoaffinity probes to demonstrate the direct interaction of 2′3′-cGAMP with purified target proteins in vitro or endogenous target proteins in 293T cells. For complete details on the use and execution of this profile, please refer to Hou et al. (2021)., Graphical abstract, Highlights • Strategy for identification of 2′3′-cGAMP interactome by photoaffinity probes • Purification of EF1A1 in EF1 complex and validation of its binding with 2′3′-cGAMP • Validate 2′3′-cGAMP interactors in vitro and in 293T cells using photoaffinity probes, Mammalian cyclic dinucleotide 2′3′-cGAMP functions as a second messenger in innate immune response. Here, we report a protocol to utilize 2′3′-cGAMP photoaffinity probes to capture 2′3′-cGAMP-binding or 2′3′-cGAMP-interacting proteins from HeLa cell lysate for in-gel visualization by fluorescent imaging or identification by SILAC–based quantitative MS. Further validation is also executed using photoaffinity probes to demonstrate the direct interaction of 2′3′-cGAMP with purified target proteins in vitro or endogenous target proteins in 293T cells.

Breast cancer is one of the most common cancers in women worldwide. In the past decades, many advances have been made in understanding and treating breast cancer. However, due to the highly heterogeneous nature of this disease, a precise characterization of breast cancer on the molecular level is of great importance but not yet readily available. In the present study, we systematically profiled proteomes and N-glycoproteomes of cancerous, paracancerous, and distal noncancerous tissues from patients with breast cancer. The data revealed distinct proteomic and N-glycoproteomic landscapes between different tissues, showing biological insights obtained from the two data sets were complementary. Specifically, the complement and angiogenesis pathways in the paracancerous tissues were activated. Taken together, the changes that occurred in paracancer tissue and N-glycoproteomics are important complements to the conventional proteomic analysis of cancer tissue. Their combination provides more precise and sensitive molecular correlates of breast cancer. Our data and strategy shed light on precisely defining breast cancer, providing valuable information for individual patient diagnosis and treatment. The MS data of this study have been deposited under the accession number IPX0001924000 at iProX.

Aspirin, or acetylsalicylic acid (ASA), is the most widely used medication to relieve pain, fever, and inflammation. Recent studies have revealed new benefits of aspirin, including reduction of heart attack and stroke, anticancer, and life extension. Despite the profound effects of aspirin, the mechanism of its action remains to be elucidated. Here, we used deuterium-labeled aspirin (D-aspirin) together with mass spectrometry-based acetylomic analysis, termed DAcMS, to investigate the landscape of protein acetylation induced by aspirin. The DAcMS revealed the acetylomes of lipopolysaccharide-induced inflammatory BV2 cells and colon cancer HCT116 cells. The acetylation level was substantially induced upon aspirin treatment in both cell lines. In total, we identified 17,003 acetylation sites on 4623 proteins in BV2 cells and 16,366 acetylated sites corresponding to 4702 acetylated proteins in HCT116 cells. Importantly, functional analyses of these aspirin-induced acetylated proteins suggested that they were highly enriched in many key biological categories, which function importantly in inflammatory response. We further demonstrated that aspirin acetylates proteins through both acetyl-CoA-dependent and acetyl-CoA-independent pathways, and the accessible lysine residues at the protein surface are major acetylation targets of aspirin. Hence, our study provides the comprehensive atlas of aspirin-induced acetylome under disease conditions. This knowledge proffers new insight into the aspirin-directed acetylome and perhaps new drug target sites relevant to human cancer and inflammatory diseases. The MS data of this study have been deposited under the accession number IPX0001923000 at iProX.

Selenocysteine (Sec, U) is the 21st amino acid, and proteins with selenocysteine are defined as selenoproteins. The currently known selenoproteins are all featured by the presence of selenocysteine insertion sequence (SECIS) on their mRNA, and SECIS plays an essential role in the selenocysteine insertion mechanism. However, due to the extremely low occurrences of selenoproteins in a whole proteome (e.g., only 25 selenoproteins in the human proteome) and the low sequence conservation of SECIS, analysis of selenoproteins and discovery of new selenoproteins exclusively on SECIS are intrinsically challenging. To this end, the selenocysteine-specific mass spectrometry (SecMS) and SECIS-independent selenoprotein (SIS) database are developed, showing abilities to profile whole selenoproteomes sensitively and to discover potential new selenoproteins. Here, we detail the SecMS strategy and propose it will advance the exploration for new selenoproteins and functional studies of selenoproteins.

α-Synuclein (α-Syn) can form different fibril strains with distinct polymorphs and neuropathologies, which is associated with the clinicopathological variability in synucleinopathies. How different α-syn fibril strains are produced and selected under disease conditions remains poorly understood. In this study, we show that the hereditary mutation G51D induces α-syn to form a distinct fibril strain in vitro. The cryogenic electron microscopy (cryo-EM) structure of the G51D fibril strain was determined at 2.96 Å resolution. The G51D fibril displays a relatively small and extended serpentine fold distinct from other α-syn fibril structures. Moreover, we show by cryo-EM that wild-type (WT) α-syn can assembly into the G51D fibril strain via cross-seeding with G51D fibrils. Our study reveals a distinct structure of G51D fibril strain triggered by G51D mutation but feasibly adopted by both WT and G51D α-syn, which suggests the cross-seeding and strain selection of WT and mutant α-syn in familial Parkinson’s disease (fPD)., G51D mutation of α-synuclein (α-syn) causes a subset of familial Parkinson’s disease that is characterized by an early onset and rapid progression of the disease. Here, the authors present the cryo-EM structure of full-length G51D α-syn fibrils that is distinct from other known α-syn fibril structures, and they show that G51D fibrils can cross-seed wild-type (WT) α-syn and that these cross-seeded WT fibrils replicate the G51D fibril structure.

Background Bone tissue engineering is a new concept bringing hope for the repair of large bone defects, which remains a major clinical challenge. The formation of vascularized bone is key for bone tissue engineering. Growth of specialized blood vessels termed type H is associated with bone formation. In vivo and in vitro studies have shown that low level laser therapy (LLLT) promotes angiogenesis, fracture healing, and osteogenic differentiation of stem cells by increasing reactive oxygen species (ROS). However, whether LLLT can couple angiogenesis and osteogenesis, and the underlying mechanisms during bone formation, remains largely unknown. Methods Mouse bone marrow mesenchymal stem cells (BMSCs) combined with biphasic calcium phosphate (BCP) grafts were implanted into C57BL/6 mice to evaluate the effects of LLLT on the specialized vessel subtypes and bone regeneration in vivo. Furthermore, human BMSCs and human umbilical vein endothelial cells (HUVECs) were co-cultured in vitro. The effects of LLLT on cell proliferation, angiogenesis, and osteogenesis were assessed. Results LLLT promoted the formation of blood vessels, collagen fibers, and bone tissue and also increased CD31hiEMCNhi-expressing type H vessels in mBMSC/BCP grafts implanted in mice. LLLT significantly increased both osteogenesis and angiogenesis, as well as related gene expression (HIF-1α, VEGF, TGF-β) of grafts in vivo and of co-cultured BMSCs/HUVECs in vitro. An increase or decrease of ROS induced by H2O2 or Vitamin C, respectively, resulted in an increase or decrease of HIF-1α, and a subsequent increase and decrease of VEGF and TGF-β in the co-culture system. The ROS accumulation induced by LLLT in the co-culture system was significantly decreased when HIF-1α was inhibited with DMBPA and was followed by decreased expression of VEGF and TGF-β. Conclusions LLLT enhanced vascularized bone regeneration by coupling angiogenesis and osteogenesis. ROS/HIF-1α was necessary for these effects of LLLT. LLLT triggered a ROS-dependent increase of HIF-1α, VEGF, and TGF-β and resulted in subsequent formation of type H vessels and osteogenic differentiation of mesenchymal stem cells. As ROS also was a target of HIF-1α, there may be a positive feedback loop between ROS and HIF-1α, which further amplified HIF-1α induction via the LLLT-mediated ROS increase. This study provided new insight into the effects of LLLT on vascularization and bone regeneration in bone tissue engineering.

Membrane protein clients of endoplasmic reticulum (ER)-associated degradation must be retrotranslocated from the ER membrane by the AAA-ATPase p97 for proteasomal degradation. Before direct engagement with p97, client transmembrane domains (TMDs) that have partially or fully crossed the membrane must be constantly shielded to avoid non-native interactions. How client TMDs are seamlessly escorted from the membrane to p97 is unknown. Here, we identified ER-anchored TMUB1 as a TMD-specific escortase. TMUB1 interacts with the TMD of clients within the membrane and holds ∼10-14 residues of a hydrophobic sequence that is exposed out of membrane, using its transmembrane and cytosolic regions, respectively. The ubiquitin-like domain of TMUB1 recruits p97, which can pull client TMDs from bound TMUB1 into the cytosol. The disruption of TMUB1 escortase activity impairs retrotranslocation and stabilizes retrotranslocating intermediates of client proteins within the ER membrane. Thus, TMUB1 promotes TMD segregation by safeguarding the TMD movement from the membrane to p97.

The long-lived proteome constitutes a pool of exceptionally stable proteins with limited turnover. Previous studies on ubiquitin-mediated protein degradation primarily focused on relatively short-lived proteins; how ubiquitylation modifies the long-lived proteome and its regulatory effect on adult lifespan is unclear. Here we profile the age-dependent dynamics of long-lived proteomes in Drosophila by mass spectrometry using stable isotope switching coupled with antibody-enriched ubiquitylome analysis. Our data describe landscapes of long-lived proteins in somatic and reproductive tissues of Drosophila during adult lifespan, and reveal a preferential ubiquitylation of older long-lived proteins. We identify an age-modulated increase of ubiquitylation on long-lived histone 2A protein in Drosophila, which is evolutionarily conserved in mouse, monkey, and human. A reduction of ubiquitylated histone 2A in mutant flies is associated with longevity and healthy lifespan. Together, our data reveal an evolutionarily conserved biomarker of aging that links epigenetic modulation of the long-lived histone protein to lifespan., Post-translational protein modifications can affect lifespan and aging but age-dependent ubiquitylation changes have not yet been systematically characterized. Here, the authors analyze age-related proteome and ubiquitylome dynamics in Drosophila and identify increasing H2A ubiquitylation as a conserved aging marker.

Mutations in RNA-binding proteins (RBPs) localized in ribonucleoprotein (RNP) granules, such as hnRNP A1 and TDP-43, promote aberrant protein aggregation, which is a pathological hallmark of various neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Protein posttranslational modifications (PTMs) are known to regulate RNP granules. In this study, we investigate the function of poly(ADP-ribosyl)ation (PARylation), an important PTM involved in DNA damage repair and cell death, in RNP granule-related neurodegeneration. We reveal that PARylation levels are a major regulator of the assembly-disassembly dynamics of RNP granules containing disease-related RBPs, hnRNP A1 and TDP-43. We find that hnRNP A1 can both be PARylated and bind to PARylated proteins or poly(ADP-ribose) (PAR). We further uncover that PARylation of hnRNP A1 at K298 controls its nucleocytoplasmic transport, whereas PAR-binding via the PAR-binding motif (PBM) of hnRNP A1 regulates its association with stress granules. Moreover, we reveal that PAR not only dramatically enhances the liquid-liquid phase separation of hnRNP A1, but also promotes the co-phase separation of hnRNP A1 and TDP-43 in vitro and their interaction in vivo. Finally, both genetic and pharmacological inhibition of PARP mitigates hnRNP A1- and TDP-43-mediated neurotoxicity in cell and Drosophila models of ALS. Together, our findings suggest a novel and crucial role for PARylation in regulating the dynamics of RNP granules, and that dysregulation in PARylation and PAR levels may contribute to ALS disease pathogenesis by promoting protein aggregation.

Summary Rapamycin is widely recognized as an inhibitor of mTOR, and has been approved for clinical use as an immunosuppressant. Its potencies in anti-cancer, anti-aging, and neurodegenerative diseases are emergingly established. The exploration of other targets of rapamycin will further elucidate its underlying mechanisms of action. In this study, we use a chemical proteomics strategy that has identified STAT3, a transcription factor considered to be undruggable, as a direct functional protein target of rapamycin. Together with other multi-dimensional proteomics data, we show that rapamycin treatment in cell culture significantly inhibits c-Myc-regulated gene expression. Furthermore, we show that rapamycin suppresses tumor growth along with a decreased expression of STAT3 and c-Myc in an in vivo xenograft mouse model for hepatocellular carcinoma. Our data suggest that rapamycin acts directly on STAT3 to decrease its transcription activity, providing important information for the pharmacological and pharmaceutical development of STAT3 inhibitors for cancer therapy.

Summary 2′3′-cyclic GMP-AMP (2′3′-cGAMP), generated by cyclic GMP-AMP synthase (cGAS) under activation by cytosolic DNA, has a vital role in innate immune response via its receptor protein stimulator of interferon genes (STING) to fight viral infections and tumors. In order to have a complete understanding of biological functions of 2′3′-cGAMP, it is important to find out whether 2′3′-cGAMP has other unrevealed binding proteins present in mammalian cells and executes unknown functions. Here we report the 2′3′-cGAMP-based photoaffinity probes that capture and isolate 2′3′-cGAMP-binding proteins. These probes enable the identification of some potential 2′3′-cGAMP-binding proteins from HeLa cells. EF1A1, an essential protein regulating protein synthesis, is further validated to associate with 2′3′-cGAMP in vitro and in cells to impede protein synthesis. Thus, our studies provide a powerful approach to enable identification of the 2′3′-cGAMP interactome, discover unknown functions of 2′3′-cGAMP, and understand its physiological/pathological roles in tumor immunity and immune-related diseases.

Funder: Science and Technology Commission of Shanghai Municipality; FundRef: http://dx.doi.org/10.13039/501100003399, Funder: Dr. John T. MacDonald Foundation; FundRef: http://dx.doi.org/10.13039/100010239, Tau hyper-phosphorylation and deposition into neurofibrillary tangles have been found in brains of patients with Alzheimer's disease (AD) and other tauopathies. Molecular chaperones are involved in regulating the pathological aggregation of phosphorylated Tau (pTau) and modulating disease progression. Here, we report that nicotinamide mononucleotide adenylyltransferase (NMNAT), a well-known NAD+ synthase, serves as a chaperone of pTau to prevent its amyloid aggregation in vitro as well as mitigate its pathology in a fly tauopathy model. By combining NMR spectroscopy, crystallography, single-molecule and computational approaches, we revealed that NMNAT adopts its enzymatic pocket to specifically bind the phosphorylated sites of pTau, which can be competitively disrupted by the enzymatic substrates of NMNAT. Moreover, we found that NMNAT serves as a co-chaperone of Hsp90 for the specific recognition of pTau over Tau. Our work uncovers a dedicated chaperone of pTau and suggests NMNAT as a key node between NAD+ metabolism and Tau homeostasis in aging and neurodegeneration.

Mixed-lineage kinase domain-like protein (MLKL) is known as the terminal executor of necroptosis. However, its function outside of necroptosis is still not clear. Herein, we demonstrate that MLKL promotes vascular inflammation by regulating the expression of adhesion molecules ICAM1, VCAM1, and E-selectin in endothelial cells (EC). MLKL deficiency suppresses the expression of these adhesion molecules, thereby reducing EC-leukocyte interaction in vitro and in vivo. Mechanistically, we show that MLKL interacts with RBM6 to promote the mRNA stability of adhesion molecules. In conclusion, this study identified a novel role of MLKL in regulating endothelial adhesion molecule expression and local EC-leukocyte interaction during acute inflammation.

Valosin-containing protein (VCP)/p97 is an AAA-ATPase that extracts polyubiquitinated substrates from multimeric macromolecular complexes and biological membranes for proteasomal degradation. During p97-mediated extraction, the substrate is largely deubiquitinated as it is threaded through the p97 central pore. How p97-extracted substrates are targeted to the proteasome with few or no ubiquitins is unknown. Here, we report that p97-extracted membrane proteins undergo a second round of ubiquitination catalyzed by the cytosolic ubiquitin ligase RNF126. RNF126 interacts with transmembrane-domain-specific chaperone BAG6, which captures p97-liberated substrates. RNF126 depletion in cells diminishes the ubiquitination of extracted membrane proteins, slows down their turnover, and dramatically stabilizes otherwise transient intermediates in the cytosol. We reconstitute the reubiquitination of a p97-extracted, misfolded multispanning membrane protein with purified factors. Our results demonstrate that p97-extracted substrates need to rapidly engage ubiquitin ligase-chaperone pairs that rebuild the ubiquitin signal for proteasome targeting to prevent harmful accumulation of unfolded intermediates.

The CRISPR-Cas9 system is a genomic editing tool widely used in basic research and under investigation for potential applications in gene therapies for human diseases. To accomplish genomic editing, the system requires the expression of a prokaryotic DNA endonuclease enzyme, Cas9, in host cells. Previous studies have mainly focused on the specificity of Cas9 on the host genome, and thus it is unclear whether this bacterium-derived enzyme affects the protein homeostasis of host cells. Here we applied multi-omic analyses, including transcriptome, proteome, phosphoproteome, Cas9-associated protein interactome, protein synthesis, and histone epigenetic modification, to investigate the cellular response of human cells upon the expression of Cas9. We demonstrate that Cas9 has minimal impact on host cells. Our assessment of intracellular effects of Cas9 paves a path for its broad applications in biological studies and potential clinical translations.

International audience; Multiple sclerosis (MS), a common neurodegenerative disease of the CNS, is characterized by the loss of oligodendrocytes and demyelination. Tumor necrosis factor α (TNF-α), a proinflammatory cytokine implicated in MS, can activate necroptosis, a necrotic cell death pathway regulated by RIPK1 and RIPK3 under caspase-8-deficient conditions. Here, we demonstrate defective caspase-8 activation, as well as activation of RIPK1, RIPK3, and MLKL, the hallmark mediators of necroptosis, in the cortical lesions of human MS pathological samples. Furthermore, we show that MS pathological samples are characterized by an increased insoluble proteome in common with other neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson’s disease (PD), and Huntington's disease (HD). Finally, we show that necroptosis mediates oligodendrocyte degeneration induced by TNF-α and that inhibition of RIPK1 protects against oligodendrocyte cell death in two animal models of MS and in culture. Our findings demonstrate that necroptosis is involved in MS and suggest that targeting RIPK1 may represent a therapeutic strategy for MS.

CKS proteins are small (9-kDa) polypeptides that bind to a subset of the cyclin-dependent kinases. The two paralogs expressed in mammals, Cks1 and Cks2, share an overlapping function that is essential for early development. However, both proteins are frequently overexpressed in human malignancy. It has been shown that CKS protein overexpression overrides the replication stress checkpoint, promoting continued origin firing. This finding has led to the proposal that CKS protein-dependent checkpoint override allows premalignant cells to evade oncogene stress barriers, providing a causal link to oncogenesis. Here, we provide mechanistic insight into how overexpression of CKS proteins promotes override of the replication stress checkpoint. We show that CKS proteins greatly enhance the ability of Cdk2 to phosphorylate the key replication initiation protein treslin in vitro. Furthermore, stimulation of treslin phosphorylation does not occur by the canonical adapter mechanism demonstrated for other substrates, as cyclin-dependent kinase (CDK) binding-defective mutants are capable of stimulating treslin phosphorylation. This effect is recapitulated in vivo, where silencing of Cks1 and Cks2 decreases treslin phosphorylation, and overexpression of wild-type or CDK binding-defective Cks2 prevents checkpoint-dependent dephosphorylation of treslin. Finally, we provide evidence that the role of CKS protein-dependent checkpoint override involves recovery from checkpoint-mediated arrest of DNA replication.

N-glycosylation is a crucial post-translational modification (PTM) and plays essential roles in biological processes. Several methods have been developed for the relative quantification of N-glycosylation at the proteome scale. However, the proportion of N-glycosylated forms in a total protein population, or the "N-glycosylation stoichiometry", varies greatly among proteins or cellular states and is frequently missing due to the lack of robust technologies. In the present study, we developed a data-independent acquisition (DIA)-based strategy that enabled the in-depth measurement of N-glycosylation stoichiometry. A spectral library containing 3,509 N-glycosylated peptides and 17,525 fragment ions from human embryonic kidney cells 293 (HEK-293) cells was established from which the stoichiometries of 1,186 N-glycosites were calculated. These stoichiometric values differ greatly among different glycosites, and many glycosites tend to occur with low stoichiometry. We then investigated the N-glycosylation changes induced by tunicamycin in HEK-293 cells and by a temperature shift in Chinese hamster ovary (CHO) cells. Quantifying the proteome, N-glycoproteome, and N-glycosylation stoichiometry demonstrated that the regulation of N-glycosylation is primarily achieved by adjusting the N-glycosylation stoichiometry. In total, the stoichiometries of 2,274 glycosites were determined in the current study. Notably, our approach can be applied to other biological systems and other types of PTMs.

Members of the 2-aminobenzamide class of histone deacetylase (HDAC) inhibitors show promise as therapeutics for the neurodegenerative diseases Friedreich's ataxia (FRDA) and Huntington's disease (HD). While it is clear that HDAC3 is one of the important targets of the 2-aminobenzamide HDAC inhibitors, inhibition of other class I HDACs (HDACs 1 and 2) may also be involved in the beneficial effects of these compounds in FRDA and HD, and other HDAC interacting proteins may be impacted by the compound. To this end, we synthesized activity-based profiling probe (ABPP) versions of one of our HDAC inhibitors (compound 106), and in the present study we used a quantitative proteomic method coupled with multidimensional protein identification technology (MudPIT) to identify the proteins captured by the ABPP 106 probe. Nuclear proteins were extracted from FRDA patient iPSC-derived neural stem cells, and then were reacted with control and ABPP 106 probe. After reaction, the bound proteins were digested on the beads, and the peptides were modified using stable isotope-labeled formaldehyde to form dimethyl amine. The selectively bound proteins determined by mass spectrometry were subjected to functional and pathway analysis. Our findings suggest that the targets of compound 106 are involved not only in transcriptional regulation but also in posttranscriptional processing of mRNA.

Regulatory T (Treg) cells, which maintain immune homeostasis and self-tolerance, form an immunological synapse (IS) with antigen-presenting cells (APCs). However, signaling events at the Treg cell IS remain unknown. Here we show that the kinase PKC-η associated with CTLA-4 and was recruited to the Treg cell IS. PKC-η-deficient Treg cells displayed defective suppressive activity, including suppression of tumor immunity but not of autoimmune colitis. Phosphoproteomic and biochemical analysis revealed an association between CTLA-4-PKC-η and the GIT2-αPIX-PAK complex, an IS-localized focal adhesion complex. Defective activation of this complex in PKC-η-deficient Treg cells was associated with reduced depletion of CD86 from APCs by Treg cells. These results reveal a CTLA-4-PKC-η signaling axis required for contact-dependent suppression and implicate this pathway as a potential cancer immunotherapy target.

Selenoproteins, defined by the presence of selenocysteines (Sec), play important roles in a wide range of biological processes. All known selenoproteins are marked by the presence of Sec insertion sequence (SECIS) at their mRNA. The lack of an effective analytical method has hindered our ability to explore the selenoproteome and new selenoproteins beyond SECIS. Here, we develop a Sec-specific mass spectrometry-based technique, termed "SecMS," which allows the systematic profiling of selenoproteomes by selective alkylation of Sec. Using SecMS, we quantitatively characterized the age- and stress-regulated selenoproteomes for nine tissues from mice of different ages and mammalian cells, demonstrating tissue-specific selenoproteomes and an age-dependent decline in specific selenoproteins in brains and hearts. We established an integrated platform using SecMS and SECIS-independent selenoprotein (SIS) database and further identified five candidate selenoproteins. The application of this integrated platform provides an effective strategy to explore the selenoproteome independent of SECIS.

Although glucose uniquely stimulates proinsulin biosynthesis in β cells, surprisingly little is known of the underlying mechanism(s). Here, we demonstrate that glucose activates the unfolded protein response transducer inositol-requiring enzyme 1 alpha (IRE1α) to initiate X-box-binding protein 1 (Xbp1) mRNA splicing in adult primary β cells. Using mRNA sequencing (mRNA-Seq), we show that unconventional Xbp1 mRNA splicing is required to increase and decrease the expression of several hundred mRNAs encoding functions that expand the protein secretory capacity for increased insulin production and protect from oxidative damage, respectively. At 2 wk after tamoxifen-mediated Ire1α deletion, mice develop hyperglycemia and hypoinsulinemia, due to defective β cell function that was exacerbated upon feeding and glucose stimulation. Although previous reports suggest IRE1α degrades insulin mRNAs, Ire1α deletion did not alter insulin mRNA expression either in the presence or absence of glucose stimulation. Instead, β cell failure upon Ire1α deletion was primarily due to reduced proinsulin mRNA translation primarily because of defective glucose-stimulated induction of a dozen genes required for the signal recognition particle (SRP), SRP receptors, the translocon, the signal peptidase complex, and over 100 other genes with many other intracellular functions. In contrast, Ire1α deletion in β cells increased the expression of over 300 mRNAs encoding functions that cause inflammation and oxidative stress, yet only a few of these accumulated during high glucose. Antioxidant treatment significantly reduced glucose intolerance and markers of inflammation and oxidative stress in mice with β cell-specific Ire1α deletion. The results demonstrate that glucose activates IRE1α-mediated Xbp1 splicing to expand the secretory capacity of the β cell for increased proinsulin synthesis and to limit oxidative stress that leads to β cell failure., Each time one eats, the endoplasmic reticulum of the pancreatic beta cells is forced to produce more insulin, thereby activating the IRE1α branch of the unfolded protein response pathway., Author Summary One of the most remarkable features of the pancreatic beta cells—those that produce and secrete insulin to regulate glucose homeostasis—is their capacity to increase the synthesis of proinsulin (the insulin precursor) up to 10-fold after glucose stimulation. This dramatic increase in the synthesis of proinsulin is a challenge to the proximal secretory pathway and triggers an adaptive stress response, the unfolded protein response, which is coordinated by the IRE1α enzyme and the X-box-binding protein 1 (XBP1) transcription factor. Deletion of IRE1α specifically from the pancreatic beta cells in adult mice resulted in overt diabetic phenotypes such as high blood glucose. mRNA analysis revealed several hundred genes whose expression was coordinately regulated by glucose and IRE1α and whose functions are important for the beta cell secretory pathway. Furthermore, IRE1α also regulates the expression of mRNAs involved in the production of reactive oxygen species (ROS), and we could show that, in fact, oxidative stress is a primary mechanism that causes beta cell failure upon collapse of the secretory pathway. Finally, in experiments with murine and human islets (the regions of the pancreas where secretory beta cells are located), we observed that while IRE1α does not regulate the expression of the gene encoding insulin, it determines final insulin levels by controlling translation of proinsulin mRNA.

We have compared the proteome, transcriptome, and metabolome of two cell lines: the human breast epithelial line MCF-10A and its mutant descendant MCF-10A-H1047R. These cell lines are derived from the same parental stock and differ by a single amino acid substitution (H1047R) caused by a single nucleotide change in one allele of the PIK3CA gene, which encodes the catalytic subunit p110α of PI3K (phosphatidylinositol 3-kinase). They are considered isogenic. The H1047R mutation of PIK3CA is one of the most frequently encountered somatic cancer-specific mutations. In MCF-10A, this mutation induces an extensive cellular reorganization that far exceeds the known signaling activities of PI3K. The changes are highly diverse, with examples in structural protein levels, the DNA repair machinery, and sterol synthesis. Gene set enrichment analysis reveals a highly significant concordance of the genes differentially expressed in MCF-10A-H1047R cells and the established protein and RNA signatures of basal breast cancer. No such concordance was found with the specific gene signatures of other histological types of breast cancer. Our data document the power of a single base mutation, inducing an extensive remodeling of the cell toward the phenotype of a specific cancer.

Hexokinase II (HK2), a key enzyme involved in glucose metabolism, is regulated by growth factor signaling and is required for initiation and maintenance of tumors. Here we show that metabolic stress triggered by perturbation of receptor tyrosine kinase FLT3 in non-acute myeloid leukemia cells sensitizes cancer cells to autophagy inhibition and leads to excessive activation of chaperone-mediated autophagy (CMA). Our data demonstrate that FLT3 is an important sensor of cellular nutritional state and elucidate the role and molecular mechanism of CMA in metabolic regulation and mediating cancer cell death. Importantly, our proteome analysis revealed that HK2 is a CMA substrate and that its degradation by CMA is regulated by glucose availability. We reveal a new mechanism by which excessive activation of CMA may be exploited pharmacologically to eliminate cancer cells by inhibiting both FLT3 and autophagy. Our study delineates a novel pharmacological strategy to promote the degradation of HK2 in cancer cells.

Parkinson’s disease (PD) is characterized by loss of A9 dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). An association has been reported between PD and exposure to mitochondrial toxins, including environmental pesticides paraquat, maneb, and rotenone. Here, using a robust, patient-derived stem cell model of PD allowing comparison of A53T α-synuclein (α-syn) mutant cells and isogenic mutation-corrected controls, we identify mitochondrial toxin-induced perturbations in A53T α-syn A9 DA neurons (hNs). We report a pathway whereby basal and toxin-induced nitrosative/oxidative stress results in S-nitrosylation of transcription factor MEF2C in A53T hNs compared to corrected controls. This redox reaction inhibits the MEF2C-PGC1α transcriptional network, contributing to mitochondrial dysfunction and apoptotic cell death. Our data provide mechanistic insight into gene-environmental interaction (GxE) in the pathogenesis of PD. Furthermore, using small-molecule high-throughput screening, we identify the MEF2C-PGC1α pathway as a therapeutic target to combat PD., Parkinson Society Canada (Fellowship), United Mitochondrial Disease Foundation (grant), National Institutes of Health (U.S.) (NIH grant P01 HD29587), National Institutes of Health (U.S.) (NIH grant P01 ES016738), National Institutes of Health (U.S.) (NIH grant P30 NS076411), National Institutes of Health (U.S.) (NIH grant R37 CA084198)