TMUB1 is an endoplasmic reticulum-resident escortase that promotes the p97-mediated extraction of membrane proteins for degradation.

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.

A TRAF-like motif of the inducible costimulator ICOS controls development of germinal center TFH cells via the kinase TBK1.

Signaling via the inducible costimulator ICOS fuels the stepwise development of follicular helper T cells (TFH cells). However, a signaling pathway unique to ICOS has not been identified. We found here that the kinase TBK1 associated with ICOS via a conserved motif, IProx, that shares homology with the tumor-necrosis-factor receptor (TNFR)-associated factors TRAF2 and TRAF3. Disruption of this motif abolished the association of TBK1 with ICOS, TRAF2 and TRAF3, which identified a TBK1-binding consensus. Alteration of this motif in ICOS or depletion of TBK1 in T cells severely impaired the differentiation of germinal center (GC) TFH cells and the development of GCs, interfered with B cell differentiation and disrupted the development of antibody responses, but the IProx motif and TBK1 were dispensable for the early differentiation of TFH cells. These results reveal a previously unknown ICOS-TBK1 signaling pathway that specifies the commitment of GC TFH cells.

Isogenic human iPSC Parkinson's model shows nitrosative stress-induced dysfunction in MEF2-PGC1α transcription.

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.

RNF126-Mediated Reubiquitination Is Required for Proteasomal Degradation of p97-Extracted Membrane Proteins.

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.

Protein kinase C-η controls CTLA-4-mediated regulatory T cell function.

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.

Loss of TDP-43 function underlies hippocampal and cortical synaptic deficits in TDP-43 proteinopathies.

TDP-43 proteinopathy is linked to neurodegenerative diseases that feature synaptic loss in the cortex and hippocampus, although it remains unclear how TDP-43 regulates mature synapses. We report that, in adult mouse hippocampus, TDP-43 knockdown, but not overexpression, induces robust structural and functional damage to excitatory synapses, supporting a role for TDP-43 in maintaining mature synapses. Dendritic spine loss induced by TDP-43 knockdown is rescued by wild-type TDP-43, but not ALS/FTLD-associated mutants, suggesting a common TDP-43 functional deficiency in neurodegenerative diseases. Interestingly, M337V and A90V mutants also display dominant negative activities against WT TDP-43, partially explaining why M337V transgenic mice develop hippocampal degeneration similar to that in excitatory neuronal TDP-43 knockout mice, and why A90V mutation is associated with Alzheimer's disease. Further analyses reveal that a TDP-43 knockdown-induced reduction in GluN2A contributes to synaptic loss. Our results show that loss of TDP-43 function underlies hippocampal and cortical synaptic degeneration in TDP-43 proteinopathies.

Quantitative Acetylomics Reveals Dynamics of Protein Lysine Acetylation in Mouse Livers During Aging and Upon the Treatment of Nicotinamide Mononucleotide.

Lysine acetylation is a reversible and dynamic post-translational modification that plays vital roles in regulating multiple cellular processes including aging. However, acetylome-wide analysis in the aging process remains poorly studied in mammalian tissues. Nicotinamide adenine dinucleotide (NAD+), a hub metabolite, benefits health span at least in part due to the activation of Sirtuins, a family of NAD+-consuming deacetylases, indicating changes in acetylome. Here, we combine two antibodies for the enrichment of acetylated peptides and perform label-free quantitative acetylomic analysis of mouse livers during natural aging and upon the treatment of beta-nicotinamide mononucleotide (NMN), a NAD+ booster. Our study describes previously unknown acetylation sites and reveals the acetylome-wide dynamics with age as well as upon the treatment of NMN. We discover protein acetylation events as potential aging biomarkers. We demonstrate that the life-beneficial effect of NMN could be partially reflected by the changes in age-related protein acetylation. Our quantitative assessment indicates that NMN has mild effects on acetylation sites previously reported as substrates of Sirtuins. Collectively, our data analyze protein acetylation with age, laying critical foundation for the functional study of protein post-translational modification essential for healthy aging and perhaps disease conditions.

Quantitative Acetylomics Reveals Substrates of Lysine Acetyltransferase GCN5 in Adult and Aging Drosophila.

Protein lysine acetylation is a dynamic post-translational modification (PTM) that regulates a wide spectrum of cellular events including aging. General control nonderepressible 5 (GCN5) is a highly conserved lysine acetyltransferase (KAT). However, the acetylation substrates of GCN5 in vivo remain poorly studied, and moreover, how lysine acetylation changes with age and the contribution of KATs to aging remain to be addressed. Here, using Drosophila, we perform label-free quantitative acetylomic analysis, identifying new substrates of GCN5 in the adult and aging process. We further characterize the dynamics of protein acetylation with age, which exhibits a trend of increase. Since the expression of endogenous fly Gcn5 progressively increases during aging, we reason that, by combining the substrate analysis, the increase in acetylation with age is triggered, at least in part, by GCN5. Collectively, our study substantially expands the atlas of GCN5 substrates in vivo, provides a resource of protein acetylation that naturally occurs with age, and demonstrates how individual KAT contributes to the aging acetylome.

The mouse nicotinamide mononucleotide adenylyltransferase chaperones diverse pathological amyloid client proteins.

Molecular chaperones safeguard cellular protein homeostasis and obviate proteotoxicity. In the process of aging, as chaperone networks decline, aberrant protein amyloid aggregation accumulates in a mechanism that underpins neurodegeneration, leading to pathologies such as Alzheimer's disease and Parkinson's disease. Thus, it is important to identify and characterize chaperones for preventing such protein aggregation. In this work, we identified that the NAD+ synthase-nicotinamide mononucleotide adenylyltransferase (NMNAT) 3 from mouse (mN3) exhibits potent chaperone activity to antagonize aggregation of a wide spectrum of pathological amyloid client proteins including α-synuclein, Tau (K19), amyloid ß, and islet amyloid polypeptide. By combining NMR spectroscopy, cross-linking mass spectrometry, and computational modeling, we further reveal that mN3 uses different region of its amphiphilic surface near the active site to directly bind different amyloid client proteins. Our work demonstrates a client recognition mechanism of NMNAT via which it chaperones different amyloid client proteins against pathological aggregation and implies a potential protective role for NMNAT in different amyloid-associated diseases.

A Simple and Effective Physical Ball-Milling Strategy to Prepare Super-Tough and Stretchable PVA@MXene@PPy Hydrogel for Flexible Capacitive Electronics.

Biomimetic flexible electronics for E-skin have received increasing attention, due to their ability to sense various movements. However, the development of smart skin-mimic material remains a challenge. Here, a simple and effective approach is reported to fabricate super-tough, stretchable, and self-healing conductive hydrogel consisting of polyvinyl alcohol (PVA), Ti3 C2 Tx MXene nanosheets, and polypyrrole (PPy) (PMP hydrogel). The MXene nanosheets and Fe3+ serve as multifunctional cross-linkers and effective stress transfer centers, to facilitate a considerable high conductivity, super toughness, and ultra-high stretchability (elongation up to 4300%) for the PMP hydrogel with. The hydrogels also exhibit rapid self-healing and repeatable self-adhesive capacity because of the presence of dynamic borate ester bond. The flexible capacitive strain sensor made by PMP hydrogel shows a relatively broad range of strain sensing (up to 400%), with a self-healing feature. The sensor can precisely monitor various human physiological signals, including joint movements, facial expressions, and pulse waves. The PMP hydrogel-based supercapacitor is demonstrated with a high capacitance retention of ≈92.83% and a coulombic efficiency of ≈100%.

KANK4 Promotes Arteriogenesis by Potentiating VEGFR2 Signaling in a TALIN-1-Dependent Manner.

BACKGROUND: Arteriogenesis plays a critical role in maintaining adequate tissue blood supply and is related to a favorable prognosis in arterial occlusive diseases. Strategies aimed at promoting arteriogenesis have thus far not been successful because the factors involved in arteriogenesis remain incompletely understood. Previous studies suggest that evolutionarily conserved KANK4 (KN motif and ankyrin repeat domain-containing proteins 4) might involve in vertebrate vessel development. However, how the KANK4 regulates vessel function remains unknown. We aim to determine the role of endothelial cell-specifically expressed KANK4 in arteriogenesis. METHODS: The role of KANK4 in regulating arteriogenesis was evaluated using Kank4-/- and KANK4iECOE mice. Molecular mechanisms underlying KANK4-potentiated arteriogenesis were investigated by employing RNA transcriptomic profiling and mass spectrometry analysis. RESULTS: By analyzing Kank4-EGFP reporter mice, we showed that KANK4 was specifically expressed in endothelial cells. In particular, KANK4 displayed a dynamic expression pattern from being ubiquitously expressed in all endothelial cells of the developing vasculature to being explicitly expressed in the endothelial cells of arterioles and arteries in matured vessels. In vitro microfluidic chip-based vascular morphology analysis and in vivo hindlimb ischemia assays using Kank4-/- and KANK4iECOE mice demonstrated that deletion of KANK4 impaired collateral artery growth and the recovery of blood perfusion, whereas KANK4 overexpression leads to increased vessel caliber and blood perfusion. Bulk RNA sequencing and Co-immunoprecipitation/mass spectrometry (Co-IP/MS) analysis identified that KANK4 promoted EC proliferation and collateral artery remodeling through coupling VEGFR2 (vascular endothelial growth factor receptor 2) to TALIN-1, which augmented the activation of the VEGFR2 signaling cascade. CONCLUSIONS: This study reveals a novel role for KANK4 in arteriogenesis in response to ischemia. KANK4 links VEGFR2 to TALIN-1, resulting in enhanced VEGFR2 activation and increased EC proliferation, highlighting that KANK4 is a potential therapeutic target for promoting arteriogenesis for arterial occlusive diseases.

Carboxypeptidase Y Assisted Disulfide-Bond Identification with Linearized Database Search.

A disulfide bond is an important protein post-translational modification and plays a key role in regulating protein oxidation status, protein structure, and stability. Analysis of a disulfide bond using mass spectrometry is challenging because there lacks an efficient method to separate the disulfide-linked peptides from a complex protein digest, and the MS data requires sophisticated interpretation. Here, we developed a novel disulfide bond identification strategy, termed as "carboxypeptidase Y assisted disulfide-bond identification (CADI)". CADI is able to significantly reduce sample complexity by depleting ∼90% of the linear peptides while keeping the disulfide-bonded peptides. Furthermore, all CADI data can be directly analyzed by widely used protein database search engines, such as Mascot and MaxQuant. Our data show that CADI is able to sensitively identify disulfide bonds in peptides and proteins. However, CADI has not yet achieved a satisfied in-depth coverage on complex mammalian cell lysates due to the limited enzymatic activity of carboxypeptidase Y and low occurrences of disulfide bonds in a proteome. Altogether, CADI is a useful method that can get disulfide-linked peptides enriched and analyzed with regular search engines. CADI holds great potentials to deepen the analysis of disulfide bond and other types of cross-linked peptides on the proteome scale.

A Simple and Efficient Method to Generate Dual Site-Specific Conjugation ADCs with Cysteine Residue and an Unnatural Amino Acid.

Antibody-drug conjugates (ADCs) are complex pharmaceutical molecules that combine monoclonal antibodies with biologically active drugs through chemical linkers. ADCs are designed to specifically kill disease cells by utilizing the target specificity of antibodies and the cytotoxicity of chemical drugs. However, the traditional ADCs were only applied to a few disease targets because of some limitations such as the huge molecular weight, the uncontrollable coupling reactions, and a single mechanism of action. Here we report a simple, one-pot, successive reaction method to produce dual payload conjugates with the site-specifically engineered cysteine and p-acetyl-phenylalanine using Herceptin (trastuzumab), an anti-HER2 antibody drug widely used for breast cancer treatment, as a tool molecule. This strategy enables antibodies to conjugate with two mechanistically distinct cytotoxic drugs through different functional groups sequentially, therefore, rendering the newly designed ADCs with functional diversity and the potential to overcome drug resistance and enhance the therapeutic efficacy.

Multidimensional Proteomics Identifies Declines in Protein Homeostasis and Mitochondria as Early Signals for Normal Aging and Age-associated Disease in Drosophila.

Aging is characterized by a gradual deterioration in proteome. However, how protein dynamics that changes with normal aging and in disease is less well understood. Here, we profiled the snapshots of aging proteome in Drosophila, from head and muscle tissues of post-mitotic somatic cells, and the testis of mitotically-active cells. Our data demonstrated that dysregulation of proteome homeostasis, or proteostasis, might be a common feature associated with age. We further used pulsed metabolic stable isotope labeling analysis to characterize protein synthesis. Interestingly, this study determined an age-modulated decline in protein synthesis with age, particularly in the pathways related to mitochondria, neurotransmission, and proteostasis. Importantly, this decline became dramatically accelerated in Pink1 mutants, a Drosophila model of human age-related Parkinson's disease. Taken together, our multidimensional proteomic study revealed tissue-specific protein dynamics with age, highlighting mitochondrial and proteostasis-related proteins. We suggest that declines in proteostasis and mitochondria early in life are critical signals prior to the onset of aging and aging-associated diseases.

Integrated Proteomic and N-Glycoproteomic Analyses of Human Breast Cancer.

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 Reshapes Acetylomes in Inflammatory and Cancer Cells via CoA-Dependent and CoA-Independent Pathways.

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.

Differential Sensitivity of Target Genes to Translational Repression by miR-17~92.

MicroRNAs (miRNAs) are thought to exert their functions by modulating the expression of hundreds of target genes and each to a small degree, but it remains unclear how small changes in hundreds of target genes are translated into the specific function of a miRNA. Here, we conducted an integrated analysis of transcriptome and translatome of primary B cells from mutant mice expressing miR-17~92 at three different levels to address this issue. We found that target genes exhibit differential sensitivity to miRNA suppression and that only a small fraction of target genes are actually suppressed by a given concentration of miRNA under physiological conditions. Transgenic expression and deletion of the same miRNA gene regulate largely distinct sets of target genes. miR-17~92 controls target gene expression mainly through translational repression and 5'UTR plays an important role in regulating target gene sensitivity to miRNA suppression. These findings provide molecular insights into a model in which miRNAs exert their specific functions through a small number of key target genes.

Multi-omic Analyses Reveal Minimal Impact of the CRISPR-Cas9 Nuclease on Cultured Human Cells.

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.

Antagonistic roles of Nibbler and Hen1 in modulating piRNA 3' ends in Drosophila.

In eukaryotes, aberrant expression of transposable elements (TEs) is detrimental to the host genome. Piwi-interacting RNAs (piRNAs) of ∼23 to 30 nucleotides bound to PIWI clade Argonaute proteins silence transposons in a manner that is strictly dependent on their sequence complementarity. Hence, a key goal in understanding piRNA pathways is to determine mechanisms that modulate piRNA sequences. Here, we identify a protein-protein interaction between the 3'-to-5' exoribonuclease Nibbler (Nbr) and Piwi that links Nbr activity with piRNA pathways. We show that there is a delicate balance in the interplay between Nbr and Hen1, a methyltransferase involved in 2'-O-methylation at the 3' terminal nucleotides of piRNAs, thus connecting two genes with opposing activities in the biogenesis of piRNA 3' ends. With age, piRNAs become shorter and fewer in number, which is coupled with the derepression of select TEs. We demonstrate that activities of Nbr and Hen1 inherently contribute to TE silencing and age-dependent profiles of piRNAs. We propose that antagonistic roles of Nbr and Hen1 define a mechanism to modulate piRNA 3' ends.