Long-lived proteins and DNA as candidate predictive biomarkers for tissue associated diseases.

Protein turnover is an important mechanism to maintain proteostasis. Long-lived proteins (LLPs) are vulnerable to lose their function due to time-accumulated damages. In this study we employed in vivo stable isotope labeling in mice from birth to postnatal day 89. Quantitative proteomics analysis of ten tissues and plasma identified 2113 LLPs, including widespread and tissue-specific ones. Interestingly, a significant percentage of LLPs was detected in plasma, implying a potential link to age-related cardiovascular diseases. LLPs identified in brains were related to neurodegenerative diseases. In addition, the relative quantification of DNA-derived deoxynucleosides from the same tissues provided information about cellular DNA renewal and showed good correlation with LLPs in the brain. The combined data reveal tissue-specific maps of mouse LLPs that may be involved in pathology due to a low renewal rate and an increased risk of damage. Tissue-derived peripheral LLPs hold promise as biomarkers for aging and age-related diseases.

N-acetylation of α-synuclein enhances synaptic vesicle clustering mediated by α-synuclein and lysophosphatidylcholine.

Post-translational modifications (PTMs) of α-synuclein (α-syn) such as acetylation and phosphorylation play important yet distinct roles in regulating α-syn conformation, membrane binding, and amyloid aggregation. However, how PTMs regulate α-syn function in presynaptic terminals remains unclear. Previously, we reported that α-syn clusters synaptic vesicles (SV) 1, and neutral phospholipid lysophosphatidylcholine (LPC) can mediate this clustering 2. Here, based on our previous findings, we further demonstrate that N-terminal acetylation, which occurs under physiological condition and is irreversible in mammalian cells, significantly enhances the functional activity of α-syn in clustering SVs. Mechanistic studies reveal that this enhancement is caused by the N-acetylation-promoted insertion of α-syn's N-terminus and increased intermolecular interactions on the LPC-containing membrane. Our work demonstrates that N-acetylation fine-tunes α-syn-LPC interaction for mediating α-syn's function in SV clustering.

Reduction of DHHC5-mediated beclin 1 S-palmitoylation underlies autophagy decline in aging.

Autophagy is a lysosome-dependent degradation pathway essential for cellular homeostasis, which decreases with age. However, it is unclear how aging induces autophagy decline. Here we show the role of protein S-palmitoylation in autophagy. We identify the palmitoyl acyltransferase DHHC5 as a regulator of autophagy by mediating the palmitoylation of beclin 1, which in turn promotes the formation of ATG14L-containing class III phosphatidylinositol-3-kinase complex I and its lipid kinase activity by promoting the hydrophobic interactions between beclin 1 and adapter proteins ATG14L and VPS15. In aging brains of human and nonhuman primate, the levels of DHHC5 exhibit a marked decrease in expression. We show that DHHC5 deficiency in neurons leads to reduced cellular protein homeostasis in two established murine models of Alzheimer's disease, which exaggerates neurodegeneration in an autophagy-dependent manner. These findings identify reduction of DHHC5-mediated beclin 1 S-palmitoylation as an underlying mechanism by which aging induces autophagy decline.

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.

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%.

Creatine kinase brain-type regulates BCAR1 phosphorylation to facilitate DNA damage repair.

Creatine kinase (CK) is an essential metabolic enzyme mediating creatine/phosphocreatine interconversion and shuttle to replenish ATP for energy needs. Ablation of CK causes a deficiency in energy supply that eventually results in reduced muscle burst activity and neurological disorders in mice. Besides the well-established role of CK in energy-buffering, the mechanism underlying the non-metabolic function of CK is poorly understood. Here we demonstrate that creatine kinase brain-type (CKB) may function as a protein kinase to regulate BCAR1 Y327 phosphorylation that enhances the association between BCAR1 and RBBP4. Then the complex of BCAR1 and RPPB4 binds to the promoter region of DNA damage repair gene RAD51 and activates its transcription by modulating histone H4K16 acetylation to ultimately promote DNA damage repair. These findings reveal the possible role of CKB independently of its metabolic function and depict the potential pathway of CKB-BCAR1-RBBP4 operating in DNA damage repair.

Proximity labeling reveals OTUD3 as a DNA-binding deubiquitinase of cGAS.

Cyclic GMP-AMP synthase (cGAS), as the major DNA sensor, initiates DNA-stimulated innate immune responses and is essential for a healthy immune system. Although some regulators of cGAS have been reported, it still remains largely unclear how cGAS is precisely and dynamically regulated and how many potential regulators govern cGAS. Here we carry out proximity labeling of cGAS with TurboID in cells and identify a number of potential cGAS-interacting or -adjacent proteins. Deubiquitinase OTUD3, one candidate identified in cytosolic cGAS-DNA complex, is further validated to not only stabilize cGAS but also enhance cGAS enzymatic activity, which eventually promotes anti-DNA virus immune response. We show that OTUD3 can directly bind DNA and is recruited to the cytosolic DNA complex, increasing its association with cGAS. Our findings reveal OTUD3 as a versatile cGAS regulator and find one more layer of regulatory mechanism in DNA-stimulated innate immune responses.

Site-specific labeling of antibodies with quantum dots could promote to retain the antigen binding capacity of antibodies.

A major concern with antibody labeling is the decreased antigen affinity binding capacity of antibodies, owing mainly to the randomly oriented binding of the marker. Herein, a universal approach for site-specific photocrosslinking of quantum dots (QDs) to the Fc-terminal of antibodies was investigated utilizing antibody Fc-terminal affinity proteins. Results showed that the QDs only bound to the heavy chain of the antibody. Further comparative tests confirmed that the site-specific directed labeling approach maximizes the retention of the antigen-binding capacity of the natural antibody. Compared with the commonly employed random orientation labeling approach, the directional labeling approach allows the labeled antibody showed 6 times greater binding affinity to antigen. QDs-labeled monoclonal antibodies were applied to fluorescent immunochromatographic test strips for the detection of shrimp tropomyosin (TM). The established procedure has a detection limit of 0.054 µg/mL. Thus, the site-specific labeling approach significantly improves the antigen binding capacity of the labeled antibody.

Benchmarking commonly used software suites and analysis workflows for DIA proteomics and phosphoproteomics.

A plethora of software suites and multiple classes of spectral libraries have been developed to enhance the depth and robustness of data-independent acquisition (DIA) data processing. However, how the combination of a DIA software tool and a spectral library impacts the outcome of DIA proteomics and phosphoproteomics data analysis has been rarely investigated using benchmark data that mimics biological complexity. In this study, we create DIA benchmark data sets simulating the regulation of thousands of proteins in a complex background, which are collected on both an Orbitrap and a timsTOF instruments. We evaluate four commonly used software suites (DIA-NN, Spectronaut, MaxDIA and Skyline) combined with seven different spectral libraries in global proteome analysis. Moreover, we assess their performances in analyzing phosphopeptide standards and TNF-α-induced phosphoproteome regulation. Our study provides a practical guidance on how to construct a robust data analysis pipeline for different proteomics studies implementing the DIA technique.

Targeting soluble epoxide hydrolase promotes osteogenic-angiogenic coupling via activating SLIT3/HIF-1α signalling pathway.

Type H vessels have recently been identified to modulate osteogenesis. Epoxyeicostrioleic acids (EETs) have an essential contribution to vascular homeostasis. However, whether increased EETs with soluble epoxide hydrolase (sEH) inhibitor TPPU enhance the coupling of angiogenesis and osteogenesis remains largely unknown. The effects of TPPU on cross-talk between co-cultured human umbilical vein endothelial cells (HUVECs) and human dental pulp stem cells (hDPSCs), and on long bone growth and calvarial defect repair in mice were investigated in vitro and in vivo. TPPU enhanced osteogenic differentiation of co-cultured HUVECs and hDPSCs in vitro and increased type H vessels, and long bone growth and bone repair of calvarial defect. Mechanistically, TPPU promoted cell proliferation and angiogenesis, reclined cell apoptosis, and significantly increased CD31hi EMCNhi endothelial cells (ECs) and SLIT3 and HIF-1α expression levels in co-cultured HUVECs and hDPSCs. Knockdown of Slit3 in hDPSCs or Hif-1α in HUVECs impaired the formation of CD31hi EMCNhi ECs and reversed TPPU-induced osteogenesis. We defined a previously unidentified effect of TPPU coupling angiogenesis and osteogenesis. TPPU induced type H vessels by upregulating the expression of hDPSCs-derived SLIT3, which resulted in the activation of ROBO1/YAP1/HIF-1α signalling pathway in ECs. Targeting metabolic pathways of EETs represents a new strategy to couple osteogenesis and angiogenesis, sEH is a promising therapeutic target for bone regeneration and repair.

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.

Specific binding of Hsp27 and phosphorylated Tau mitigates abnormal Tau aggregation-induced pathology.

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.

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.

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.

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.

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.

SecMS analysis of selenoproteins with selenocysteine insertion sequence and beyond.

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.

Protocol for identification and validation of 2'3'-cGAMP-binding proteins by 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. For complete details on the use and execution of this profile, please refer to Hou et al. (2021).

microRNA-252 and FoxO repress inflammaging by a dual inhibitory mechanism on Dawdle-mediated TGF-ß pathway in Drosophila.

Inflammaging refers to low-grade, chronically activated innate immunity that has deleterious effects on healthy lifespan. However, little is known about the intrinsic signaling pathway that elicits innate immune genes during aging. Here, using Drosophila melanogaster, we profile the microRNA targetomes in young and aged animals, and reveal Dawdle, an activin-like ligand of the TGF-ß pathway, as a physiological target of microRNA-252. We show that microRNA-252 cooperates with Forkhead box O, a conserved transcriptional factor implicated in aging, to repress Dawdle. Unopposed Dawdle triggers hyperactivation of innate immune genes coupled with a decline in organismal survival. Using adult muscle tissues, single-cell sequencing analysis describes that Dawdle and its downstream innate immune genes are expressed in distinct cell types, suggesting a cell nonautonomous mode of regulation. We further determine the genetic cascade by which Dawdle signaling leads to increased Kenny/IKKγ protein, which in turn activates Relish/NF-κB protein and consequentially innate immune genes. Finally, transgenic increase of microRNA-252 and Forkhead box O pathway factors in wild-type Drosophila extends lifespan and mitigates the induction of innate immune genes in aging. Together, we propose that microRNA-252 and Forkhead box O promote healthy longevity by cooperative inhibition on Dawdle-mediated inflammaging.