Loss of proteostasis is a hallmark of aging that underlies many age-related diseases. Different cell compartments experience distinctive challenges in maintaining protein quality control, but how aging regulates subcellular proteostasis remains underexplored. Here, by targeting the misfolding-prone Fluc luciferase to the cytoplasm, mitochondria, and nucleus, we established transgenic sensors to examine subcellular proteostasis in Drosophila.
View Article and Find Full Text PDFDisruption of the circadian clock in skeletal muscle worsens local and systemic health, leading to decreased muscle strength, metabolic dysfunction, and aging-like phenotypes. Whole-body knockout mice that lack Bmal1, a key component of the molecular clock, display premature aging. Here, by using adeno-associated viruses, we rescued Bmal1 expression specifically in the skeletal muscle fibers of Bmal1-KO mice and found that this engaged the circadian clock and clock output gene expression, contributing to extended lifespan.
View Article and Find Full Text PDFUbiquitin controls many cellular processes via its posttranslational conjugation onto substrates. Its use is highly variable due to its ability to form poly-ubiquitin chains with various topologies. Among them, linear chains have emerged as important regulators of immune responses and protein degradation.
View Article and Find Full Text PDFUbiquitin controls many cellular processes via its post-translational conjugation onto substrates. Its use is highly variable due to its ability to form poly-ubiquitin with various topologies. Among them, linear chains have emerged as important regulators of immune responses and protein degradation.
View Article and Find Full Text PDFUbiquitin-conjugating enzymes (E2s) are key for regulating protein function and turnover via ubiquitination but it remains undetermined which E2s maintain proteostasis during aging. Here, we find that E2s have diverse roles in handling a model aggregation-prone protein (huntingtin-polyQ) in the retina: while some E2s mediate aggregate assembly, UBE2D/effete (eff) and other E2s are required for huntingtin-polyQ degradation. UBE2D/eff is key for proteostasis also in skeletal muscle: eff protein levels decline with aging, and muscle-specific eff knockdown causes an accelerated buildup in insoluble poly-ubiquitinated proteins (which progressively accumulate with aging) and shortens lifespan.
View Article and Find Full Text PDFUbiquitination is a post-translational modification initiated by the E1 enzyme UBA1, which transfers ubiquitin to ~35 E2 ubiquitin-conjugating enzymes. While UBA1 loss is cell lethal, it remains unknown how partial reduction in UBA1 activity is endured. Here, we utilize deep-coverage mass spectrometry to define the E1-E2 interactome and to determine the proteins that are modulated by knockdown of UBA1 and of each E2 in human cells.
View Article and Find Full Text PDF53BP1 is a well-established DNA damage repair factor recently shown to regulate gene expression and critically influence tumor suppression and neural development. For gene regulation, how 53BP1 is regulated remains unclear. Here, we showed that 53BP1-serine 25 phosphorylation by ATM is required for neural progenitor cell proliferation and neuronal differentiation in cortical organoids.
View Article and Find Full Text PDFSkeletal muscle atrophy is a debilitating condition that occurs with aging and disease, but the underlying mechanisms are incompletely understood. Previous work determined that common transcriptional changes occur in muscle during atrophy induced by different stimuli. However, whether this holds true at the proteome level remains largely unexplored.
View Article and Find Full Text PDFRecent evidence indicates that the composition of the ribosome is heterogeneous and that multiple types of specialized ribosomes regulate the synthesis of specific protein subsets. In Drosophila, we find that expression of the ribosomal RpS28 protein variants RpS28a and RpS28-like preferentially occurs in the germline, a tissue resistant to aging and that it significantly declines in skeletal muscle during aging. Muscle-specific overexpression of RpS28a at levels similar to those seen in the germline decreases early mortality and promotes the synthesis of a subset of proteins with known anti-aging roles, some of which have preferential expression in the germline.
View Article and Find Full Text PDFUSP7, which encodes a deubiquitylating enzyme, is among the most frequently mutated genes in pediatric T-ALL, with somatic heterozygous loss-of-function mutations (haploinsufficiency) predominantly affecting the subgroup that has aberrant TAL1 oncogene activation. Network analysis of > 200 T-ALL transcriptomes linked USP7 haploinsufficiency with decreased activities of E-proteins. E-proteins are also negatively regulated by TAL1, leading to concerted down-regulation of E-protein target genes involved in T-cell development.
View Article and Find Full Text PDFSarcopenia is a degenerative condition that consists in age-induced atrophy and functional decline of skeletal muscle cells (myofibers). A common hypothesis is that inducing myofiber hypertrophy should also reinstate myofiber contractile function but such model has not been extensively tested. Here, we find that the levels of the ubiquitin ligase UBR4 increase in skeletal muscle with aging, and that UBR4 increases the proteolytic activity of the proteasome.
View Article and Find Full Text PDFTandem mass tag (TMT)-based mass spectrometry (MS) enables deep proteomic profiling of more than 10,000 proteins in complex biological samples but requires up to 100 μg protein in starting materials during a standard analysis. Here, we present a streamlined protocol to quantify more than 9000 proteins with 0.5 μg protein per sample by 16-plex TMT coupled with two-dimensional liquid chromatography and tandem mass spectrometry (LC/LC-MS/MS).
View Article and Find Full Text PDFIsobaric tandem mass tag (TMT) labeling is widely used in proteomics because of its high multiplexing capacity and deep proteome coverage. Recently, an expanded 16-plex TMT method has been introduced, which further increases the throughput of proteomic studies. In this manuscript, we present an optimized protocol for 16-plex TMT-based deep-proteome profiling, including protein sample preparation, enzymatic digestion, TMT labeling reaction, two-dimensional reverse-phase liquid chromatography (LC/LC) fractionation, tandem mass spectrometry (MS/MS), and computational data processing.
View Article and Find Full Text PDFChromatin modifiers affect spatiotemporal gene expression programs that underlie organismal development. The Polycomb repressive complex 2 (PRC2) is a crucial chromatin modifier in executing neurodevelopmental programs. Here, we find that PRC2 interacts with the nucleic acid-binding protein Ybx1.
View Article and Find Full Text PDFAlzheimer's disease (AD) displays a long asymptomatic stage before dementia. We characterize AD stage-associated molecular networks by profiling 14,513 proteins and 34,173 phosphosites in the human brain with mass spectrometry, highlighting 173 protein changes in 17 pathways. The altered proteins are validated in two independent cohorts, showing partial RNA dependency.
View Article and Find Full Text PDFHigh throughput omics approaches provide an unprecedented opportunity for dissecting molecular mechanisms in cancer biology. Here we present deep profiling of whole proteome, phosphoproteome and transcriptome in two high-grade glioma (HGG) mouse models driven by mutated RTK oncogenes, PDGFRA and NTRK1, analyzing 13,860 proteins and 30,431 phosphosites by mass spectrometry. Systems biology approaches identify numerous master regulators, including 41 kinases and 23 transcription factors.
View Article and Find Full Text PDFSkeletal muscle cell (myofiber) atrophy is a detrimental component of aging and cancer that primarily results from muscle protein degradation via the proteasome and ubiquitin ligases. Transcriptional upregulation of some ubiquitin ligases contributes to myofiber atrophy, but little is known about the role that most other ubiquitin ligases play in this process. To address this question, we have used RNAi screening in Drosophila to identify the function of > 320 evolutionarily conserved ubiquitin ligases in myofiber size regulation in vivo.
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