Anatomy and Neuroscience - Research Publications

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    Glycoproteomics
    Bagdonaite, I ; Malaker, SA ; Polasky, DA ; Riley, NM ; Schjoldager, K ; Vakhrushev, SY ; Halim, A ; Aoki-Kinoshita, KF ; Nesvizhskii, AI ; Bertozzi, CR ; Wandall, HH ; Parker, BL ; Thaysen-Andersen, M ; Scott, NE (SPRINGERNATURE, 2022-06-23)
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    Phosphoproteomics of three exercise modalities identifies canonical signaling and C18ORF25 as anAMPK substrate regulating skeletal muscle function
    Blazev, R ; Carl, CS ; Ng, Y-K ; Molendijk, J ; Voldstedlund, CT ; Zhao, Y ; Xiao, D ; Kueh, AJ ; Miotto, PM ; Haynes, VR ; Hardee, JP ; Chung, JD ; McNamara, JW ; Qian, H ; Gregorevic, P ; Oakhill, JS ; Herold, MJ ; Jensen, TE ; Lisowski, L ; Lynch, GS ; Dodd, GT ; Watt, MJ ; Yang, P ; Kiens, B ; Richter, EA ; Parker, BL (CELL PRESS, 2022-10-04)
    Exercise induces signaling networks to improve muscle function and confer health benefits. To identify divergent and common signaling networks during and after different exercise modalities, we performed a phosphoproteomic analysis of human skeletal muscle from a cross-over intervention of endurance, sprint, and resistance exercise. This identified 5,486 phosphosites regulated during or after at least one type of exercise modality and only 420 core phosphosites common to all exercise. One of these core phosphosites was S67 on the uncharacterized protein C18ORF25, which we validated as an AMPK substrate. Mice lacking C18ORF25 have reduced skeletal muscle fiber size, exercise capacity, and muscle contractile function, and this was associated with reduced phosphorylation of contractile and Ca2+ handling proteins. Expression of C18ORF25 S66/67D phospho-mimetic reversed the decreased muscle force production. This work defines the divergent and canonical exercise phosphoproteome across different modalities and identifies C18ORF25 as a regulator of exercise signaling and muscle function.
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    Proteome-wide systems genetics identifies UFMylation as a regulator of skeletal muscle function
    Molendijk, J ; Blazev, R ; Mills, RJ ; Ng, Y-K ; Watt, K ; Chau, D ; Gregorevic, P ; Crouch, PJ ; Hilton, JBW ; Lisowski, L ; Zhang, P ; Reue, K ; Lusis, AJ ; Hudson, JE ; James, DE ; Seldin, MM ; Parker, BL (eLIFE SCIENCES PUBL LTD, 2022-12-06)
    Improving muscle function has great potential to improve the quality of life. To identify novel regulators of skeletal muscle metabolism and function, we performed a proteomic analysis of gastrocnemius muscle from 73 genetically distinct inbred mouse strains, and integrated the data with previously acquired genomics and >300 molecular/phenotypic traits via quantitative trait loci mapping and correlation network analysis. These data identified thousands of associations between protein abundance and phenotypes and can be accessed online (https://muscle.coffeeprot.com/) to identify regulators of muscle function. We used this resource to prioritize targets for a functional genomic screen in human bioengineered skeletal muscle. This identified several negative regulators of muscle function including UFC1, an E2 ligase for protein UFMylation. We show UFMylation is up-regulated in a mouse model of amyotrophic lateral sclerosis, a disease that involves muscle atrophy. Furthermore, in vivo knockdown of UFMylation increased contraction force, implicating its role as a negative regulator of skeletal muscle function.
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    Temporal profiling of the breast tumour microenvironment reveals collagen XII as a driver of metastasis
    Papanicolaou, M ; Parker, AL ; Yam, M ; Filipe, EC ; Wu, SZ ; Chitty, JL ; Wyllie, K ; Tran, E ; Mok, E ; Nadalini, A ; Skhinas, JN ; Lucas, MC ; Herrmann, D ; Nobis, M ; Pereira, BA ; Law, AMK ; Castillo, L ; Murphy, KJ ; Zaratzian, A ; Hastings, JF ; Croucher, DR ; Lim, E ; Oliver, BG ; Mora, FV ; Parker, BL ; Gallego-Ortega, D ; Swarbrick, A ; O'Toole, S ; Timpson, P ; Cox, TR (NATURE PORTFOLIO, 2022-08-06)
    The tumour stroma, and in particular the extracellular matrix (ECM), is a salient feature of solid tumours that plays a crucial role in shaping their progression. Many desmoplastic tumours including breast cancer involve the significant accumulation of type I collagen. However, recently it has become clear that the precise distribution and organisation of matrix molecules such as collagen I is equally as important in the tumour as their abundance. Cancer-associated fibroblasts (CAFs) coexist within breast cancer tissues and play both pro- and anti-tumourigenic roles through remodelling the ECM. Here, using temporal proteomic profiling of decellularized tumours, we interrogate the evolving matrisome during breast cancer progression. We identify 4 key matrisomal clusters, and pinpoint collagen type XII as a critical component that regulates collagen type I organisation. Through combining our proteomics with single-cell transcriptomics, and genetic manipulation models, we show how CAF-secreted collagen XII alters collagen I organisation to create a pro-invasive microenvironment supporting metastatic dissemination. Finally, we show in patient cohorts that collagen XII may represent an indicator of breast cancer patients at high risk of metastatic relapse.
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    SILAC kinase screen identifies potential MASTL substrates
    Marzec, KA ; Rogers, S ; McCloy, R ; Parker, BL ; James, DE ; Watkins, DN ; Burgess, A (NATURE PORTFOLIO, 2022-06-22)
    Microtubule-associated serine/threonine kinase-like (MASTL) has emerged as a critical regulator of mitosis and as a potential oncogene in a variety of cancer types. To date, Arpp-19/ENSA are the only known substrates of MASTL. However, with the roles of MASTL expanding and increased interest in development of MASTL inhibitors, it has become critical to determine if there are additional substrates and what the optimal consensus motif for MASTL is. Here we utilized a whole cell lysate in vitro kinase screen combined with stable isotope labelling of amino acids in cell culture (SILAC) to identify potential substrates and the residue preference of MASTL. Using the related AGC kinase family members AKT1/2, the kinase screen identified several known and new substrates highly enriched for the validated consensus motif of AKT. Applying this method to MASTL identified 59 phospho-sites on 67 proteins that increased in the presence of active MASTL. Subsequent in vitro kinase assays suggested that MASTL may phosphorylate hnRNPM, YB1 and TUBA1C under certain in vitro conditions. Taken together, these data suggest that MASTL may phosphorylate several additional substrates, providing insight into the ever-increasing biological functions and roles MASTL plays in driving cancer progression and therapy resistance.
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    Trafficking regulator of GLUT4-1 (TRARG1) is a GSK3 substrate
    Duan, X ; Norris, DM ; Humphrey, SJ ; Yang, P ; Cooke, KC ; Bultitude, WP ; Parker, BL ; Conway, OJ ; Burchfield, JG ; Krycer, JR ; Brodsky, FM ; James, DE ; Fazakerley, DJ (PORTLAND PRESS LTD, 2022-06)
    Trafficking regulator of GLUT4-1, TRARG1, positively regulates insulin-stimulated GLUT4 trafficking and insulin sensitivity. However, the mechanism(s) by which this occurs remain(s) unclear. Using biochemical and mass spectrometry analyses we found that TRARG1 is dephosphorylated in response to insulin in a PI3K/Akt-dependent manner and is a novel substrate for GSK3. Priming phosphorylation of murine TRARG1 at serine 84 allows for GSK3-directed phosphorylation at serines 72, 76 and 80. A similar pattern of phosphorylation was observed in human TRARG1, suggesting that our findings are translatable to human TRARG1. Pharmacological inhibition of GSK3 increased cell surface GLUT4 in cells stimulated with a submaximal insulin dose, and this was impaired following Trarg1 knockdown, suggesting that TRARG1 acts as a GSK3-mediated regulator in GLUT4 trafficking. These data place TRARG1 within the insulin signaling network and provide insights into how GSK3 regulates GLUT4 trafficking in adipocytes.
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    Time-resolved phosphoproteome and proteome analysis reveals kinase signaling on master transcription factors during myogenesis
    Xiao, D ; Caldow, M ; Kim, HJ ; Blazev, R ; Koopman, R ; Manandi, D ; Parker, BL ; Yang, P (CELL PRESS, 2022-06-17)
    Myogenesis is governed by signaling networks that are tightly regulated in a time-dependent manner. Although different protein kinases have been identified, knowledge of the global signaling networks and their downstream substrates during myogenesis remains incomplete. Here, we map the myogenic differentiation of C2C12 cells using phosphoproteomics and proteomics. From these data, we infer global kinase activity and predict the substrates that are involved in myogenesis. We found that multiple mitogen-activated protein kinases (MAPKs) mark the initial wave of signaling cascades. Further phosphoproteomic and proteomic profiling with MAPK1/3 and MAPK8/9 specific inhibitions unveil their shared and distinctive roles in myogenesis. Lastly, we identified and validated the transcription factor nuclear factor 1 X-type (NFIX) as a novel MAPK1/3 substrate and demonstrated the functional impact of NFIX phosphorylation on myogenesis. Altogether, these data characterize the dynamics, interactions, and downstream control of kinase signaling networks during myogenesis on a global scale.
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    Mechanism of Bloom syndrome complex assembly required for double Holliday junction dissolution and genome stability
    Hodson, C ; Low, JKK ; van Twest, S ; Jones, SE ; Swuec, P ; Murphy, V ; Tsukada, K ; Fawkes, M ; Bythell-Douglas, R ; Davies, A ; Holien, JK ; O'Rourke, JJ ; Parker, BL ; Glaser, A ; Parker, MW ; Mackay, JP ; Blackford, AN ; Costa, A ; Deans, AJ (NATL ACAD SCIENCES, 2022-02-08)
    The RecQ-like helicase BLM cooperates with topoisomerase IIIα, RMI1, and RMI2 in a heterotetrameric complex (the "Bloom syndrome complex") for dissolution of double Holliday junctions, key intermediates in homologous recombination. Mutations in any component of the Bloom syndrome complex can cause genome instability and a highly cancer-prone disorder called Bloom syndrome. Some heterozygous carriers are also predisposed to breast cancer. To understand how the activities of BLM helicase and topoisomerase IIIα are coupled, we purified the active four-subunit complex. Chemical cross-linking and mass spectrometry revealed a unique architecture that links the helicase and topoisomerase domains. Using biochemical experiments, we demonstrated dimerization mediated by the N terminus of BLM with a 2:2:2:2 stoichiometry within the Bloom syndrome complex. We identified mutations that independently abrogate dimerization or association of BLM with RMI1, and we show that both are dysfunctional for dissolution using in vitro assays and cause genome instability and synthetic lethal interactions with GEN1/MUS81 in cells. Truncated BLM can also inhibit the activity of full-length BLM in mixed dimers, suggesting a putative mechanism of dominant-negative action in carriers of BLM truncation alleles. Our results identify critical molecular determinants of Bloom syndrome complex assembly required for double Holliday junction dissolution and maintenance of genome stability.
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    Community evaluation of glycoproteomics informatics solutions reveals high-performance search strategies for serum glycopeptide analysis (vol 18, pg 1304, 2021)
    Kawahara, R ; Chernykh, A ; Alagesan, K ; Bern, M ; Cao, W ; Chalkley, RJ ; Cheng, K ; Choo, MS ; Edwards, N ; Goldman, R ; Hoffmann, M ; Hu, Y ; Huang, Y ; Kim, JY ; Kletter, D ; Liquet, B ; Liu, M ; Mechref, Y ; Meng, B ; Neelamegham, S ; Nguyen-Khuong, T ; Nilsson, J ; Pap, A ; Park, GW ; Parker, BL ; Pegg, CL ; Penninger, JM ; Phung, TK ; Pioch, M ; Rapp, E ; Sakalli, E ; Sanda, M ; Schulz, BL ; Scott, NE ; Sofronov, G ; Stadlmann, J ; Vakhrushev, SY ; Woo, CM ; Wu, H-Y ; Yang, P ; Ying, W ; Zhang, H ; Zhang, Y ; Zhao, J ; Zaia, J ; Haslam, SM ; Palmisano, G ; Yoo, JS ; Larson, G ; Khoo, K-H ; Medzihradszky, KF ; Kolarich, D ; Packer, NH ; Thaysen-Andersen, M (NATURE PORTFOLIO, 2022-01)
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    Dynamic Changes to the Skeletal Muscle Proteome and Ubiquitinome Induced by the E3 Ligase, ASB2β (vol 20, 10050, 2021)
    Goodman, CAA ; Davey, JRR ; Hagg, A ; Parker, BLL ; Gregorevic, P (ELSEVIER, 2022-02)