Medical Biology - Research Publications

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    Defining the molecular architecture, metal dependence, and distribution of metal-dependent class II sulfofructose-1-phosphate aldolases
    Sharma, M ; Kaur, A ; Madiedo Soler, N ; Lingford, J ; Epa, R ; Goddard-Borger, E ; Davies, G ; Williams, S ( 2023-08-08)
    Sulfoquinovose (SQ or 6-deoxy-6-sulfoglucose) is a sulfosugar that is the anionic head group of plant and cyanobacterial sulfolipids: sulfoquinovosyl diacylglycerols. SQ is produced within photosynthetic tissues, forms a major terrestrial reservoir of biosulfur, and is an important species within the biogeochemical sulfur cycle. A major pathway for the breakdown of SQ is the sulfoglycolytic Embden-Meyerhof-Parnas (sulfo-EMP) pathway, which involves cleavage of the 6-carbon chain of the intermediate sulfofructose-1-phosphate (SFP) into dihydroxyacetone and sulfolactaldehyde, catalyzed by class I or II SFP aldolases. While the molecular basis of catalysis is well studied for class I SFP aldolases, comparatively little is known about class II SFP aldolases. Here, we report the molecular architecture and biochemical basis of catalysis of two metal-dependent class II SFP aldolases from Hafnia paralvei and Yersinia aldovae. 3D X-ray structures in complex with the substrate SFP and product DHAP reveal a dimer-of-dimers (tetrameric) assembly, and identify the sulfonate binding pocket that defines the substrate specificity of these enzymes, two metal binding sites, and flexible loops that are implicated in catalysis. Both enzymes were metal dependent and exhibited high KM values for SFP, consistent with their role in a unidirectional nutrient acquisition pathway. Bioinformatic analysis identified a range of sulfo-EMP gene clusters containing class I/II SFP aldolases. The class I and II SFP aldolases occur exclusively within Actinobacteria and Firmicutes phyla, respectively, while both classes of enzyme occur within Proteobacteria. This work emphasizes the importance of SQ as a nutrient for diverse bacterial phyla and the different chemical strategies they use to harvest carbon from this sulfosugar.
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    Trabid patient mutations impede the axonal trafficking of adenomatous polyposis coli to disrupt neurite growth
    Frank, D ; Bergamasco, M ; Mlodzianoski, M ; Kueh, A ; Tsui, E ; Hall, C ; Kastrappis, G ; Voss, AK ; McLean, C ; Faux, M ; Rogers, K ; Tran, B ; Vincan, E ; Komander, D ; Dewson, G ; Tran, H ( 2023-07-19)
    Abstract Trabid/ZRANB1missense mutations have been identified in children diagnosed with a range of congenital disorders including reduced brain size, but how Trabid regulates neurodevelopment is not understood. We have characterised these patient mutations in cells and mice to identify a key role for Trabid in the regulation of neurite growth. One of the patient mutations flanked the catalytic cysteine of Trabid and its deubiquitylating (DUB) activity was abrogated. The second variant retained DUB activity, but failed to bind STRIPAK, a large multiprotein assembly implicated in cytoskeleton organisation and neural development.Trabid/ZRANB1knock-in mice harbouring either of these patient mutations exhibited reduced neuronal and glial cell densities in the brain and a motor deficit consistent with fewer dopaminergic neurons and projections. Mechanistically, both DUB-impaired and STRIPAK-binding-deficient Trabid variants impeded the trafficking of adenomatous polyposis coli (APC) to microtubule plus-ends. Consequently, the formation of neuronal growth cones and the trajectory of neurite outgrowth from mutant midbrain progenitors were severely compromised. We propose that STRIPAK recruits Trabid to deubiquitylate APC, and that in cells with mutant Trabid, APC becomes hyperubiquitylated and mislocalised causing impaired organisation of the cytoskeleton that underlie the neuronal and developmental phenotypes.
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    Mind bomb 2 limits inflammatory dermatitis in Sharpin mutant mice independently of cell death
    Simpson, DS ; Anderton, H ; Yousef, J ; Vaibhav, V ; Cobbold, SA ; Bandala-Sanchez, E ; Kueh, AJ ; Dagley, LF ; Herold, MJ ; Silke, J ; Vince, JE ; Feltham, R ; Gutkind, JS (OXFORD UNIV PRESS, 2023-12-21)
    Skin inflammation is a complex process implicated in various dermatological disorders. The chronic proliferative dermatitis (cpd) phenotype driven by the cpd mutation (cpdm) in the Sharpin gene is characterized by dermal inflammation and epidermal abnormalities. Tumour necrosis factor (TNF) and caspase-8-driven cell death causes the pathogenesis of Sharpincpdm mice; however, the role of mind bomb 2 (MIB2), a pro-survival E3 ubiquitin ligase involved in TNF signaling, in skin inflammation remains unknown. Here, we demonstrate that MIB2 antagonizes inflammatory dermatitis in the context of the cpd mutation. Surprisingly, the role of MIB2 in limiting skin inflammation is independent of its known pro-survival function and E3 ligase activity. Instead, MIB2 enhances the production of wound-healing molecules, granulocyte colony-stimulating factor, and Eotaxin, within the skin. This discovery advances our comprehension of inflammatory cytokines and chemokines associated with cpdm pathogenesis and highlights the significance of MIB2 in inflammatory skin disease that is independent of its ability to regulate TNF-induced cell death.
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    Structures of the interleukin 11 signalling complex reveal gp130 dynamics and the inhibitory mechanism of a cytokine variant
    Metcalfe, RD ; Hanssen, E ; Fung, KY ; Aizel, K ; Kosasih, CC ; Zlatic, CO ; Doughty, L ; Morton, CJ ; Leis, AP ; Parker, MW ; Gooley, PR ; Putoczki, TL ; Griffin, MDW (NATURE PORTFOLIO, 2023-11-20)
    Interleukin (IL-)11, an IL-6 family cytokine, has pivotal roles in autoimmune diseases, fibrotic complications, and solid cancers. Despite intense therapeutic targeting efforts, structural understanding of IL-11 signalling and mechanistic insights into current inhibitors are lacking. Here we present cryo-EM and crystal structures of the human IL-11 signalling complex, including the complex containing the complete extracellular domains of the shared IL-6 family β-receptor, gp130. We show that complex formation requires conformational reorganisation of IL-11 and that the membrane-proximal domains of gp130 are dynamic. We demonstrate that the cytokine mutant, IL-11 Mutein, competitively inhibits signalling in human cell lines. Structural shifts in IL-11 Mutein underlie inhibition by altering cytokine binding interactions at all three receptor-engaging sites and abrogating the final gp130 binding step. Our results reveal the structural basis of IL-11 signalling, define the molecular mechanisms of an inhibitor, and advance understanding of gp130-containing receptor complexes, with potential applications in therapeutic development.
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    Trabid patient mutations impede the axonal trafficking of adenomatous polyposis coli to disrupt neurite growth
    Frank, D ; Bergamasco, M ; Mlodzianoski, MJ ; Kueh, A ; Tsui, E ; Hall, C ; Kastrappis, G ; Voss, AK ; McLean, C ; Faux, M ; Rogers, KL ; Tran, B ; Vincan, E ; Komander, D ; Dewson, G ; Tran, H (eLIFE SCIENCES PUBL LTD, 2023-12-15)
    ZRANB1 (human Trabid) missense mutations have been identified in children diagnosed with a range of congenital disorders including reduced brain size, but how Trabid regulates neurodevelopment is not understood. We have characterized these patient mutations in cells and mice to identify a key role for Trabid in the regulation of neurite growth. One of the patient mutations flanked the catalytic cysteine of Trabid and its deubiquitylating (DUB) activity was abrogated. The second variant retained DUB activity, but failed to bind STRIPAK, a large multiprotein assembly implicated in cytoskeleton organization and neural development. Zranb1 knock-in mice harboring either of these patient mutations exhibited reduced neuronal and glial cell densities in the brain and a motor deficit consistent with fewer dopaminergic neurons and projections. Mechanistically, both DUB-impaired and STRIPAK-binding-deficient Trabid variants impeded the trafficking of adenomatous polyposis coli (APC) to microtubule plus-ends. Consequently, the formation of neuronal growth cones and the trajectory of neurite outgrowth from mutant midbrain progenitors were severely compromised. We propose that STRIPAK recruits Trabid to deubiquitylate APC, and that in cells with mutant Trabid, APC becomes hyperubiquitylated and mislocalized causing impaired organization of the cytoskeleton that underlie the neuronal and developmental phenotypes.
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    Three-dimensional genome architecture coordinates key regulators of lineage specification in mammary epithelial cells
    Milevskiy, MJG ; Coughlan, HD ; Kane, SR ; Johanson, TM ; Kordafshari, S ; Chan, WF ; Tsai, M ; Surgenor, E ; Wilcox, S ; Allan, RS ; Chen, Y ; Lindeman, GJ ; Smyth, GK ; Visvader, JE (ELSEVIER, 2023-11-08)
    Although lineage-specific genes have been identified in the mammary gland, little is known about the contribution of the 3D genome organization to gene regulation in the epithelium. Here, we describe the chromatin landscape of the three major epithelial subsets through integration of long- and short-range chromatin interactions, accessibility, histone modifications, and gene expression. While basal genes display exquisite lineage specificity via distal enhancers, luminal-specific genes show widespread promoter priming in basal cells. Cell specificity in luminal progenitors is largely mediated through extensive chromatin interactions with super-enhancers in gene-body regions in addition to interactions with polycomb silencer elements. Moreover, lineage-specific transcription factors appear to be controlled through cell-specific chromatin interactivity. Finally, chromatin accessibility rather than interactivity emerged as a defining feature of the activation of quiescent basal stem cells. This work provides a comprehensive resource for understanding the role of higher-order chromatin interactions in cell-fate specification and differentiation in the adult mouse mammary gland.
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    Stuttering associated with a pathogenic variant in the chaperone protein cyclophilin 40
    Morgan, AT ; Scerri, TS ; Vogel, AP ; Reid, CA ; Quach, M ; Jackson, VE ; McKenzie, C ; Burrows, EL ; Bennett, MF ; Turner, SJ ; Reilly, S ; Horton, SE ; Block, S ; Kefalianos, E ; Frigerio-Domingues, C ; Sainz, E ; Rigbye, KA ; Featherby, TJ ; Richards, KL ; Kueh, A ; Herold, MJ ; Corbett, MA ; Gecz, J ; Helbig, I ; Thompson-Lake, DGY ; Liegeois, FJ ; Morell, RJ ; Hung, A ; Drayna, D ; Scheffer, IE ; Wright, DK ; Bahlo, M ; Hildebrand, MS (OXFORD UNIV PRESS, 2023-12-01)
    Stuttering is a common speech disorder that interrupts speech fluency and tends to cluster in families. Typically, stuttering is characterized by speech sounds, words or syllables which may be repeated or prolonged and speech that may be further interrupted by hesitations or 'blocks'. Rare variants in a small number of genes encoding lysosomal pathway proteins have been linked to stuttering. We studied a large four-generation family in which persistent stuttering was inherited in an autosomal dominant manner with disruption of the cortico-basal-ganglia-thalamo-cortical network found on imaging. Exome sequencing of three affected family members revealed the PPID c.808C>T (p.Pro270Ser) variant that segregated with stuttering in the family. We generated a Ppid p.Pro270Ser knock-in mouse model and performed ex vivo imaging to assess for brain changes. Diffusion-weighted MRI in the mouse revealed significant microstructural changes in the left corticospinal tract, as previously implicated in stuttering. Quantitative susceptibility mapping also detected changes in cortico-striatal-thalamo-cortical loop tissue composition, consistent with findings in affected family members. This is the first report to implicate a chaperone protein in the pathogenesis of stuttering. The humanized Ppid murine model recapitulates network findings observed in affected family members.
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    Ang II Promotes Cardiac Autophagy and Hypertrophy via Orai1/STIM1.
    Zheng, C-B ; Gao, W-C ; Xie, M ; Li, Z ; Ma, X ; Song, W ; Luo, D ; Huang, Y ; Yang, J ; Zhang, P ; Huang, Y ; Yang, W ; Yao, X (Frontiers Media S.A., 2021)
    The pathophysiology of cardiac hypertrophy is complex and multifactorial. Both the store-operated Ca2+ entry (SOCE) and excessive autophagy are the major causative factors for pathological cardiac hypertrophy. However, it is unclear whether these two causative factors are interdependent. In this study, we examined the functional role of SOCE and Orai1 in angiotensin II (Ang II)-induced autophagy and hypertrophy using in vitro neonatal rat cardiomyocytes (NRCMs) and in vivo mouse model, respectively. We show that YM-58483 or SKF-96365 mediated pharmacological inhibition of SOCE, or silencing of Orai1 with Orail-siRNA inhibited Ang II-induced cardiomyocyte autophagy both in vitro and in vivo. Also, the knockdown of Orai1 attenuated Ang II-induced pathological cardiac hypertrophy. Together, these data suggest that Ang II promotes excessive cardiomyocyte autophagy through SOCE/Orai1 which can be the prime contributing factors in cardiac hypertrophy.
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    Trajectory of immune evasion and cancer progression in hepatocellular carcinoma.
    Nguyen, PHD ; Wasser, M ; Tan, CT ; Lim, CJ ; Lai, HLH ; Seow, JJW ; DasGupta, R ; Phua, CZJ ; Ma, S ; Yang, J ; Suthen, SDO ; Tam, WL ; Lim, TKH ; Yeong, J ; Leow, WQ ; Pang, YH ; Soon, G ; Loh, TJ ; Wan, WK ; Chan, CY ; Cheow, PC ; Toh, HC ; Kow, A ; Dan, YY ; Kam, JH ; Iyer, S ; Madhavan, K ; Chung, A ; Bonney, GK ; Goh, BKP ; Fu, N ; Yu, VC ; Zhai, W ; Albani, S ; Chow, PKH ; Chew, V (Nature Portfolio, 2022-03-17)
    Immune evasion is key to cancer initiation and later at metastasis, but its dynamics at intermediate stages, where potential therapeutic interventions could be applied, is undefined. Here we show, using multi-dimensional analyses of resected tumours, their adjacent non-tumour tissues and peripheral blood, that extensive immune remodelling takes place in patients with stage I to III hepatocellular carcinoma (HCC). We demonstrate the depletion of anti-tumoural immune subsets and accumulation of immunosuppressive or exhausted subsets along with reduced tumour infiltration of CD8 T cells peaking at stage II tumours. Corresponding transcriptomic modification occur in the genes related to antigen presentation, immune responses, and chemotaxis. The progressive immune evasion is validated in a murine model of HCC. Our results show evidence of ongoing tumour-immune co-evolution during HCC progression and offer insights into potential interventions to reverse, prevent or limit the progression of the disease.
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    Sequential genome-wide CRISPR-Cas9 screens identify genes regulating cell-surface expression of tetraspanins
    Yang, J ; Guo, F ; San Chin, H ; Chen, GB ; Ang, CH ; Lin, Q ; Hong, W ; Fu, NY (CELL PRESS, 2023-02-28)
    Tetraspanins, a superfamily of membrane proteins, mediate diverse biological processes through tetraspanin-enriched microdomains in the plasma membrane. However, how their cell-surface presentation is controlled remains unclear. To identify the regulators of tetraspanin trafficking, we conduct sequential genome-wide loss-of-function CRISPR-Cas9 screens based on cell-surface expression of a tetraspanin member, TSPAN8. Several genes potentially involved in endoplasmic reticulum (ER) targeting, different biological processes in the Golgi apparatus, and protein trafficking are identified and functionally validated. Importantly, we find that biantennary N-glycans generated by MGAT1/2, but not more complex glycan structures, are important for cell-surface tetraspanin expression. Moreover, we unravel that SPPL3, a Golgi intramembrane-cleaving protease reported previously to act as a sheddase of multiple glycan-modifying enzymes, controls cell-surface tetraspanin expression through a mechanism associated with lacto-series glycolipid biosynthesis. Our study provides critical insights into the molecular regulation of cell-surface presentation of tetraspanins with implications for strategies to manipulate their functions, including cancer cell invasion.