Cell biology
Abstract
Biallelic loss-of-function variants in the adaptor protein complex 4 (AP-4) disrupt trafficking of transmembrane proteins at the trans-Golgi network, including the autophagy-related protein 9A (ATG9A), leading to childhood-onset hereditary spastic paraplegia (AP-4-HSP). AP-4-HSP is characterized by features of both a neurodevelopmental and degenerative neurological disease. To investigate the molecular mechanisms underlying AP-4-HSP and identify potential therapeutic targets, we conducted an arrayed CRISPR/Cas9 loss-of-function screen of 8,478 genes, targeting the ‘druggable genome’, in a human neuronal model of AP-4 deficiency. Through this phenotypic screen and subsequent experiments, key modulators of ATG9A trafficking were identified, and complementary pathway analyses provided insights into the regulatory landscape of ATG9A transport. Knockdown of ANPEP and NPM1 enhanced ATG9A availability outside the trans-Golgi network, suggesting they regulate ATG9A localization. These findings deepen our understanding of ATG9A trafficking in the context of AP-4 deficiency and offer a framework for the development of targeted interventions for AP-4-HSP.
Authors
Marvin Ziegler, Cedric Günter, Julian E. Alecu, Xutong Xue, Hyo-Min Kim, Afshin Saffari, Alexandra K. Davies, Mustafa Sahin, Darius Ebrahimi-Fakhari
Abstract
Idiopathic pulmonary fibrosis (IPF) is characterized by parenchymal scarring reflecting an imbalance between collagen deposition by myofibroblasts (MFs) and its turnover. Although collagen clearance is essential for fibrosis resolution, this process and its potential for therapeutic modulation in IPF are poorly understood. Here we evaluated internalization of degraded collagen and the role of its requisite endocytic receptor mannose receptor C-type 2 (MRC2), in lung tissue and MFs from IPF patients and bleomycin-injured mice. Fibrotic human and murine lung tissue exhibited an accumulation of degraded collagen, highlighting a failure of its clearance. MFs from fibrotic lung demonstrated a reduced capacity to internalize extracellular degraded collagen, with a concomitant reduction in MRC2 expression and endolysosomal activity. Both diminished collagen uptake and MRC2 expression recovered to baseline levels during spontaneous resolution of bleomycin fibrosis. In vitro treatment of IPF or TGF-β-elicited MFs with a variety of mechanistically distinct agents known to effect phenotypic dedifferentiation restored defective collagen internalization. Although enhanced uptake was MRC2-dependent, it involved increased endolysosomal activity rather than increased MRC2 expression. These results implicate defective MRC2-dependent collagen internalization and endolysosomal function in MFs as important factors contributing to fibrosis that may be therapeutically targeted to promote resolution.
Authors
Natalie M. Walker, Sean M. Fortier, Jennifer Speth, Steven K. Huang, Sergey Gutor, Timothy S. Blackwell, Marc Peters-Golden
Abstract
Ubiquitin-Specific Protease 18 (USP18) is a deISGylation enzyme and antineoplastic target. To develop USP18 inhibitors, an enzymatically active human recombinant USP18 protein was engineered suitable for high-throughput screening of ~80,000 chemical compounds. Three of them substantially inhibited USP18 enzymatic activity with β-lapachone having prominent antineoplastic activity. Independent β-lapachone treatments of murine and human lung cancer cell lines statistically-significantly reduced proliferation and increased apoptosis. Gain of USP18 expression antagonized these effects. β-lapachone treatments statistically-significantly repressed lung cancer xenograft growth. β-lapachone increased reactive oxygen species (ROS), but antineoplastic effects occurred at dosages with negligible ROS production. ROS scavenger treatments did not rescue β-lapachone effects at these concentrations, consistent with an ROS-independent mechanism. Interferon-Stimulated Response Element (ISRE) reporter assays following β-lapachone treatment activated this reporter. USP18 co-transfection antagonized this activity. β-lapachone treatments increased global ISGylation. RNA sequencing of lung cancer cells engineered with or without enhanced USP18 expression showed specific pathways affected by β-lapachone treatment. Proteomic analysis of these treated cells revealed known and new ISGylated proteins. In silico modeling identified a unique USP18 pocket where these USP18 inhibitors bind. Engineered mutation of this pocket disrupted β-lapachone activity. Taken together, β-lapachone is an antineoplastic tool compound useful for USP18 inhibitor development.
Authors
Blessing O. Ogunlade, Kevin N. Dalby, Samuel C. Okpechi, Eun Jeong Cho, Liliya Tyutyunyk-Massey, Zibo Chen, Xiuxia Liu, Joseph Ivanic, Brian Luke, Shyamal D. Desai, Yair Alfaro, Ashwini K. Devkota, Rae M. Sammons, Gilbert G. Privé, Xi Liu, Ethan Dmitrovsky
Abstract
Ischemia/reperfusion (IR) enhances oxidative stress, leading to myocardial injury. Although Perm1 promotes cytoprotective mechanisms, the underlying mechanisms are poorly understood. Cysteine oxidation of Keap1 alleviates Cul3-mediated ubiquitination/degradation of Nrf2 and promotes antioxidant transcription. Here we show that Perm1 activates Nrf2 through cysteine oxidation of Keap1 and stabilization of Nrf2. Endogenous Perm1 was downregulated during IR, whereas the rescue of Perm1 reduced IR injury. Downregulation of Perm1 exacerbated oxidative stress, whereas upregulation of Perm1 alleviated it, accompanied by downregulation and upregulation of Nrf2-regulated antioxidant genes, respectively. Perm1 promoted oxidation of cysteine residues in Keap1, possibly through thiol-disulfide exchange reactions, which decreases Keap1-Nrf2 interaction and inhibits Cul3-mediated degradation of Nrf2. We identified Cys121 and Cys746 in Perm1 as critical for Keap1 oxidation and cardioprotection. Thus, Perm1 induces cysteine oxidation of Keap1, thereby conferring myocardial resistance to IR injury by inducing Nrf2 stabilization and transcriptional activation of antioxidant genes.
Authors
Shin-ichi Oka, Chun-Yang Huang, Masato Matsushita, Allen Sam Titus, Yasuki Nakada, Risa Mukai, Samta Veera, Youssef Mourad, Ghassan Yehia, Peter Romanienko, Yimin Tian, Peiyong Zhai, Junichi Sadoshima
Abstract
Methyltransferase-like 5 (METTL5) is a methyltransferase responsible for rRNA N6-methyladenosine (m6A) modification, mutations in which are associated with skeletal abnormalities and cognitive deficits. Despite METTL5’s clinical relevance, the molecular mechanisms underlying METTL5-related genetic disorders remain poorly understood. In this study, we demonstrated that Mettl5 KO led to reduced bone mass and smaller body size in mice and impaired the osteogenic differentiation of mesenchymal stem cells. Mechanistically, Mettl5 deficiency decreased the translation efficiency of oxidative stress–responsive serine-rich protein 1 mRNA, downregulated the expression of key antioxidant genes, and diminished antioxidant capacity. Importantly, administration of the antioxidant N-acetylcysteine (NAC) partially rescued skeletal defects in Mettl5-KO mice. These findings reveal a critical role for METTL5 in antioxidant defense and suggest that NAC supplementation may represent a promising therapeutic strategy for METTL5-related disorders.
Authors
Kexin Lei, Qi Yin, Qiwen Li, Qian Wang, Zhong Zhang, Fei Xue, Ruoshi Xu, Xinyi Zhou, Lin Peng, Shoichiro Kokabu, Shuibin Lin, Quan Yuan
Abstract
Lysyl oxidase (LOX) is a copper-dependent monoamine oxidase whose primary function is the covalent cross-linking of collagen and elastin in the extracellular matrix (ECM). However, the regulation of LOX activity in renal fibrosis is not well understood. Here, our study showed that (a) LOX expression and ECM cross-linking were markedly increased in fibrotic kidneys. Reduction of copper levels in the Golgi apparatus by treatment with the copper chelator tetrathiomolybdate or by specific knockdown of copper transporter 1 (CTR1) decreased LOX activity and ameliorated renal fibrosis. (b) Overexpression of ATP7A caused an elevation of copper ions within the Golgi apparatus, resulting in increased LOX activity and enhanced ECM crosslinking, thereby promoting the progression of renal fibrosis. Knockdown of ATP7A showed the opposite result. (c) FBLN4 was essential for the ATP7A-mediated transfer of copper to LOX and formed a ternary complex of ATP7A-FBLN4-LOX. Our research revealed that high ATP7A expression induced copper overload in the Golgi apparatuses. FBLN4 then assisted ATP7A in transporting this excess copper to LOX, resulting in LOX overactivation. This, in turn, catalyzed the cross-linking of ECM components, thereby accelerating renal fibrosis.
Authors
Wenqian Zhou, Yan Zheng, Yuqing Liu, Jing Liu, Yiguo Liu, Yangyang Niu, Ying Yu, Xiaoqin Zhang, Yingying Zhang, Chen Yu
Abstract
The lungs have a remarkable capacity to undergo homoeostatic repair and regeneration after injury, which often occurs in patients with acute respiratory distress syndrome (ARDS) and in the single-dose bleomycin mouse model. Fibroblasts are critical mediators of fibrotic disease and RNA sequencing has identified significant heterogeneity within pulmonary fibroblast populations. However, the contribution of distinct fibroblast subsets to the repair process has been understudied compared to their role in fibrosis initiation and progression. Therefore, we sought to define the transcriptional landscape of three phenotypically-defined fibroblast subsets that occupy discrete spatial locations in naïve lungs. Using TdTomato-lineage tracing approaches, we identified and interrogated collagen1a1+ (Col1a1) fibroblasts, perilipin 2+ (Plin2) alveolar fibroblasts, and a-smooth muscle actin+ (Acta2) myofibroblasts during fibrosis development and resolution after single-dose bleomycin. Quantification of fibroblast numbers showed that all three subsets expand during fibrosis and contract towards naïve levels with resolution. Principal component and gene-set enrichment analyses indicated that each subset undergoes major transcriptomic shifts during fibrosis development, converging on a similar pro-fibrotic transcriptional profile. However, during resolution, Plin2+ and Acta2+ fibroblasts revert towards a pre-fibrotic transcriptional state, whereas Col1a1+ fibroblasts acquire a distinct program that suggests suggesting an active role in mediating the repair processes.
Authors
Daniel G. Foster, Nomin Javkhlan, Bart P. Black, Brian E. Vestal, David W.H. Riches, Elizabeth F. Redente
Development and characterization of triazole-based WDR5 inhibitors for the treatment of glioblastoma
Abstract
Glioblastoma (GBM) cancer stem cells (CSCs) contribute to tumor recurrence, treatment resistance, and dismal clinical outcomes. Genetic and pharmacological evidence suggests that the nuclear scaffolding protein WD-repeat containing protein 5 (WDR5) is a therapeutic vulnerability of the CSC population. However, previously reported WDR5 inhibitors display low permeability and are unable to penetrate the blood-brain barrier (BBB), limiting their utility in GBM. Herein, we report the structure-guided development of a novel series of triazole-based WDR5 WIN-site inhibitors designed to increase passive brain penetration. We identified triazole-based WDR5 inhibitors that are potent, passively permeable, and in some cases more brain penetrant than other scaffolds. We phenotypically assessed our novel WDR5 inhibitors in a panel of patient-derived CSC models and uncovered unique WDR5-regulated metabolic genes in GBM. We also evaluated their antiproliferative activity against CSCs both in vitro and in vivo. Finally, to identify novel combination opportunities, we screened a 2,100-compound chemical probe library and identified that the ATAD2 inhibitor BAY-850 synergizes with WDR5 inhibitors to enhance CSC killing. Our work diversifies the chemical matter targeting WDR5, clarifies the in vitro consequences of WIN-site inhibition in CSCs, and encourages the future development of next-generation WDR5 inhibitors with the potential to achieve in vivo efficacy in the brain.
Authors
Jesse A. Coker, Steven R. Martinez, Sang Hoon Han, Anthony R. Sloan, Amit Kumar Gupta, George Bukenya, Paul Polzer, James H. Ramos, Emma G. Rico, Annabella Rico, A. Abigail Lindsey, Tanvi Navadgi, Natalie Reitz, Todd Romigh, Jonathan Macdonald, Dhiraj Sonawane, Christopher M. Goins, Christopher G. Hubert, Nancy S. Wang, Feixiong Cheng, Joseph Alvarado, Samuel A. Sprowls, Justin D. Lathia, Shaun R Stauffer
Abstract
Abdominal aortic aneurysm (AAA) lacks effective pharmacological therapies. Here, we investigate transcription factor 7-like 2 (TCF7L2), a genetic locus associated with both thoracic and abdominal aortic aneurysms, to elucidate its role in AAA pathogenesis. Integrating summary-data-based Mendelian randomization (SMR) with single-cell RNA sequencing (scRNA-seq) of human and mouse aortas, we identify TCF7L2 as a gene enriched in vascular smooth muscle cells (VSMCs) and causally linked to AAA development. Smooth muscle cell-specific TCF7L2 knockout significantly attenuates AAA formation across three distinct murine models (Ang II infusion-, BAPN/Ang II co-administration-, and elastase-induced AAA), independent of systemic blood pressure or lipid levels. Mechanistic studies reveal that TCF7L2 directly upregulates MMP14 and downregulates TIMP3 expression in vitro and in vivo, driving MMP2-mediated extracellular matrix (ECM) degradation. Concurrently, TCF7L2 represses integrin β1 (ITGB1) expression, reducing VSMC adhesion to the ECM. Collectively, these findings identify TCF7L2 as a key driver of pathological vascular remodeling in AAA, suggesting that targeting TCF7L2 may offer a novel therapeutic strategy for limiting AAA progression.
Authors
Yongjie Deng, Yaozhong Liu, Yang Zhao, Hongyu Liu, Guizhen Zhao, Zhenguo Wang, Xu Zhang, Chao Xue, Wei Huang, Tianqing Zhu, Haocheng Lu, Yanhong Guo, Lin Chang, Ida Surakka, Y. Eugene Chen, Jifeng Zhang
Abstract
Aortic dissection or rupture is a leading cause of mortality in vascular Ehlers-Danlos syndrome (VEDS), a disorder caused by mutations in the COL3A1 gene. Col3a1G938D/+ mice recapitulate features of VEDS, including high risk of aortic rupture. As in people with VEDS, aortic risk in this model accelerates at the onset of puberty, especially in males. We identify developmentally regulated gene programs associated with this vulnerability and that are targeted by treatments that mitigate aortic risk. Both genetic and pharmacological inhibition of the androgen receptor (AR) eliminated survival differences between sexes, while treatment with a dual AR and mineralocorticoid receptor (MR) antagonist provided near-complete and durable protection in both sexes. Pathways targeted by dual AR/MR inhibition, including those related to extracellular matrix (ECM) organization and cell-ECM interactions, largely overlapped with those also modulated by isolated MR antagonism. Selective targeting of MR signaling emerged as an effective therapeutic strategy in both sexes that avoids sexual side effects in males.
Authors
Emily E. Juzwiak, Caitlin J. Bowen, Rhiannon Edwards, Leda Restrepo, Serena Lee, Cassie A. Parks, Anthony Zeng, Maya M. Black, Oscar E. Reyes Gaido, Emily E. Bramel, Dustin T. Shigaki, Michael A. Beer, Chiara Bellini, Harry C. Dietz, Elena Gallo MacFarlane
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