Linear ubiquitin chains, which are generated specifically by the linear ubiquitin assembly complex (LUBAC) ubiquitin ligase, play crucial roles in immune signaling, including NF-κB activation. LUBAC comprises catalytic large isoform of heme-oxidized iron regulatory protein 2 ubiquitin ligase 1 (HOIL-1L) interacting protein (HOIP), accessory HOIL-1L, and SHANK-associated RH domain-interacting protein (SHARPIN). Deletion of the ubiquitin ligase activity of HOIL-1L, an accessory ligase of LUBAC, augments LUBAC functions by enhancing LUBAC-mediated linear ubiquitination, which is catalyzed by HOIP. Here, we show that HOIL-1L ΔRING1 mice, which exhibit augmented LUBAC functions upon loss of the HOIL-1L ligase, developed systemic lupus erythematosus (SLE) and Sjögren’s syndrome in a female-dominant fashion. Augmented LUBAC activity led to hyperactivation of both lymphoid and myeloid cells. In line with the findings in mice, we sought to identify missense single nucleotide polymorphisms/variations of the RBCK1/HOIL-1L gene in humans that attenuate HOIL-1L ligase activity. We found that the R464H variant, which is encoded by rs774507518 within the RBCK1/HOIL-1L gene, attenuated HOIL-1L ligase activity and augmented LUBAC-mediated immune signaling, including that mediated by Toll-like receptors. We also found that rs774507518 was enriched significantly in patients with SLE, strongly suggesting that RBCK1/HOIL-1L is an SLE susceptibility gene and that augmented linear ubiquitin signaling generated specifically by LUBAC underlies the pathogenesis of this prototype systemic autoimmune disease.
Yasuhiro Fuseya, Keiichiro Kadoba, Xiaoxi Liu, Hiroyuki Suetsugu, Takeshi Iwasaki, Koichiro Ohmura, Takayuki Sumida, Yuta Kochi, Akio Morinobu, Chikashi Terao, Kazuhiro Iwai
The glucocerebrosidase (GCase) encoded by the GBA1 gene hydrolyzes glucosylceramide (GluCer) to ceramide and glucose in lysosomes. Homozygous or compound heterozygous GBA1 mutations cause the lysosomal storage disease Gaucher disease (GD) due to severe loss of GCase activity. Loss-of-function variants in the GBA1 gene are also the most common genetic risk factor for Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Restoring lysosomal GCase activity represents an important therapeutic approach for GBA1-associated diseases. We hypothesized that increasing the stability of lysosomal GCase protein could correct deficient GCase activity in these conditions. However, it remains unknown how GCase stability is regulated in the lysosome. We found that cathepsin L, a lysosomal cysteine protease, cleaves GCase and regulates its stability. In support of these data, GCase protein was elevated in the brain of cathepsin L–KO mice. Chemical inhibition of cathepsin L increased both GCase levels and activity in fibroblasts from patients with GD. Importantly, inhibition of cathepsin L in dopaminergic neurons from a patient GBA1-PD led to increased GCase levels and activity as well as reduced phosphorylated α-synuclein. These results suggest that targeting cathepsin L–mediated GCase degradation represents a potential therapeutic strategy for GCase deficiency in PD and related disorders that exhibit decreased GCase activity.
Myung Jong Kim, Soojin Kim, Thomas Reinheckel, Dimitri Krainc
High grade serous carcinoma (HGSC) is the most lethal gynecological malignancy in the United States. Late diagnosis and the emergence of chemoresistance have prompted studies into how the tumor microenvironment, and more recently tumor innervation, may be leveraged for HGSC prevention and interception. In addition to biobehavioral sources, concentrations of the sympathetic neurotransmitter norepinephrine (NE) in the ovary increase during ovulation and after menopause. Importantly, NE exacerbates advanced HGSC progression. However, little is known about the role of NE in early disease pathogenesis. Here, we investigated the role of NE in instigating anchorage independence and micrometastasis of preneoplastic lesions from the fallopian tube epithelium (FTE) to the ovary, an essential step in HGSC onset. We found that in the presence of NE, FTE cell lines are able to survive in ultra-low attachment (ULA) culture in a beta-adrenergic receptor (β-AR)-dependent manner. Importantly, spheroid formation and cell viability conferred by treatment with physiological sources of NE can be abrogated using the beta-adrenergic receptor blocker propranolol. We have also identified that NE-mediated anoikis resistance may be attributable to downregulation of colony stimulating factor 2 (CSF2). These findings provide mechanistic insight and identify targets that may be regulated by ovarian-derived NE in early HGSC.
Hunter D. Reavis, Stefan M. Gysler, Grace B. McKenney, Matthew Knarr, Hannah J. Lusk, Priyanka Rawat, Hannah S. Rendulich, Marilyn A. Mitchell, Dara S. Berger, Jamie S. Moon, Suyeon Ryu, Monica Mainigi, Marcin P. Iwanicki, Dave S.B. Hoon, Laura M. Sanchez, Ronny Drapkin
Vascular calcification is a severe complication of cardiovascular diseases. Previous studies demonstrated that endothelial lineage cells transitioned into osteoblast-like cells and contributed to vascular calcification. Here, we found that inhibition of cyclin-dependent kinase (CDK) prevented endothelial lineage cells from transitioning to osteoblast-like cells and reduced vascular calcification. We identified a robust induction of CDK1 in endothelial cells (ECs) in calcified arteries and showed that endothelial-specific gene deletion of CDK1 decreased the calcification. We found that limiting CDK1 induced E-twenty-six specific sequence variant 2 (ETV2), which was responsible for blocking endothelial lineage cells from undergoing osteoblast differentiation. We also found that inhibition of CDK1 reduced vascular calcification in a diabetic mouse model. Together, the results highlight the importance of CDK1 suppression and suggest CDK1 inhibition as a potential option for treating vascular calcification.
Yan Zhao, Yang Yang, Xiuju Wu, Li Zhang, Xinjiang Cai, Jaden Ji, Sydney Chen, Abigail Vera, Kristina I. Boström, Yucheng Yao
Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a monogenic disorder accounting for approximately 5% of patients with renal failure. Yet, therapeutics for the treatment of ADPKD remain limited. ADPKD tissues display abnormalities in the biogenesis of the centrosome, a defect that can cause genome instability, aberrant ciliary signaling, and secretion of pro-inflammatory factors. Cystic cells form excess centrosomes via a process termed centrosome amplification (CA), which causes abnormal multipolar spindle configurations, mitotic catastrophe, and reduced cell viability. However, cells with CA can suppress multipolarity via “centrosome clustering,” a key mechanism by which cells circumvent apoptosis. Here, we demonstrate that inhibiting centrosome clustering can counteract the proliferation of renal cystic cells with high incidences of CA. Using ADPKD human cells and mouse models, we show that preventing centrosome clustering with two inhibitors, CCB02 and PJ34, blocks cyst initiation and growth in vitro and in vivo. Inhibiting centrosome clustering activates a p53-mediated surveillance mechanism leading to apoptosis, reduced cyst expansion, interstitial fibrosis, and improved kidney function. Transcriptional analysis of kidneys from treated mice identified pro-inflammatory signaling pathways implicated in CA-mediated cystogenesis and fibrosis. Our results demonstrate that centrosome clustering is a cyst-selective target for the improvement of renal morphology and function in ADPKD.
Tao Cheng, Aruljothi Mariappan, Ewa Langner, Kyuhwan Shim, Jay Gopalakrishnan, Moe R. Mahjoub
Cutaneous neurofibromas (cNFs) are benign Schwann cell (SC) tumors arising from subepidermal glia. Neurofibromatosis Type 1 (NF1) individuals may develop thousands of cNFs, greatly affecting their quality of life. cNF growth is driven by the proliferation of NF1(-/-) SCs and their interaction with NF1(+/-) microenvironment. We analyzed the crosstalk between human cNF-derived SCs and fibroblasts (FBs), identifying an expression signature specific to SC-FB interaction. We validated the secretion of proteins involved in immune cell migration, suggesting a role of SC-FB crosstalk in immune cell recruitment. The signature also captured components of developmental signaling pathways, including the cAMP-elevator G protein-coupled receptor 68 (GPR68). Activation of Gpr68 by Ogerin in combination with the MEKi Selumetinib reduced viability and induced differentiation and death of human cNF-derived primary SCs, a result corroborated using an iPSC-derived 3D neurofibromasphere model. Similar results were obtained using other Gpr68 activators or cAMP analogs/adenylyl cyclase activators in combination with Selumetinib. Interestingly, whereas primary SC cultures reset proliferation after removal of single Selumetib treatment, Ogerin-Selumetinib combo elicited a permanent halt on SC expansion, also after drug removal. These results indicate that unbalancing the Ras and cAMP pathways by combining MEKi and cAMP elevators arises as a potential treatment for cNFs.
Helena Mazuelas, Miriam Magallón-Lorenz, Itziar Uriarte-Arrazola, Alejandro Negro, Inma Rosas, Ignacio Blanco, Elisabeth Castellanos, Conxi Lázaro, Bernat Gel, Meritxell Carrió, Eduard Serra
Secondary lymphedema occurs in up to 20% of patients after lymphadenectomy performed for the surgical management of tumors involving the breast, prostate, uterus, and skin. Patients develop progressive edema of the affected extremity due to retention of protein-rich lymphatic fluid. Despite compression therapy, patients progress to chronic lymphedema in which noncompressible fibrosis and adipose tissue are deposited within the extremity. The presence of fibrosis led to our hypothesis that rosiglitazone, a PPARγ agonist that inhibits fibrosis, would reduce fibrosis in a mouse model of secondary lymphedema after hind limb lymphadenectomy. In vivo, rosiglitazone reduced fibrosis in the hind limb after lymphadenectomy. Our findings verified that rosiglitazone reestablished the adipogenic features of TGF-β1–treated mesenchymal cells in vitro. Despite this, rosiglitazone led to a reduction in adipose tissue deposition. Single-cell RNA-Seq data obtained from human tissues and flow cytometric and histological evaluation of mouse tissues demonstrated increased presence of PDGFRα+ cells in lymphedema; human tissue analysis verified these cells have the capacity for adipogenic and fibrogenic differentiation. Upon treatment with rosiglitazone, we noted a reduction in the overall quantity of PDGFRα+ cells and LipidTOX+ cells. Our findings provide a framework for treating secondary lymphedema as a condition of fibrosis and adipose tissue deposition, both of which, paradoxically, can be prevented with a pro-adipogenic agent.
Ziyu Chen, Soheila Ali Akbari Ghavimi, Mengfan Wu, John McNamara, Olga Barreiro, David Maridas, Radomir Kratchmarov, Ashley Siegel, Sarah Djeddi, Maria Gutierrez-Arcelus, Patrick J. Brennan, Timothy P. Padera, Ulrich von Andrian, Babak Mehrara, Arin K. Greene, C. Ronald Kahn, Dennis P. Orgill, Indranil Sinha, Vicki Rosen, Shailesh Agarwal
Obscurins are giant cytoskeletal proteins with structural and regulatory roles. Obscurin-B (~870 kDa), the largest known isoform, contains two enzymatically active Ser/Thr kinase (kin) domains, kin1 and kin2, which belong to the myosin light chain kinase (MLCK) family. Kin1 binds to and phosphorylates N-cadherin, a major component of the intercalated disc (ICD), the unique sarcolemmal microdomain that mediates the mechanochemical coupling of adjacent cardiomyocytes. Obscurin-B containing kin1 and N-cadherin co-localize at cell junctions in embryonic rat ventricular myocytes (ERVM), and their co-distribution is regulated by Ca2+. Phosphoproteomics analysis revealed that obscurin-kin1 phosphorylates N-cadherin at Ser-788 located within the juxtamembrane region of its cytoplasmic domain with an apparent Kcat of ~5.05 min-1. Overexpression of obscurin-kin1 or phosphomimic-Ser-788-Glu N-cadherin in ERVM markedly increases cell adhesion and chemical coupling. Importantly, phosphomimic-Ser-788-Glu N-cadherin exhibits significantly reduced binding to p120-catenin, while overexpression of phosphoablated-Ser-788-Ala N-cadherin increases RhoA activity. Consistent with an essential role of the obscurin-kin1/N-cadherin axis in cardiomyocyte coupling, it is deregulated in end-stage human heart failure. Given the nearly ubiquitous expression of obscurin and N-cadherin, our findings may have broad applicability in deciphering the obscurin-kin1/N-cadherin axis that likely mediates cell coupling in diverse tissues and organs.
Li Wang, Panagiotis Tsakiroglou, Rex R. Gonzales, Suhan Cho, Amy Li, Cristobal dos Remedios, Nathan T. Wright, Aikaterini Kontrogianni-Konstantopoulos
Patients with cholangiocarcinoma have poor clinical outcomes due to late diagnoses, poor prognoses, and limited treatment strategies. To identify drug combinations for this disease, we have conducted a genome-wide CRISPR screen anchored on the bromodomain and extraterminal domain (BET) PROTAC degrader ARV825, from which we identified anti-cancer synergy when combined with genetic ablation of members of the mTOR pathway. This combination effect was validated using multiple pharmacological BET and mTOR inhibitors, accompanied by increased levels of apoptosis and cell cycle arrest. In a xenograft model, combined BET degradation and mTOR inhibition induced tumor regression. Mechanistically, the two inhibitor classes converged on H3K27ac-marked epigenetic suppression of the serine glycine one carbon (SGOC) metabolism pathway, including the key regulators PHGDH and PSAT1. Knockdown of PSAT1 was sufficient to replicate synergy with single agent inhibition of either BET or mTOR. Our results tie together epigenetic regulation, metabolism, and apoptosis induction as key therapeutic targets for further exploration in this underserved disease.
Yan Zhu, Dengyong Zhang, Pooja Shukla, Young-Ho Jung, Prit Benny Malgulwar, Sharmeen Chagani, Medina Colic, Sarah Benjamin, John A. Copland III, Lin Tan, Philip L. Lorenzi, Milind Javle, Jason T. Huse, Jason Roszik, Traver Hart, Lawrence N. Kwong
The use of patient-derived organoids (PDOs) to characterize therapeutic sensitivity and resistance is a promising precision medicine approach, and its potential to inform clinical decisions is now being tested in several large multi-institutional clinical trials. PDOs are cultivated in extracellular matrix from basement membrane extracts (BMEs) that are most commonly acquired commercially. Each clinical site utilizes distinct BME lots and may be restricted due to the availability of commercial BME sources. However, the impact of different sources of BMEs on organoid drug response is unknown. Here, we tested the impact of BME source on proliferation, drug response, and gene expression in mouse and human pancreatic ductal adenocarcinoma (PDA) organoids. Both human and mouse organoids displayed increased proliferation in Matrigel (Corning) compared to Cultrex (RnD) and UltiMatrix (RnD). However, we observed no substantial impact on drug response when organoids were cultured in Matrigel, Cultrex, or UltiMatrix. We also did not observe major shifts in gene expression across the different BME sources, and PDOs maintained their Classical or Basal-like designation. Overall, we find that BME source (Matrigel, Cultrex, UltiMatrix) does not shift PDO dose-response curves and drug testing results, indicating that PDO pharmacotyping is a robust approach for precision medicine.
Jan C. Lumibao, Shira R. Okhovat, Kristina L. Peck, Xiaoxue Lin, Kathryn Lande, Shira Yomtoubian, Isabella Ng, Herve Tiriac, Andrew M. Lowy, Jingjing Zou, Dannielle D. Engle
No posts were found with this tag.