scholarly journals Loss-of-function mutations in PTPRJ cause a new form of inherited thrombocytopenia

Blood ◽  
2019 ◽  
Vol 133 (12) ◽  
pp. 1346-1357 ◽  
Author(s):  
Caterina Marconi ◽  
Christian A. Di Buduo ◽  
Kellie LeVine ◽  
Serena Barozzi ◽  
Michela Faleschini ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Unknown Origin ◽  
Bleeding Tendency ◽  
Loss Of Function ◽  
Spontaneous Bleeding ◽  
Protein Levels ◽  
New Form ◽  
Functional Defects

Abstract Inherited thrombocytopenias (ITs) are a heterogeneous group of disorders characterized by low platelet count that may result in bleeding tendency. Despite progress being made in defining the genetic causes of ITs, nearly 50% of patients with familial thrombocytopenia are affected with forms of unknown origin. Here, through exome sequencing of 2 siblings with autosomal-recessive thrombocytopenia, we identified biallelic loss-of-function variants in PTPRJ. This gene encodes for a receptor-like PTP, PTPRJ (or CD148), which is expressed abundantly in platelets and megakaryocytes. Consistent with the predicted effects of the variants, both probands have an almost complete loss of PTPRJ at the messenger RNA and protein levels. To investigate the pathogenic role of PTPRJ deficiency in hematopoiesis in vivo, we carried out CRISPR/Cas9-mediated ablation of ptprja (the ortholog of human PTPRJ) in zebrafish, which induced a significantly decreased number of CD41+ thrombocytes in vivo. Moreover, megakaryocytes of our patients showed impaired maturation and profound defects in SDF1-driven migration and formation of proplatelets in vitro. Silencing of PTPRJ in a human megakaryocytic cell line reproduced the functional defects observed in patients’ megakaryocytes. The disorder caused by PTPRJ mutations presented as a nonsyndromic thrombocytopenia characterized by spontaneous bleeding, small-sized platelets, and impaired platelet responses to the GPVI agonists collagen and convulxin. These platelet functional defects could be attributed to reduced activation of Src family kinases. Taken together, our data identify a new form of IT and highlight a hitherto unknown fundamental role for PTPRJ in platelet biogenesis.

scholarly journals TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

BMC Cancer ◽  
2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Chengwu Xiao ◽  
Wei Zhang ◽  
Meimian Hua ◽  
Huan Chen ◽  
Bin Yang ◽  
...  
Keyword(s):  
Cell Lines ◽  
Prognostic Indicator ◽  
The Cancer Genome Atlas ◽  
Mrna Levels ◽  
Loss Of Function ◽  
Protein Levels ◽  
Kaplan Meier ◽  
Cancer Genome Atlas

Abstract Background The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC. Methods The mRNA levels of TRIM27 and Kaplan–Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression. Results We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation. Conclusions Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

scholarly journals Conformational dynamics is key to understanding loss-of-function of NQO1 cancer-associated polymorphisms and its correction by pharmacological ligands

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Encarnación Medina-Carmona ◽  
Rogelio J. Palomino-Morales ◽  
Julian E. Fuchs ◽  
Esperanza Padín-Gonzalez ◽  
Noel Mesa-Torres ◽  
...  
Keyword(s):  
Protein Dynamics ◽  
Protein Function ◽  
Conformational Dynamics ◽  
Loss Of Function ◽  
Quinone Oxidoreductase ◽  
Protein Levels ◽  
Terminal Domain ◽  
Directional Preference

Abstract Protein dynamics is essential to understand protein function and stability, even though is rarely investigated as the origin of loss-of-function due to genetic variations. Here, we use biochemical, biophysical, cell and computational biology tools to study two loss-of-function and cancer-associated polymorphisms (p.R139W and p.P187S) in human NAD(P)H quinone oxidoreductase 1 (NQO1), a FAD-dependent enzyme which activates cancer pro-drugs and stabilizes several oncosuppressors. We show that p.P187S strongly destabilizes the NQO1 dimer in vitro and increases the flexibility of the C-terminal domain, while a combination of FAD and the inhibitor dicoumarol overcome these alterations. Additionally, changes in global stability due to polymorphisms and ligand binding are linked to the dynamics of the dimer interface, whereas the low activity and affinity for FAD in p.P187S is caused by increased fluctuations at the FAD binding site. Importantly, NQO1 steady-state protein levels in cell cultures correlate primarily with the dynamics of the C-terminal domain, supporting a directional preference in NQO1 proteasomal degradation and the use of ligands binding to this domain to stabilize p.P187S in vivo. In conclusion, protein dynamics are fundamental to understanding loss-of-function in p.P187S and to develop new pharmacological therapies to rescue this function.

Upregulation of SGOL2 Predicts Poor Prognosis and Facilitates Hepatocellular Carcinoma Progression via Dysregulating the Cell Cycle by Directly Activating MAD2

2021 ◽  
Author(s):  
Qingqing Hu ◽  
Xiaochu Hu ◽  
Yalei Zhao ◽  
Lingjian Zhang ◽  
Ya Yang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Cycle ◽  
The Cancer Genome Atlas ◽  
Loss Of Function ◽  
Protein Levels ◽  
Cancer Genome Atlas ◽  
Centromeric Protein ◽  
Hcc Cells

Abstract Background: Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Shugoshin-like protein 2 (SGOL2) is a centromeric protein that ensures the correct and orderly process of mitosis by protecting and maintaining centripetal adhesions during meiosis and mitosis. However, the role of SGOL2 in cancer is not well understood. Methods: The mRNA and protein levels of SGOL2 and survival analysis were conducted in The Cancer Genome Atlas (TCGA) and further validated in 2 independent cohorts. Differential genes correlated with SGOL2 and mitotic arrest deficient 2 like 1 (MAD2) were obtained using LinkedOmics. Subsequently, loss-of-function and rescue assays were carried out in vitro and in vivo to assess the functions of SGOL2 in hepatic tumorigenisis. Findings: We found that SGOL2 was significantly overexpressed in HCC and predicted unfavorable overall survival in HCC patients. Next, we identified 47 differentially expressed genes positively correlated with both SGOL2 and MAD2 to be mainly involved in the cell cycle. In addition, SGOL2 downregulation suppressed the migration, invasion, proliferation, stemness and EMT of HCC cells and inhibited tumorigenesis in vivo. Furthermore, SGOL2 promoted tumor proliferation by activating MAD2-induced cell cycle dysregulation, which could be reversed by the MAD2 inhibitor M2I-1. We also proved that SGOL2 activated MAD2 by directly binding with MAD2. Conclusions: The results of this study showed that SGOL2 acts as an oncogene in HCC cells by directly activating MAD2 and then dysregulating the cell cycle, thereby providing a potential target for HCC patients in the future.

scholarly journals Evaluation of biomarkers for in vitro prediction of drug-induced nephrotoxicity in RPTEC/TERT1 cells

2020 ◽  
Vol 9 (2) ◽  
pp. 91-100 ◽  
Author(s):  
Xuan Qiu ◽  
Yufa Miao ◽  
Xingchao Geng ◽  
Xiaobing Zhou ◽  
Bo Li
Keyword(s):  
Transcriptional Activation ◽  
Messenger Rna ◽  
Aristolochic Acid ◽  
Kidney Injury ◽  
Drug Induced ◽  
Vitro Assay ◽  
Protein Levels ◽  
Aristolochic Acid I

Abstract There have been intensive efforts to identify in vivo biomarkers that can be used to monitor drug-induced kidney damage before significant impairment occurs. Kidney injury molecule-1, neutrophil gelatinase-associated lipocalin, clusterin, β2-microglobulin and cystatin C (CysC) have been validated as clinical or preclinical biomarkers in urinary and plasma predictive of acute and chronic kidney injuries and diseases. A high-throughput in vitro assay predictive of nephrotoxicity could potentially be implemented in early drug discovery stage to reduce attrition at later stages of drug development. To assess the potential of these known in vivo biomarkers for in vitro evaluation of drug-induced nephrotoxicity, we selected four nephrotoxic agents (cisplatin, cyclosporin, aristolochic acid I and gentamicin) and detected their effects on the protein levels of nephrotoxic biomarkers in RPTEC/TERT1 cells. The protein levels of clusterin, CysC, GSTπ and TIMP-1 significantly increased in the conditioned media of RPTEC/TERT1 cells treated with cisplatin, cyclosporin, aristolochic acid I and gentamicin. The messenger RNA levels of clusterin, CysC, GSTπ and TIMP-1 also increased in RPTEC/TERT1 cells treated with cisplatin, cyclosporin, aristolochic acid I and gentamicin, indicating that drug-induced upregulation involves transcriptional activation. Taken together, the results clearly demonstrate that among the known in vivo nephrotoxic biomarkers, clusterin, CysC, GSTπ and TIMP-1 can be effectively used as in vitro biomarkers for drug-induced nephrotoxicity in RPTEC/TERT1 cells.

scholarly journals TDP-43 stabilizes transcripts encoding stress granule protein G3BP1: potential relevance to ALS/FTD

2020 ◽  
Author(s):  
Hadjara Sidibé ◽  
Yousra Khalfallah ◽  
Shangxi Xiao ◽  
Nicolás B. Gómez ◽  
Elizabeth M.H. Tank ◽  
...  
Keyword(s):  
Regulatory Element ◽  
Stress Granule ◽  
Loss Of Function ◽  
Cytoplasmic Inclusions ◽  
Protein Levels ◽  
Assembly Factor ◽  
Direct Binding ◽  
Lateral Sclerosis

ABSTRACTTDP-43 nuclear depletion and concurrent cytoplasmic accumulation in vulnerable neurons is a hallmark feature of progressive neurodegenerative proteinopathies such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cellular stress signalling and stress granule dynamics are now recognized to play a role in ALS/FTD pathogenesis. Defective stress granule assembly is associated with increased cellular vulnerability and death. G3BP1 (Ras-GAP SH3-domain-binding protein 1) is a critical stress granule assembly factor. Here, we define that TDP-43 stabilizes G3BP1 transcripts via direct binding of a highly conserved cis regulatory element within the 3’UTR. Moreover, we show in vitro and in vivo that nuclear TDP-43 depletion is sufficient to reduce G3BP1 protein levels. Finally, we establish that G3BP1 transcripts are reduced in ALS/FTD patient neurons bearing TDP-43 cytoplasmic inclusions/nuclear depletion. Thus, our data suggest that, in ALS/FTD, there is a compromised stress granule response in disease-affected neurons due to impaired G3BP1 mRNA stability caused by TDP-43 nuclear depletion. These data implicate TDP-43 and G3BP1 loss of function as contributors to disease.

scholarly journals METTL3 Regulates Angiogenesis by Modulating let-7e-5p and miRNA-18a-5p Expression in Endothelial Cells

2021 ◽  
Author(s):  
Aránzazu Chamorro-Jorganes ◽  
Walid K. Sweaad ◽  
Rajesh Katare ◽  
Marie Besnier ◽  
Maryam Anwar ◽  
...  
Keyword(s):  
Cancer Biology ◽  
Limb Ischemia ◽  
Fine Tuning ◽  
Loss Of Function ◽  
New Approach ◽  
Rna Methylation ◽  
Angiogenic Potential ◽  
Protein Levels

Objective: Postnatal angiogenesis is critical in vascular homeostasis and repair. m 6 A RNA methylation is emerging as a new layer for fine-tuning gene expression. Although the contribution of the m 6 A-catalyzing enzyme, METTL3 (methyltransferase-like 3), in cancer biology has been described, its role in endothelial cell (EC) function, particularly during angiogenesis, remains unclear. Approach and Results: To characterize the relevance of METTL3 in angiogenesis regulation, we performed gain- and loss-of-function studies in vitro. We demonstrated that depletion of METTL3 in ECs reduced the level of m 6 A and impaired EC function, whereas adenovirus-mediated METTL3 overexpression increased angiogenesis. Mechanistically, we showed that METTL3 depletion in ECs decreased mature angiogenic microRNAs let-7e-5p and the miR-17-92 cluster, and increased the expression of their common target, Tsp1 (thrombospondin 1). Conversely, Ad.METTL3 increased the expression of let-7e-5p and miR-17-92 cluster and reduced protein levels of Tsp1 in ECs. Moreover, overexpression of let-7e-5p and miR-18a-5p restored the angiogenic potential of METTL3-depleted ECs. We corroborated our data in vivo employing 3 mouse models. When tested in an in vivo Matrigel plug assay, METTL3-depleted ECs had diminished ability to vascularize the plug, whereas overexpression of METTL3 promoted angiogenesis. Local Ad.METTL3 gene transfer increased postischemic neovascularization in mice with either unilateral limb ischemia or myocardial infarction. Conclusions: METTL3 regulates m 6 A RNA methylation in ECs. Endogenous METTL3 is essential for EC function and angiogenesis, potentially through influencing let-7e and miR-17-92 cluster processing. Thus, the therapeutic modulation of METTL3 should be considered as a new approach for controlling angiogenic responses in the clinical setting.

scholarly journals Deficiency of Mettl3 in Bladder Cancer Stem Cells Inhibits Bladder Cancer Progression and Angiogenesis

2021 ◽  
Vol 9 ◽  
Author(s):  
Ganping Wang ◽  
Yarong Dai ◽  
Kang Li ◽  
Maosheng Cheng ◽  
Gan Xiong ◽  
...  
Keyword(s):  
Bladder Cancer ◽  
Cancer Progression ◽  
Messenger Rna ◽  
Conditional Knockout ◽  
Stem Cell Population ◽  
Protein Levels ◽  
Rna Immunoprecipitation ◽  
Endothelial Growth Factor

RNA N6-methyladenosine is a key step of posttranscriptional modulation that is involved in governing gene expression. The m6A modification catalyzed by Mettl3 has been widely recognized as a critical epigenetic regulation process for tumorigenic properties in various cancer cell lines, including bladder cancer. However, the in vivo function of Mettl3 in bladder cancer remains largely unknown. In our study, we found that ablation of Mettl3 in bladder urothelial attenuates the oncogenesis and tumor angiogenesis of bladder cancer using transgenic mouse model. In addition, conditional knockout of Mettl3 in K14+ bladder cancer stem cell population leads to inhibition of bladder cancer progression. Coupled with the global transcriptome sequencing and methylated RNA immunoprecipitation sequencing results, we showed that deletion of Mettl3 leads to the suppression of tyrosine kinase endothelial (TEK) and vascular endothelial growth factor A (VEGF-A) through reduced abundance of m6A peaks on a specific region. In addition, the depletion of Mettl3 results in the decrease in both messenger RNA (mRNA) and protein levels of TEK and VEGF-A in vitro. Taken together, Mettl3-mediated m6A modification is required for the activation of TEK–VEGF-A-mediated tumor progression and angiogenesis. Our findings may provide theoretical basis for bladder cancer treatment targeting Mettl3.

scholarly journals Long noncoding RNA GAS5 inhibits progression of colorectal cancer by interacting with and triggering YAP phosphorylation and degradation and is negatively regulated by the m6A reader YTHDF3

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Wen Ni ◽  
Su Yao ◽  
Yunxia Zhou ◽  
Yuanyuan Liu ◽  
Piao Huang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cancer Progression ◽  
Noncoding Rna ◽  
Function Analysis ◽  
Loss Of Function ◽  
Functional Link ◽  
Protein Levels ◽  
Lncrna Gas5

Abstract Background YAP activation is crucial for cancer development including colorectal cancer (CRC). Nevertheless, it remains unclear whether N6-Methyladenosine (m6A) modified transcripts of long noncoding RNAs (lncRNAs) can regulate YAP activation in cancer progression. We investigated the functional link between lncRNAs and the m6A modification in YAP signaling and CRC progression. Methods YAP interacting lncRNAs were screened by RIP-sequencing, RNA FISH and immunofluorescence co-staining assays. Interaction between YAP and lncRNA GAS5 was studied by biochemical methods. MeRIP-sequencing combined with lncRNA-sequencing were used to identify the m6A modified targets of YTHDF3 in CRC. Gain-of-function and Loss-of-function analysis were performed to measure the function of GAS5-YAP-YTHDF3 axis in CRC progression in vitro and in vivo. Results GAS5 directly interacts with WW domain of YAP to facilitate translocation of endogenous YAP from the nucleus to the cytoplasm and promotes phosphorylation and subsequently ubiquitin-mediated degradation of YAP to inhibit CRC progression in vitro and in vivo. Notably, we demonstrate the m6A reader YTHDF3 not only a novel target of YAP but also a key player in YAP signaling by facilitating m6A-modified lncRNA GAS5 degradation, which profile a new insight into CRC progression. Clinically, lncRNA GAS5 expressions is negatively correlated with YAP and YTHDF3 protein levels in tumors from CRC patients. Conclusions Our study uncovers a negative functional loop of lncRNA GAS5-YAP-YTHDF3 axis, and identifies a new mechanism for m6A-induced decay of GAS5 on YAP signaling in progression of CRC which may offer a promising approach for CRC treatment.

scholarly journals A familial hemorrhagic diathesis in a Dutch family: an inherited deficiency of alpha 2-antiplasmin

Blood ◽  
1982 ◽  
Vol 59 (6) ◽  
pp. 1169-1180
Author(s):  
C Kluft ◽  
E Vellenga ◽  
EJ Brommer ◽  
G Wijngaards
Keyword(s):  
Factor Xiii ◽  
Family Members ◽  
Recessive Gene ◽  
Coagulation System ◽  
Hemorrhagic Diathesis ◽  
Bleeding Tendency ◽  
Spontaneous Bleeding ◽  
Normal Plasma

This study concerns a case of congenital homozygous deficiency in alpha 2-antiplasmin associated with a severe hemorrhagic diathesis. Heterozygous family members also show a mild bleeding tendency. The propositus is a 17-yr-old male born of white parents and showing a severe hemorrhagic diathesis characterized by spontaneous bleeding in the joints since his early childhood. He was originally suspected of having factor XIII deficiency but was found to have normal functions of the coagulation system and the platelets. Except for alpha 2- antiplasmin, all protease inhibitors showed normal plasma values. With the immediate plasmin inhibition test (synthetic substrate), only 2% of normal functional inhibition was detected, while no reaction with monospecific antisera for alpha 2-antiplasmin was observed. Inhibition of activator-induced fibrinolysis in vitro was reduced. No enhanced spontaneous in vitro fibrinolysis was detected nor were there signs of increased in vivo fibrinolysis during an asymptomatic period. During recovery from a hemorrhagic episode, signs of previous consumption of antithrombin III, alpha 2-macroglobulin, factor XIII, and inter-alpha- trypsin inhibitor were noted. After the diagnosis was made, treatment with tranexamic acid (4 daily doses of 1 g) was effective for about 2 yr. Among the 37 family members studied, a separate group of 16 individuals (including the father and mother of the propositus) with approximately one-half normal plasma levels of alpha 2-antiplasmin both functionally (59% +/- 6%) and immunologically 48% +/- 8%) was discovered. The defect appeared to be inherited as an autosomal recessive gene; no ancestral consanguinity could be shown. The group of apparent heterozygotes as a whole showed increased levels of alpha 1- antitrypsin (142% +/- 39%; p less than 0.01), indicating systemic consequences of the deficiency and reduced binding (+/- 50%) of alpha 2- antiplasmin to fibrin. Six exhibited a mild hemorrhagic diathesis for which no explanation was provided by routine screening of coagulation and platelet functions; also, within the group of heterozygotes, the occurrence of the bleeding tendency did not correlate with differences in residual alpha 2-antiplasmin levels and functions. It is concluded that not only the absence of alpha 2-antiplasmin but also a reduction in its plasma level to +/- 60% of normal may predispose to a hemorrhagic diathesis.

scholarly journals Metadherin enhances vulnerability of cancer cells to ferroptosis

2019 ◽  
Vol 10 (10) ◽  
Author(s):  
Jianling Bi ◽  
Shujie Yang ◽  
Long Li ◽  
Qun Dai ◽  
Nicholas Borcherding ◽  
...  
Keyword(s):  
Cancer Therapy ◽  
Cancer Cells ◽  
Messenger Rna ◽  
Underlying Mechanism ◽  
Lipid Hydroperoxides ◽  
Protein Levels ◽  
Enhanced Sensitivity ◽  
Regulated Cell Death

Abstract Ferroptosis is an iron-dependent, non-apoptotic form of regulated cell death driven by lipid hydroperoxides within biological membranes. Although therapy-resistant mesenchymal-high cancers are particularly vulnerable to ferroptosis inducers, especially phospholipid glutathione peroxidase 4 (GPx4) inhibitors, the underlying mechanism is yet to be deciphered. As such, the full application of GPx4 inhibitors in cancer therapy remains challenging. Here we demonstrate that metadherin (MTDH) confers a therapy-resistant mesenchymal-high cell state and enhanced sensitivity to inducers of ferroptosis. Mechanistically, MTDH inhibited GPx4, as well as the solute carrier family 3 member 2 (SLC3A2, a system Xc− heterodimerization partner), at both the messenger RNA and protein levels. Our metabolomic studies demonstrated that MTDH reduced intracellular cysteine, but increased glutamate levels, ultimately decreasing levels of glutathione and setting the stage for increased vulnerability to ferroptosis. Finally, we observed an enhanced antitumor effect when we combined various ferroptosis inducers both in vitro and in vivo; the level of MTDH correlated with the ferroptotic effect. We have demonstrated for the first time that MTDH enhances the vulnerability of cancer cells to ferroptosis and may serve as a therapeutic biomarker for future ferroptosis-centered cancer therapy.

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