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.

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.

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 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 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 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 Long Non-Coding RNA FOXD1-AS1 Promotes the Progression and Glycolysis of Nasopharyngeal Carcinoma by Sustaining FOXD1 Expression

2020 ◽  
Author(s):  
Zhanwang Wang ◽  
Dong He ◽  
Yuxing Zhu ◽  
Xueying Hu ◽  
Yi Jin ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Molecular Mechanisms ◽  
Bioinformatics Analysis ◽  
Regulatory Role ◽  
Loss Of Function ◽  
Over Expression ◽  
Protein Levels ◽  
Non Coding Rna

Abstract Background: Increasing evidence have emphasized the importance of long non-coding RNAs (lncRNAs) in various human cancers progression. Forkhead box D1 antisense RNA1 (FOXD1-AS1) is a novel lncRNA and plays vital regulatory role in diverse biological processes of cancers. However, the biological function, molecular mechanism and clinical significance of FOXD1-AS1 in nasopharyngeal carcinoma is still unknown.Methods: Comprehensive bioinformatics analysis and qRT-PCR was conducted to detect the expression level of FOXD1-AS1. Loss-of-function and gain-of-function experiments were performed to verify the functions of FOXD1-AS1 in proliferation, migration, invasion, apoptosis and glycolysis of nasopharyngeal carcinoma in vitro and in vivo. Further bioinformatics analysis and experiments were carried out to explore the underlying molecular mechanisms of FOXD1-AS1. Results: FOXD1-AS1 was significantly overexpressed in nasopharyngeal carcinoma and associated with poor survival in patients. Knockdown of FOXD1-AS1 significantly inhibited cell proliferation, migration, invasion and glycolysis, and promotes apoptosis in nasopharyngeal carcinoma, whereas over-expression of FOXD1-AS1 has the opposite effect. Mechanistically, we found that FOXD1-AS1 could upregulate the expression of FOXD1 through stabilizing the FOXD1 expression at mRNA and protein levels, and FOXD1 increased the glycolysis level by transcriptionally upregulating the expression of LDHA, PKM and ENO1, thus playing an oncogenic role in nasopharyngeal carcinoma progression. Conclusion: FOXD1-AS1 plays a critical regulatory role in nasopharyngeal carcinoma. The identified FOXD1-AS1/FOXD1 axis may serve as a potential prognostic biomarker and therapeutic target for patients with nasopharyngeal carcinoma.

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

2021 ◽  
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 Regulation of Chk1 by Its C-terminal Domain

2008 ◽  
Vol 19 (11) ◽  
pp. 4546-4553 ◽  
Author(s):  
Ana Kosoy ◽  
Matthew J. O'Connell
Keyword(s):  
Catalytic Domain ◽  
Kinase Domain ◽  
Temperature Sensitive ◽  
Loss Of Function ◽  
Terminal Domain ◽  
C Terminus ◽  
The Face ◽  
Chk1 Kinase

Chk1 is a protein kinase that is the effector molecule in the G2 DNA damage checkpoint. Chk1 homologues have an N-terminal kinase domain, and a C-terminal domain of ∼200 amino acids that contains activating phosphorylation sites for the ATM/R kinases, though the mechanism of activation remains unknown. Structural studies of the human Chk1 kinase domain show an open conformation; the activity of the kinase domain alone is substantially higher in vitro than full-length Chk1, and coimmunoprecipitation studies suggest the C-terminal domain may contain an autoinhibitory activity. However, we show that truncation of the C-terminal domain inactivates Chk1 in vivo. We identify additional mutations within the C-terminal domain that activate ectopically expressed Chk1 without the need for activating phosphorylation. When expressed from the endogenous locus, activated alleles show a temperature-sensitive loss of function, suggesting these mutations confer a semiactive state to the protein. Intragenic suppressors of these activated alleles cluster to regions in the catalytic domain on the face of the protein that interacts with substrate, suggesting these are the regions that interact with the C-terminal domain. Thus, rather than being an autoinhibitory domain, the C-terminus of Chk1 also contains domains critical for adopting an active configuration.

scholarly journals Gemin3 Is an Essential Gene Required for Larval Motor Function and Pupation in Drosophila

2009 ◽  
Vol 20 (1) ◽  
pp. 90-101 ◽  
Author(s):  
Karl B. Shpargel ◽  
Kavita Praveen ◽  
T. K. Rajendra ◽  
A. Gregory Matera
Keyword(s):  
Protein Function ◽  
Muscular Atrophy ◽  
Essential Gene ◽  
Survival Motor Neuron ◽  
Loss Of Function ◽  
Transposon Insertion ◽  
Associated Proteins ◽  
Smn Protein

The assembly of metazoan Sm-class small nuclear ribonucleoproteins (snRNPs) is an elaborate, step-wise process that takes place in multiple subcellular compartments. The initial steps, including formation of the core RNP, are mediated by the survival motor neuron (SMN) protein complex. Loss-of-function mutations in human SMN1 result in a neuromuscular disease called spinal muscular atrophy. The SMN complex is comprised of SMN and a number of tightly associated proteins, collectively called Gemins. In this report, we identify and characterize the fruitfly ortholog of the DEAD box protein, Gemin3. Drosophila Gemin3 (dGem3) colocalizes and interacts with dSMN in vitro and in vivo. RNA interference for dGem3 codepletes dSMN and inhibits efficient Sm core assembly in vitro. Transposon insertion mutations in Gemin3 are larval lethals and also codeplete dSMN. Transgenic overexpression of dGem3 rescues lethality, but overexpression of dSMN does not, indicating that loss of dSMN is not the primary cause of death. Gemin3 mutant larvae exhibit motor defects similar to previously characterized Smn alleles. Remarkably, appreciable numbers of Gemin3 mutants (along with one previously undescribed Smn allele) survive as larvae for several weeks without pupating. Our results demonstrate the conservation of Gemin3 protein function in metazoan snRNP assembly and reveal that loss of either Smn or Gemin3 can contribute to neuromuscular dysfunction.

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