scholarly journals Male infertility-linked point mutation reveals a vital binding role for the C2 domain of sperm PLCζ

2017 ◽  
Vol 474 (6) ◽  
pp. 1003-1016 ◽  
Author(s):  
Michail Nomikos ◽  
Panagiotis Stamatiadis ◽  
Jessica R. Sanders ◽  
Konrad Beck ◽  
Brian L. Calver ◽  
...  
Keyword(s):  
Male Infertility ◽  
Point Mutation ◽  
Biochemical Properties ◽  
Vital Role ◽  
Egg Activation ◽  
C2 Domain ◽  
Binding Characteristics

Sperm-specific phospholipase C zeta (PLCζ) is widely considered to be the physiological stimulus that evokes intracellular calcium (Ca2+) oscillations that are essential for the initiation of egg activation during mammalian fertilisation. A recent genetic study reported a male infertility case that was directly associated with a point mutation in the PLCζ C2 domain, where an isoleucine residue had been substituted with a phenylalanine (I489F). Here, we have analysed the effect of this mutation on the in vivo Ca2+ oscillation-inducing activity and the in vitro biochemical properties of human PLCζ. Microinjection of cRNA or recombinant protein corresponding to PLCζI489F mutant at physiological concentrations completely failed to cause Ca2+ oscillations and trigger development. However, this infertile phenotype could be effectively rescued by microinjection of relatively high (non-physiological) amounts of recombinant mutant PLCζI489F protein, leading to Ca2+ oscillations and egg activation. Our in vitro biochemical analysis suggested that the PLCζI489F mutant displayed similar enzymatic properties, but dramatically reduced binding to PI(3)P and PI(5)P-containing liposomes compared with wild-type PLCζ. Our findings highlight the importance of PLCζ at fertilisation and the vital role of the C2 domain in PLCζ function, possibly due to its novel binding characteristics.

scholarly journals Activation of MAT2A-RIP1 signaling axis reprograms monocytes in gastric cancer

2021 ◽  
Vol 9 (2) ◽  
pp. e001364
Author(s):  
Yan Zhang ◽  
Hui Yang ◽  
Jun Zhao ◽  
Ping Wan ◽  
Ye Hu ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cell Metabolism ◽  
Vital Role ◽  
Methionine Metabolism ◽  
Promoter Regions ◽  
Normal Tissues ◽  
Methionine Cycle

BackgroundThe activation of tumor-associated macrophages (TAMs) facilitates the progression of gastric cancer (GC). Cell metabolism reprogramming has been shown to play a vital role in the polarization of TAMs. However, the role of methionine metabolism in function of TAMs remains to be explored.MethodsMonocytes/macrophages were isolated from peripheral blood, tumor tissues or normal tissues from healthy donors or patients with GC. The role of methionine metabolism in the activation of TAMs was evaluated with both in vivo analyses and in vitro experiments. Pharmacological inhibition of the methionine cycle and modulation of key metabolic genes was employed, where molecular and biological analyses were performed.ResultsTAMs have increased methionine cycle activity that are mainly attributed to elevated methionine adenosyltransferase II alpha (MAT2A) levels. MAT2A modulates the activation and maintenance of the phenotype of TAMs and mediates the upregulation of RIP1 by increasing the histone H3K4 methylation (H3K4me3) at its promoter regions.ConclusionsOur data cast light on a novel mechanism by which methionine metabolism regulates the anti-inflammatory functions of monocytes in GC. MAT2A might be a potential therapeutic target for cancer cells as well as TAMs in GC.

scholarly journals The Emerging Role of Neutrophil Extracellular Traps in Arterial, Venous and Cancer-Associated Thrombosis

2021 ◽  
Vol 8 ◽  
Author(s):  
Yilu Zhou ◽  
Weimin Tao ◽  
Fuyi Shen ◽  
Weijia Du ◽  
Zhendong Xu ◽  
...  
Keyword(s):  
Current Knowledge ◽  
Neutrophil Extracellular Traps ◽  
Vital Role ◽  
Extracellular Traps ◽  
Protein Components ◽  
Cancer Associated Thrombosis ◽  
Coagulation Pathway

Neutrophils play a vital role in the formation of arterial, venous and cancer-related thrombosis. Recent studies have shown that in a process known as NETosis, neutrophils release proteins and enzymes complexed to DNA fibers, collectively called neutrophil extracellular traps (NETs). Although NETs were originally described as a way for the host to capture and kill bacteria, current knowledge indicates that NETs also play an important role in thrombosis. According to recent studies, the destruction of vascular microenvironmental homeostasis and excessive NET formation lead to pathological thrombosis. In vitro experiments have found that NETs provide skeletal support for platelets, red blood cells and procoagulant molecules to promote thrombosis. The protein components contained in NETs activate the endogenous coagulation pathway to promote thrombosis. Therefore, NETs play an important role in the formation of arterial thrombosis, venous thrombosis and cancer-related thrombosis. This review will systematically summarize and explain the study of NETs in thrombosis in animal models and in vivo experiments to provide new targets for thrombosis prevention and treatment.

scholarly journals Transcription factor Krüppel-like factor 2 plays a vital role in endothelial colony forming cells differentiation

2013 ◽  
Vol 99 (3) ◽  
pp. 514-524 ◽  
Author(s):  
Yimeng Song ◽  
Xiaoxia Li ◽  
Dawei Wang ◽  
Chenglai Fu ◽  
Zhenjiu Zhu ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Tube Formation ◽  
Vital Role ◽  
Ampk Activation ◽  
Cell Markers ◽  
Endothelial Colony Forming Cells ◽  
Angiogenesis Assay

Abstract Aims Endothelial colony forming cells (ECFCs) participate in post-natal vasculogenesis. We previously reported that vascular endothelial growth factor (VEGF) promotes human ECFC differentiation through AMP-activated protein kinase (AMPK) activation. However, the mechanisms underlying transcriptional regulation of ECFC differentiation still remain largely elusive. Here, we investigated the role of transcription factor Krüppel-like factor 2 (KLF2) in the regulation of ECFC function. Methods and results Human ECFCs were isolated from cord blood and cultured. Treatment with VEGF significantly increased endothelial markers in ECFCs and their capacity for migration and tube formation. The mRNA and protein levels of KLF2 were also significantly up-regulated. This up-regulation was abrogated by AMPK inhibition or by knockdown of KLF2 with siRNA. Furthermore, adenovirus-mediated overexpression of KLF2 promoted ECFC differentiation by enhancing expression of endothelial cell markers, reducing expression of progenitor cell markers, and increasing the capacity for tube formation in vitro, indicating the important role of KLF2 in ECFC-mediated angiogenesis. Histone deacetylase 5 (HDAC5) was phosphorylated by AMPK activity induced by VEGF and the AMPK agonist AICAR (5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide). In vivo angiogenesis assay revealed that overexpression of KLF2 in bone-marrow-derived pro-angiogenic progenitor cells promoted vessel formation when the cells were implanted in C57BL/6 mice. Conclusion Up-regulation of KLF2 by AMPK activation constitutes a novel mechanism of ECFC differentiation, and may have therapeutic value in the treatment of ischaemic heart disease.

scholarly journals The Bacterial Second Messenger Cyclic di-GMP Regulates Brucella Pathogenesis and Leads to Altered Host Immune Response

2016 ◽  
Vol 84 (12) ◽  
pp. 3458-3470 ◽  
Author(s):  
Mike Khan ◽  
Jerome S. Harms ◽  
Fernanda M. Marim ◽  
Leah Armon ◽  
Cherisse L. Hall ◽  
...  
Keyword(s):  
Immune Response ◽  
Second Messenger ◽  
Host Immune Response ◽  
Vital Role ◽  
Host Cells ◽  
Content Type ◽  
Type Iv

Brucella species are facultative intracellular bacteria that cause brucellosis, a chronic debilitating disease significantly impacting global health and prosperity. Much remains to be learned about how Brucella spp. succeed in sabotaging immune host cells and how Brucella spp. respond to environmental challenges. Multiple types of bacteria employ the prokaryotic second messenger cyclic di-GMP (c-di-GMP) to coordinate responses to shifting environments. To determine the role of c-di-GMP in Brucella physiology and in shaping host- Brucella interactions, we utilized c-di-GMP regulatory enzyme deletion mutants. Our results show that a Δ bpdA phosphodiesterase mutant producing excess c-di-GMP displays marked attenuation in vitro and in vivo during later infections. Although c-di-GMP is known to stimulate the innate sensor STING, surprisingly, the Δ bpdA mutant induced a weaker host immune response than did wild-type Brucella or the low-c-di-GMP guanylate cyclase Δ cgsB mutant. Proteomics analysis revealed that c-di-GMP regulates several processes critical for virulence, including cell wall and biofilm formation, nutrient acquisition, and the type IV secretion system. Finally, Δ bpdA mutants exhibited altered morphology and were hypersensitive to nutrient-limiting conditions. In summary, our results indicate a vital role for c-di-GMP in allowing Brucella to successfully navigate stressful and shifting environments to establish intracellular infection.

Biochemical characterization of human DNA polymerase i provides clues to its biological function

2001 ◽  
Vol 29 (2) ◽  
pp. 183-187 ◽  
Author(s):  
A. Tissier ◽  
E. G. Frank ◽  
J. P. McDonald ◽  
A. Vaisman ◽  
A. R. Fernàndez deHenestrosa Henestrosa ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Biochemical Characterization ◽  
Short Review ◽  
Biochemical Properties ◽  
Dna Polymerase I ◽  
Polymerase I

The human RAD30B gene has recently been shown to encode a novel DNA polymerase, DNA polymerase i (poli). The role of poli within the cell is presently unknown, and the only clues to its cellular function come from its biochemical characterization in vitro. The aim of this short review is, therefore, to summarize the known enzymic activities of poli and to speculate as to how these biochemical properties might relate to its in vivo function.

scholarly journals CMTM7 as a novel molecule of ATG14L-Beclin1-VPS34 complex enhances autophagy by Rab5 to regulate tumorigenicity

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Baocai Liu ◽  
Yinliang Lu ◽  
Tingting Zhang ◽  
Xinyue Yu ◽  
Qian Wang ◽  
...  
Keyword(s):  
Early Stage ◽  
Xenograft Model ◽  
Vital Role ◽  
Autophagic Flux ◽  
In Vivo Experiments ◽  
Transmission Electron ◽  
Autophagy Induction

Abstract Background CMTM7 is a tumor suppressor that positively regulates EGFR degradation by promoting Rab5 activation, and plays a vital role in tumor progression. Rab5 forms complexes with Beclin1 and VPS34, and acts in the early stage of autophagy. However, the affects of CMTM7 on autophagy and its mechanism are still unclear. Methods The effect of CMTM7 on autophagy induction was confirmed by western blotting, confocal microscopy and transmission electron microscopy. Co-immunoprecipitation was used to analyse the interaction of CMTM7 with autophagy initiation complex and Rab5. The xenograft model in nude mice was used to elucidate the function of CMTM7 in tumorigenicity and autophagy in vivo. Results In this study, we first demonstrated that CMTM7 facilitated the initiation of autophagosome formation, which consequently promoted the subsequent multistage process of autophagic flux, i.e. from autophagosome assembly till autolysosome formation and degradation. Confocal and co-immunoprecipitation showed that CMTM7 interacted with Rab5, VPS34, Beclin1, and ATG14L, but not with ULK1, UVRAG and LC3B. CMTM7 also increased the activity of ATG14L-linked VPS34 complex and its association with Rab5. Both in vitro and in vivo experiments demonstrated that knockdown of CMTM7 enhanced tumor growth by impairing autophagy. Conclusion These findings highlighted the role of CMTM7 in the regulation of autophagy and tumorigenicity, revealing it as a novel molecule that is associated with the interaction of Rab5 and ATG14L-Beclin1-VPS34 complex.

scholarly journals Regulator of calcineurin 1 gene isoform 4 in pancreatic ductal adenocarcinoma regulates the progression of tumor cells

Oncogene ◽  
2021 ◽  
Author(s):  
Mengyi Lao ◽  
Xiaozhen Zhang ◽  
Tao Ma ◽  
Jian Xu ◽  
Hanshen Yang ◽  
...  
Keyword(s):  
Pancreatic Ductal Adenocarcinoma ◽  
Tumor Cells ◽  
Vital Role ◽  
Ductal Adenocarcinoma ◽  
Migration And Invasion ◽  
Metastasis Model ◽  
Inducible Protein

AbstractTherapeutic strategies to treat pancreatic ductal adenocarcinoma (PDAC) remain unsatisfying and limited. Therefore, it is imperative to fully determine the mechanisms underlying PDAC progression. In the present study, we report a novel role of regulator of calcineurin 1, isoform 4 (RCAN1.4) in regulating PDAC progression. We demonstrated that RCAN1.4 expression was decreased significantly in PDAC tissues compared with that in para-cancerous tissues, and correlated with poor prognosis of patients with pancreatic cancer. In vitro, stable high expression of RCAN1.4 could suppress the metastasis and proliferation and angiogenesis of pancreatic tumor cells. In addition, interferon alpha inducible protein 27 (IFI27) was identified as having a functional role in RCAN1.4-mediated PDAC migration and invasion, while VEGFA play a vital role in RCAN1.4-mediated PDAC angiogenesis. Analysis of mice with subcutaneously/orthotopic implanted xenograft tumors and liver metastasis model confirmed that RCAN1.4 could modulate the growth, metastasis, and angiogenesis of tumors via IFI27/VEGFA in vivo. In conclusion, our results suggested that RCAN1.4 suppresses the growth, metastasis, and angiogenesis of PDAC, functioning partly via IFI27 and VEGFA. Importantly, our results provided possible diagnostic criteria and therapeutic targets for PDAC.

scholarly journals Male infertility-linked point mutation disrupts the Ca2+ oscillation-inducing and PIP2 hydrolysis activity of sperm PLCζ

2011 ◽  
Vol 434 (2) ◽  
pp. 211-217 ◽  
Author(s):  
Michail Nomikos ◽  
Khalil Elgmati ◽  
Maria Theodoridou ◽  
Brian L. Calver ◽  
Bevan Cumbes ◽  
...  
Keyword(s):  
Male Infertility ◽  
Point Mutation ◽  
Dominant Negative ◽  
Wild Type ◽  
Normal Pattern ◽  
Mouse Eggs ◽  
Hydrolysis Activity

A male infertility-linked human PLCζ (phospholipase Cζ) mutation introduced into mouse PLCζ completely abolishes both in vitro PIP2 (phosphatidylinositol 4,5-bisphosphate) hydrolysis activity and the ability to trigger in vivo Ca2+ oscillations in mouse eggs. Wild-type PLCζ initiated a normal pattern of Ca2+ oscillations in eggs in the presence of 10-fold higher mutant PLCζ, suggesting that infertility is not mediated by a dominant-negative mechanism.

scholarly journals LncRNA TUG1 sponges miR-204-5p to promote osteoblast differentiation through upregulating Runx2 in aortic valve calcification

2017 ◽  
Vol 114 (1) ◽  
pp. 168-179 ◽  
Author(s):  
Cong Yu ◽  
Lifu Li ◽  
Fei Xie ◽  
Shichao Guo ◽  
Fayuan Liu ◽  
...  
Keyword(s):  
Aortic Valve ◽  
Osteoblast Differentiation ◽  
Vital Role ◽  
Transcriptional Level ◽  
Calcific Aortic Valve Disease ◽  
Mirna Sponge ◽  
Lncrna Tug1

Abstract Aims Emerging evidence indicates that long non-coding RNAs (lncRNAs) play a vital role in cardiovascular physiology and pathology. Although the lncRNA TUG1 is implicated in atherosclerosis, its function in calcific aortic valve disease (CAVD) remains unknown. Methods and results In this study, we found that TUG1 was highly expressed in human aortic valves and primary valve interstitial cells (VICs). Moreover, TUG1 knockdown induced inhibition of osteoblast differentiation in CAVD both in vitro and in vivo. Mechanistically, silencing of TUG1 increased the expression of miR-204-5p and subsequently inhibited Runx2 expression at the post-transcriptional level. Importantly, TUG1 directly interacted with miR-204-5p and downregulation of miR-204-5p efficiently reversed the suppression of Runx2 induced by TUG1 short hairpin RNA (shRNA). Thus, TUG1 positively regulated the expression of Runx2, through sponging miR-204-5p, and promoted osteogenic differentiation in CAVD. Conclusion All together, the evidence generated by our study elucidates the role of lncRNA TUG1 as a miRNA sponge in CAVD, and sheds new light on lncRNA-directed diagnostics and therapeutics in CAVD.

scholarly journals Targeting ERK induced cell death and p53/ROS-dependent protective autophagy in colorectal cancer

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Wunan Mi ◽  
Chuyue Wang ◽  
Guang Luo ◽  
Jiehan Li ◽  
Yizheng Zhang ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Cell Death ◽  
Vital Role ◽  
In Vivo Studies ◽  
Dependent Manner ◽  
Treatment Groups ◽  
Combinational Treatment

AbstractIn recent years, many studies have shown that autophagy plays a vital role in the resistance of tumor chemotherapy. However, the interaction between autophagy and cell death has not yet been clarified. In this study, a new specific ERK inhibitor CC90003 was found to suppress colorectal cancer growth by inducing cell death both in vitro and in vivo. Studies have confirmed that higher concentrations of ROS leads to autophagy or cell death. In this research, the role of CC90003-induced ROS was verified. But after inhibiting ROS by two kinds of ROS inhibitors NAC and SFN, the autophagy induced by CC90003 decreased, while cell death strengthened. In parallel, protective autophagy was also induced, while in a p53-dependent manner. After silencing p53 or using the p53 inhibitor PFTα, the autophagy induced by CC90003 was weakened and the rate of cell death increases. Therefore, we confirmed that CC90003 could induce autophagy by activating ROS/p53. Furthermore, in the xenograft mouse model, the effect was obtained remarkably in the combinational treatment group of CC90003 plus CQ, comparing with that of the single treatment groups. In a word, our results demonstrated that targeting ERK leads to cell death and p53/ROS-dependent protective autophagy simultaneously in colorectal cancer, which offers new potential targets for clinical therapy.

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