scholarly journals ADAM15 participates in fertilization through a physical interaction with acrogranin

Reproduction ◽  
2014 ◽  
Vol 148 (6) ◽  
pp. 623-634 ◽  
Author(s):  
Karina Pastén ◽  
Yadira Bastian ◽  
Ana L Roa-Espitia ◽  
Deneb Maldonado-García ◽  
Guillermo Mendoza-Hernández ◽  
...  
Keyword(s):  
Cell Surface ◽  
Guinea Pig ◽  
Direct Interaction ◽  
Protein Band ◽  
Equatorial Region ◽  
Proteolytic Processing ◽  
Breast Cancer Cell Lines ◽  
Physical Interaction ◽  
Epididymal Maturation ◽  
Mammalian Fertilization

Mammalian fertilization is completed by direct interaction between sperm and egg. This process is primarily mediated by both adhesion and membrane-fusion proteins found on the gamete surface. ADAM1, 2, and 3 are members of the ADAMs protein family, and have been involved in sperm–egg binding. In this study, we demonstrate the proteolytic processing of ADAM15 during epididymal maturation of guinea pig spermatozoa to produce a mature form a size of 45 kDa. We find that the size of the mature ADAM15, 45 kDa, in cauda epididymal spermatozoa indicates that the pro-domain and metalloprotease domain are absent. In addition, using indirect immunofluorescence, ADAM15 was found throughout the acrosome, at the equatorial region and along the flagellum of guinea pig spermatozoa. After acrosome reaction, ADAM15 is lost from the acrosomal region and retained in the equatorial region and flagellum. In this study, we also report the first evidence of a complex between ADAM15 and acrogranin. By immunoprecipitation, we detected a protein band of 65 kDa which co-immunoprecipated together ADAM15. Analysis of the N-terminal sequence of this 65 kDa protein has revealed its identity as acrogranin. In addition, using cell-surface labeling, ADAM15 was found to be present on the cell surface. Assays of heterologous fertilization showed that the antibody against acrogranin inhibited the sperm–egg adhesion. Interestingly, ADAM15 and acrogranin were also found associated in two breast cancer cell lines. In conclusion, our results demonstrated that ADAM15 and acrogranin are present on and associated with the surface of guinea pig spermatozoa; besides both proteins may play a role during sperm–egg binding.

scholarly journals Raf-1 Physically Interacts with Rb and Regulates Its Function: a Link between Mitogenic Signaling and Cell Cycle Regulation

1998 ◽  
Vol 18 (12) ◽  
pp. 7487-7498 ◽  
Author(s):  
Sheng Wang ◽  
Richik N. Ghosh ◽  
Srikumar P. Chellappan
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Growth Factor ◽  
Cell Surface ◽  
Mammalian Cells ◽  
Direct Interaction ◽  
Physical Interaction ◽  
Proliferating Cells

ABSTRACT Cells initiate proliferation in response to growth factor stimulation, but the biochemical mechanisms linking signals received at the cell surface receptors to the cell cycle regulatory molecules are not yet clear. In this study, we show that the signaling molecule Raf-1 can physically interact with Rb and p130 proteins in vitro and in vivo and that this interaction can be detected in mammalian cells without overexpressing any component. The binding of Raf-1 to Rb occurs subsequent to mitogen stimulation, and this interaction can be detected only in proliferating cells. Raf-1 can inactivate Rb function and can reverse Rb-mediated repression of E2F1 transcription and cell proliferation efficiently. The region of Raf-1 involved in Rb binding spanned residues 1 to 28 at the N terminus, and functional inactivation of Rb required a direct interaction. Serum stimulation of quiescent human fibroblast HSF8 cells led to a partial translocation of Raf-1 into the nucleus, where it colocalized with Rb. Further, Raf-1 was able to phosphorylate Rb in vitro quite efficiently. We believe that the physical interaction of Raf-1 with Rb is a vital step in the growth factor-mediated induction of cell proliferation and that Raf-1 acts as a direct link between cell surface signaling cascades and the cell cycle machinery.

scholarly journals Direct Interaction of Polar Scaffolding Protein Wag31 with Nucleoid-Associated Protein Rv3852 Regulates Its Polar Localization

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1558
Author(s):  
Rajni Garg ◽  
Chinmay Anand ◽  
Sohini Ganguly ◽  
Sandhya Rao ◽  
Rinkee Verma ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Envelope ◽  
Transmembrane Helix ◽  
Ectopic Expression ◽  
Direct Interaction ◽  
Fine Tuning ◽  
Scaffolding Protein ◽  
Physical Interaction ◽  
Cell Wall Synthesis ◽  
Interaction Approach

Rv3852 is a unique nucleoid-associated protein (NAP) found exclusively in Mycobacterium tuberculosis (Mtb) and closely related species. Although annotated as H-NS, we showed previously that it is very different from H-NS in its properties and is distinct from other NAPs, anchoring to cell membrane by virtue of possessing a C-terminal transmembrane helix. Here, we investigated the role of Rv3852 in Mtb in organizing architecture or synthesis machinery of cell wall by protein–protein interaction approach. We demonstrated a direct physical interaction of Rv3852 with Wag31, an important cell shape and cell wall integrity determinant essential in Mtb. Wag31 localizes to the cell poles and possibly acts as a scaffold for cell wall synthesis proteins, resulting in polar cell growth in Mtb. Ectopic expression of Rv3852 in M. smegmatis resulted in its interaction with Wag31 orthologue DivIVAMsm. Binding of the NAP to Wag31 appears to be necessary for fine-tuning Wag31 localization to the cell poles, enabling complex cell wall synthesis in Mtb. In Rv3852 knockout background, Wag31 is mislocalized resulting in disturbed nascent peptidoglycan synthesis, suggesting that the NAP acts as a driver for localization of Wag31 to the cell poles. While this novel association between these two proteins presents one of the mechanisms to structure the elaborate multi-layered cell envelope of Mtb, it also exemplifies a new function for a NAP in mycobacteria.

scholarly journals BRG1 Controls the Activity of the Retinoblastoma Protein via Regulation of p21CIP1/WAF1/SDI

2004 ◽  
Vol 24 (3) ◽  
pp. 1188-1199 ◽  
Author(s):  
Hyeog Kang ◽  
Kairong Cui ◽  
Keji Zhao
Keyword(s):  
Transcriptional Repression ◽  
Cell Cycle Progression ◽  
Target Genes ◽  
Cellular Proliferation ◽  
Growth Arrest ◽  
Retinoblastoma Protein ◽  
Direct Interaction ◽  
Physical Interaction ◽  
Cdk Inhibitor ◽  
Regulation Of Cell Cycle

ABSTRACT The ubiquitous mammalian chromatin-remodeling SWI/SNF-like BAF complexes play critical roles in tumorigenesis. It was suggested that the direct interaction of BRG1 with the retinoblastoma protein pRB is required for regulation of cell cycle progression by pRB. We present evidence that the BRG1-containing complexes regulate the expression of the cdk inhibitor p21CIP1/WAF1/SDI. Furthermore, we show that the physical interaction between BRG1 and pRB is not required for induction of cell growth arrest and transcriptional repression of E2F target genes by pRB. Instead, BRG1 activates pRB by inducing its hypophosphorylation through up-regulation of the cdk inhibitor p21. The hypophosphorylation of pRB is reinforced by down-regulation of critical components, including cdk2, cyclin E, and cyclin D, in the pRB regulatory network. We demonstrate that up-regulation of p21 by BRG1 is necessary to induce formation of flat cells, growth arrest, and finally, cell senescence. Our results suggest that the BRG1-containing complexes control cellular proliferation and senescence by modulating the pRB pathway via multiple mechanisms.

Immunocytochemical alterations in the intra-acrosomal antigen MN7 during epididymal maturation of guinea pig spermatozoa

1998 ◽  
Vol 292 (2) ◽  
pp. 427-433 ◽  
Author(s):  
K. Yoshinaga ◽  
I. Tanii ◽  
D. K. Saxena ◽  
K. Toshimori
Keyword(s):  
Guinea Pig ◽  
Epididymal Maturation

scholarly journals Genetic and physical interaction of Drosophila Ino80 with Polycomb Responsive Element

2019 ◽  
Author(s):  
Mohsen Ghasemi ◽  
Jayant Maini ◽  
Shruti Jain ◽  
Vasanthi Dasari ◽  
Rakesh Mishra ◽  
...  
Keyword(s):  
Direct Interaction ◽  
Apparent Competition ◽  
Imaginal Discs ◽  
Physical Interaction ◽  
Homeotic Genes ◽  
Knock Down ◽  
Polycomb Proteins ◽  
Independent Functions ◽  
Regulatory Complex ◽  
Responsive Element

AbstractThe chromatin remodeling protein, dIno80 (Drosophila Ino80) regulates homeotic genes. We show that Ino80, along with Trx and ETP (Enhancer of Trithorax and Polycomb) proteins, interacts with two Polycomb/Trithorax Responsive Elements (PRE/TRE), iab-7 and bxd PRE in flies and the larval imaginal discs. In S2 cells, dIno80 localizes to the endogenous iab-7 and bxd-PREs. The localization of Ino80 and Pleiohomeotic (Pho) at the PRE is sensitive to the cellular abundance of each other; when levels of Ino80 are limiting, there is increased Pho enrichment, and Pho knock-down leads to increased enrichment of Ino80. We demonstrate that over-expression of dIno80 rescues the pupal lethality in pleiohomeotic (pho) deficient flies, which suggests that dIno80 has a role in cellular memory. The apparent competition between Pho and Ino80 for binding at the PRE indicates that Ino80 may act as a potential recruiter of the regulatory complex in addition to being a chromatin remodeler.Author SummaryThe null mutants of Pho and dIno80 show lethality at different stages of development in the fly, implying that they may function independent of each other. The observation that Pho-lethality can be rescued by overexpression of dIno80 with significant penetrance and that Ino80 has its own DNA binding domain, led us to predict that Ino80 may have Pho-independent functions, perhaps through non-canonical complexes. In the current study, we show that dIno80 interacts with bxd and iab-7 PRE in cooperation with Polycomb and Trithorax proteins and regulate the homeotic genes. The effect of knock-down or mutation of dIno80 results in altered phenotype in adult flies and rescue of Lac-Z expression in imaginal discs, in parallel with similar effect of Pho mutation or knock-down. We provide evidence of direct interaction of dIno80 with iab7- and bxd-PRE using chromatin immunoprecipitation. The dIno80 localization in and around the PRE sequence was enhanced in the absence of Pho, indicating competition between Pho and dIno80 for binding at the PRE.

Testase 1 (ADAM 24) a plasma membrane-anchored sperm protease implicated in sperm function during epididymal maturation or fertilization

2001 ◽  
Vol 114 (9) ◽  
pp. 1787-1794 ◽  
Author(s):  
G.Z. Zhu ◽  
D.G. Myles ◽  
P. Primakoff
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Acrosome Reaction ◽  
Phosphorylation Site ◽  
Equatorial Region ◽  
Proteolytic Processing ◽  
Mature Sperm ◽  
Kinase C

Plasma membrane-anchored proteases have key roles in cell signaling, migration and refashioning the cell surface and its surroundings. We report the first example of a plasma membrane-anchored protease on mature sperm, testase 1 (ADAM 24). Unlike other studied sperm ADAMs (fertilin (α) and (β), cyritestin) whose metalloprotease domains are removed during sperm development, we found testase 1 retains an active metalloprotease domain, suggesting it acts as a protease on mature sperm. Testase 1 is a glycoprotein (molecular mass 88 kDa), localized to the equatorial region of the plasma membrane of cauda epididymal sperm. Typically, proteolytic removal of the pro-domain is an initial activation step for ADAM proteases. The pro-domain of the testase 1 precursor (108 kDa) is proteolytically removed as sperm transit the caput epididymis to produce processed (mature) testase 1 (88 kDa). Testase 1 is unique among all studied ADAMs in that its proteolytic processing occurs on the sperm plasma membrane instead of at an intracellular site (the Golgi). Using GST-fusion proteins and a synthetic testase 1 C-terminal peptide, we found that the cytoplasmic tail of testase 1 could be phosphorylated in vitro by protein kinase C (PKC). Thus testase 1 apparently has a cytoplasmic PKC phosphorylation site(s). Protein kinase C is known to stimulate other ADAMs' protease activity. Because events of the acrosome reaction include PKC activation, we speculate that testase 1 protease function could be important in sperm penetration of the zona pellucida after sperm PKC is activated during the acrosome reaction.

scholarly journals Mertk Interacts with Tim-4 to Enhance Tim-4-Mediated Efferocytosis

Cells ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 1625 ◽  
Author(s):  
Byeongjin Moon ◽  
Juyeon Lee ◽  
Sang-Ah Lee ◽  
Chanhyuk Min ◽  
Hyunji Moon ◽  
...  
Keyword(s):  
Cell Surface ◽  
Molecular Mechanism ◽  
Soluble Form ◽  
Physical Interaction ◽  
Apoptotic Cells ◽  
Type Iii ◽  
Biochemical Interaction ◽  
Fibronectin Type Iii ◽  
Fibronectin Type Iii Domain ◽  
Fibronectin Type

Apoptotic cells expressing phosphatidylserine (PS) on their cell surface are directly or indirectly recognized by phagocytes through PS-binding proteins. The PS-binding protein Tim-4 secures apoptotic cells to phagocytes to facilitate the engulfment of apoptotic cells. However, the molecular mechanism by which Tim-4 transduces signals to phagocytes during Tim-4-mediated efferocytosis is incompletely understood. Here, we report that Tim-4 collaborates with Mertk during efferocytosis through a biochemical interaction with Mertk. Proximal localization between the two proteins in phagocytes was observed by immunofluorescence and proximal ligation assays. Physical association between Tim-4 and Mertk, which was mediated by an interaction between the IgV domain of Tim-4 and the fibronectin type-III domain of Mertk, was also detected with immunoprecipitation. Furthermore, the effect of Mertk on Tim-4-mediated efferocytosis was abolished by GST-MertkFnIII, a soluble form of the fibronectin type-III domain of Mertk that disrupts the interaction between Tim-4 and Mertk. Taken together, the results from our study suggest that a physical interaction between Tim-4 and Mertk is necessary for Mertk to enhance efferocytosis mediated by Tim-4.

scholarly journals MicroRNA-224 Promotes Tumorigenesis through Downregulation of Caspase-9 in Triple-Negative Breast Cancer

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Li Zhang ◽  
Xin Zhang ◽  
Xin Wang ◽  
Miao He ◽  
Shixing Qiao
Keyword(s):  
Breast Cancer ◽  
Triple Negative Breast Cancer ◽  
Triple Negative ◽  
Direct Interaction ◽  
Luciferase Reporter ◽  
Migration And Invasion ◽  
Breast Cancer Cell Lines ◽  
Protein Levels ◽  
Reporter Assays ◽  
Novel Therapeutic Strategies

Triple-negative breast cancer (TNBC) harbors genetic heterogeneity and generally has more aggressive clinical outcomes. As such, there is urgency in identifying new prognostic targets and developing novel therapeutic strategies. In this study, miR-224 was overexpressed in breast cancer cell lines and TNBC primary cancer samples. Knockdown of miR-224 in MDA-MB-231 cancer cells reduced cell proliferation, migration, and invasion. Through integrating in silico prediction algorithms with KEGG pathway and Gene Ontology analyses, CASP9 was identified to be a potential target of miR-224. miR-224 knockdown significantly increased CASP9 transcript and protein levels. Furthermore, luciferase reporter assays confirmed a direct interaction of miR-224 with CASP9. Our findings have demonstrated that the miR-224/CASP9 axis plays an important role in TNBC progression, providing evidence in support of a promising therapeutic strategy for this disease.

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