Article

Detection of a secreted metalloprotease within the nuclei of liver cells.

Laboratory of Hemostasis, Division of Hematology, Center for Biologic, Food and Drug Administration, Bethesda, Maryland 20892, USA.
Molecular BioSystems (Impact Factor: 3.18). 06/2011; 7(6):2012-8. DOI: 10.1039/c0mb00303d
Source: PubMed

ABSTRACT ADAMTS13 is a secreted zinc metalloprotease expressed by various cell types. Here, we investigate its cellular pathway in endogenously expressing liver cell lines and after transient transfection with ADAMTS13. Besides compartmentalizations of the cellular secretory system, we detected an appreciable level of endogenous ADAMTS13 within the nucleus. A positively charged amino acid cluster (R-Q-R-Q-R-Q-R-R) present in the ADAMTS13 propeptide may act as a nuclear localization signal (NLS). Fusing this NLS-containing region to eGFP greatly potentiated its nuclear localization. Bioinformatics analysis suggests that the ADAMTS13 CUB-2 domain has a double-stranded beta helix (DSBH) structural architecture characteristic of various protein-protein interaction modules like nucleoplasmins, class I collagenase, tumor necrosis factor ligand superfamily, supernatant protein factor (SPF) and the B1 domain of neuropilin-2. Based on this contextual evidence and that largely conserved polar residues could be mapped on to a template CUB domain homolog, we hypothesize that a region in the ADAMTS13 CUB-2 domain with conserved polar residues might be involved in protein-protein interaction within the nucleus.

0 Followers
 · 
105 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Coagulation factor IX (FIX) is a serine protease that plays a pivotal role in the blood coagulation cascade. FIX deficiency leads to a blood clotting disorder known as haemophilia B. FIX, synthesized as a prepro-peptide of 461 amino acids, is processed and secreted into plasma. The protein undergoes numerous modifications, including, but not limited to glycosylation, γ-carboxylation and disulphide bond formation. Upon processing and limited proteolysis, the protein is converted into an active protease. Under physiological conditions, the FIX zymogen is a monomer. The purpose of this work was to analyse the conditions that may affect FIX monomeric state and promote and/or reduce oligomerization. Using native gel electrophoresis and size exclusion chromatography, we found that under decreased pH and ionic strength conditions, the FIX zymogen can oligomerize, resulting in the formation of higher molecular weight species, with a concomitant reduction in specific activity. Similarly, FIX oligomers formed readily with low bovine serum albumin (BSA) concentrations; however, increased BSA concentrations impeded FIX oligomerization. We hypothesize that normal blood physiological conditions are critical for maintaining active FIX monomers. Under conditions of stress associated with acidosis, electrolyte imbalance and low albumin levels, FIX oligomerization is expected to take place thus leading to compromised activity. Furthermore, albumin, which is commonly used as a drug stabilizer, may enhance the efficacy of FIX biological drugs by reducing oligomerization.
    Haemophilia 01/2014; DOI:10.1111/hae.12356 · 2.47 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Epididymal sperm maturation occurs via interactions between sperm and proteins secreted by the epididymal epithelium. Although this is an important process, the genes that encode the involved proteins remain largely uncharacterized. Previous studies have demonstrated that the genes involved in sperm maturation are regulated by androgen. Spag11a is an epididymal gene that is influenced by androgen. However, little is known about the putative role of this gene in the sperm maturation process. The objective of this study was to characterize Spag11a in the mouse epididymis. In silico analyses were performed to predict signal peptides and functional domains. Spag11a expression was measured by quantitative real-time RT-PCR. Western blots and immunocytochemistry were performed to determine protein expression. SPAG11A is a member of the beta defensin protein family and constitutes a secretory protein. Spag11a was expressed exclusively in the epididymis. Moreover, it exhibited region-specific expression in the caput, which is typical for genes that are involved in creating a suitable microenvironment for sperm maturation. Mouse Spag11a was regulated by androgen. A significant decrease of Spag11a expression was observed at third day following a gonadectomy (P < 0.001). Interestingly, testosterone replacement therapy was able to maintain the expression almost at the normal level, indicating a dependency on androgen. Besides androgen, testicular factors influenced Spag11a expression in a different way. This was revealed by efferent duct ligation in which Spag11a was transiently up-regulated at the third day following the ligation before returning to the normal level at day 5. Spag11a regional expression was also observed at protein level detected by western immunoblotting which revealed a clear band in the caput but not in other regions. The prediction that SPAG11A is a secretory protein was confirmed by immunocytochemical analyses indicating cell-specific expression mainly in the caput principal cells and detection of the protein in epididymal luminal fluid and spermatozoa. Based on the characteristics of Spag11a, it is likely that this gene has a specific role in epididymal sperm maturation. Further studies using functional assays are necessary to confirm this finding.
    Reproductive Biology and Endocrinology 07/2013; 11(1):59. DOI:10.1186/1477-7827-11-59 · 2.41 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: BACKGROUND INFORMATION: Matrix metalloproteinases (MMPs) are the key enzymes responsible for the remodeling of extracellular matrix Two of them, namely MMP-2 and MMP-9 (gelatinases A and B, respectively), are expressed in skeletal muscles and are involved in their regeneration after the injury. Although MMPs are primarily known to act extracellularly, recent studies have shown that some of them are also found within the cell. In this study, we examine intracellular localization of gelatinases during myoblasts differentiation in vitro, focusing at the impact of MMPs inhibition on the myoblasts proliferation and function. RESULTS: We show that MMP-9 localizes within the S-phase nuclei of in vitro differentiating myoblasts . The inhibition of MMPs activity achieved by either doxycycline (a non-competitive inhibitor of collagenases), or TIMP-1 (Tissue Inhibitor of Metalloproteinases 1), or neutralizing anti MMP-9 antibody affects nuclear localization of this gelatinase, and impacts at myoblasts proliferation. CONCLUSIONS: During myoblasts differentiation, MMP-9 that is localized in nuclei might be involved in the processes regulating cell cycle progression.
    Biology of the Cell 05/2013; DOI:10.1111/boc.201300020 · 3.87 Impact Factor

Preview

Download
1 Download
Available from