scholarly journals Functional Characterization of Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) N- and C-Terminal Domains duringXenopus laevisDevelopment

2014 ◽  
Vol 2014 ◽  
pp. 1-10 ◽  
Author(s):  
M. A. Nieuwesteeg ◽  
J. A. Willson ◽  
M. Cepeda ◽  
M. A. Fox ◽  
S. Damjanovski
Keyword(s):  
Functional Characterization ◽  
Full Length ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Mrna Levels ◽  
Ecm Remodeling ◽  
Protein Levels ◽  
Terminal Domain ◽  
The Individual ◽  
Terminal Domains

Extracellular matrix (ECM) remodeling is essential for facilitating developmental processes. ECM remodeling, accomplished by matrix metalloproteinases (MMPs), is regulated by endogenous tissue inhibitors of metalloproteinases (TIMPs). While the TIMP N-terminal domain is involved in inhibition of MMP activity, the C-terminal domain exhibits cell-signaling activity, which is TIMP and cell type dependent. We have previously examined the distinct roles of theXenopus laevisTIMP-2 and -3 C-terminal domains during development and here examined the unique roles of TIMP-1 N- and C-terminal domains in earlyX. laevisembryos. mRNA microinjection was used to overexpress full-length TIMP-1 or its individual N- or C-terminal domains in embryos. Full-length and C-terminal TIMP-1 resulted in increased lethality compared to N-terminal TIMP-1. Overexpression of C-terminal TIMP-1 resulted in significant decreases in mRNA levels of proteolytic genes including TIMP-2, RECK, MMP-2, and MMP-9, corresponding to decreases in MMP-2 and -9 protein levels, as well as decreased MMP-2 and MMP-9 activities. These trends were not observed with the N-terminus. Our research suggests that the individual domains of TIMP-1 are capable of playing distinct roles in regulating the ECM proteolytic network during development and that the unique functions of these domains are moderated in the endogenous full-length TIMP-1 molecule.

scholarly journals The C-terminal domain of 72 kDa gelatinase A is not required for catalysis, but is essential for membrane activation and modulates interactions with tissue inhibitors of metalloproteinases

1992 ◽  
Vol 283 (3) ◽  
pp. 637-641 ◽  
Author(s):  
G Murphy ◽  
F Willenbrock ◽  
R V Ward ◽  
M I Cockett ◽  
D Eaton ◽  
...  
Keyword(s):  
Kinetic Studies ◽  
Full Length ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Gelatinase A ◽  
Truncated Form ◽  
Tissue Inhibitor ◽  
Tissue Inhibitors ◽  
Terminal Domain

Recombinant 72 kDa gelatinase A and a truncated form lacking the C-terminal domain were shown to be activated by organomercurials and to possess similar activities towards a number of substrates. The truncated proenzyme differed from the full-length gelatinase in that it could not be activated by a membrane activator and did not bind tissue inhibitor of metalloproteinase (TIMP)-2. Kinetic studies also showed that the inhibition of the activated truncated enzyme, by both TIMP-1 and TIMP-2, was considerably decreased compared with the full-length enzyme. We conclude that the C-terminal domain plays an important role in the regulation of gelatinase A by a potential physiological activator and inhibitors.

Structural and functional analysis of the C-terminal region of Streptococcus gordonii SspB

2021 ◽  
Vol 77 (9) ◽  
pp. 1206-1215
Author(s):  
Norbert Schormann ◽  
Sangeetha Purushotham ◽  
Joshua L. Mieher ◽  
Manisha Patel ◽  
Hui Wu ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Scavenger Receptor ◽  
Protein Docking ◽  
Terminal Region ◽  
Tooth Surface ◽  
Functional Difference ◽  
Streptococcus Gordonii ◽  
Viridans Streptococci ◽  
The Individual ◽  
Terminal Domains

Streptococcus gordonii is a member of the viridans streptococci and is an early colonizer of the tooth surface. Adherence to the tooth surface is enabled by proteins present on the S. gordonii cell surface, among which SspB belongs to one of the most well studied cell-wall-anchored adhesin families: the antigen I/II (AgI/II) family. The C-terminal region of SspB consists of three tandemly connected individual domains that display the DEv-IgG fold. These C-terminal domains contain a conserved Ca2+-binding site and isopeptide bonds, and they adhere to glycoprotein 340 (Gp340; also known as salivary agglutinin, SAG). Here, the structural and functional characterization of the C123 SspB domain at 2.7 Å resolution is reported. Although the individual C-terminal domains of Streptococcus mutans AgI/II and S. gordonii SspB show a high degree of both sequence and structural homology, superposition of these structures highlights substantial differences in their electrostatic surface plots, and this can be attributed to the relative orientation of the individual domains (C1, C2 and C3) with respect to each other and could reflect their specificity in binding to extracellular matrix molecules. Studies further confirmed that affinity for Gp340 or its scavenger receptor cysteine-rich (SRCR) domains requires two of the three domains of C123 SspB, namely C12 or C23, which is different from AgI/II. Using protein–protein docking studies, models for this observed functional difference between C123 SspB and C123 AgI/II in their binding to SRCR1 are presented.

scholarly journals Insulin-like Growth Factor II Signaling in Neoplastic Proliferation Is Blocked by Transgenic Expression of the Metalloproteinase Inhibitor Timp-1

1999 ◽  
Vol 146 (4) ◽  
pp. 881-892 ◽  
Author(s):  
David C. Martin ◽  
John L. Fowlkes ◽  
Bojana Babic ◽  
Rama Khokha
Keyword(s):  
Growth Factor ◽  
T Antigen ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Early Event ◽  
Mrna Levels ◽  
Insulin Like Growth Factor ◽  
Igf Binding Proteins ◽  
Protein Levels ◽  
Igf I ◽  
Igfbp 3

Insulin-like growth factor (IGF) II is overexpressed in many human cancers and is reactivated by, and crucial for viral oncogene (SV40 T antigen, [TAg])–induced tumorigenesis in several tumor models. Using a double transgenic murine hepatic tumor model, we demonstrate that tissue inhibitor of metalloproteinase 1 (TIMP-1) blocks liver hyperplasia during tumor development, despite TAg-mediated reactivation of IGF-II. Because the activity of IGFs is controlled by IGF-binding proteins (IGFBPs), we investigated whether TIMP-1 overexpression altered the IGFBP status in the transgenic liver. Ligand blotting showed that IGFBP-3 protein levels were increased in TIMP-1–overexpressing double transgenic littermates, whereas IGFBP-3 mRNA levels were not different, suggesting that TIMP-1 affects IGFBP-3 at a posttranscriptional level. IGFBP-3 proteolysis assays demonstrated that IGFBP-3 degradation was lower in TIMP-1–overexpressing livers, and zymography showed that matrix metalloproteinases (MMPs) were present in the liver homogenates and were capable of degrading IGFBP-3. As a consequence of reduced IGFBP-3 proteolysis and elevated IGFBP-3 protein levels, dissociable IGF-II levels were significantly lower in TIMP-1–overexpressing animals. This decrease in bioavailable IGF-II ultimately resulted in diminished IGF-I receptor signaling in vivo as evidenced by diminished receptor kinase activity and decreased tyrosine phosphorylation of the IGF-I receptor downstream effectors, insulin receptor substrate 1 (IRS-1), extracellular signal regulatory kinase (Erk)-1, and Erk-2. Together, these results provide evidence that TIMP-1 inhibits liver hyperplasia, an early event in TAg-mediated tumorigenesis, by reducing the activity of the tumor-inducing mitogen, IGF-II. These data implicate the control of MMP-mediated degradation of IGFBPs as a novel therapy for controlling IGF bioavailability in cancer.

scholarly journals Molecular dissection of radixin: distinct and interdependent functions of the amino- and carboxy-terminal domains.

1995 ◽  
Vol 129 (4) ◽  
pp. 1007-1022 ◽  
Author(s):  
M D Henry ◽  
C Gonzalez Agosti ◽  
F Solomon
Keyword(s):  
Full Length ◽  
Detectable Effect ◽  
Limiting Factor ◽  
Cell Physiology ◽  
Erm Proteins ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Terminal Domains

The ERM proteins--ezrin, radixin, and moesin--occur in particular cortical cytoskeletal structures. Several lines of evidence suggest that they interact with both cytoskeletal elements and plasma membrane components. Here we described the properties of full-length and truncated radixin polypeptides expressed in transfected cells. In stable transfectants, exogenous full-length radixin behaves much like endogenous ERM proteins, localizing to the same cortical structures. However, the presence of full-length radixin or its carboxy-terminal domain in cortical structures correlates with greatly diminished staining of endogenous moesin in those structures, suggesting that radixin and moesin compete for a limiting factor required for normal associations in the cell. The results also reveal distinct roles for the amino- and carboxy-terminal domains. At low levels relative to endogenous radixin, the carboxy-terminal polypeptide is associated with most of the correct cortical targets except cleavage furrows. In contrast, the amino-terminal polypeptide is diffusely localized throughout the cell. Low level expression of full-length radixin or either of the truncated polypeptides has no detectable effect on cell physiology. However, high level expression of the carboxy-terminal domain dramatically disrupts normal cytoskeletal structures and functions. At these high levels, the amino-terminal polypeptide does localize to cortical structures, but does not affect the cells. We conclude that the behavior of radixin in cells depends upon activities contributed by separate domains of the protein, but also requires modulating interactions between those domains.

scholarly journals Engineering N-terminal domain of tissue inhibitor of metalloproteinase (TIMP)-3 to be a better inhibitor against tumour necrosis factor-α-converting enzyme

2002 ◽  
Vol 364 (1) ◽  
pp. 227-234 ◽  
Author(s):  
Meng-Huee LEE ◽  
Vandana VERMA ◽  
Klaus MASKOS ◽  
Deepa NATH ◽  
Vera KNÄUPER ◽  
...  
Keyword(s):  
Full Length ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Binding Affinities ◽  
Converting Enzyme ◽  
Wild Type ◽  
Tissue Inhibitor ◽  
Terminal Domain ◽  
Tnf Α ◽  
Factor Α ◽  
Necrosis Factor

We previously reported that full-length tissue inhibitor of metalloproteinase-3 (TIMP-3) and its N-terminal domain form (N-TIMP-3) displayed equal binding affinity for tissue necrosis factor-α (TNF-α)-converting enzyme (TACE). Based on the computer graphic of TACE docked with a TIMP-3 model, we created a number of N-TIMP-3 mutants that showed significant improvement in TACE inhibition. Our strategy was to select those N-TIMP-3 residues that were believed to be in actual contact with the active-site pockets of TACE and mutate them to amino acids of a better-fitting nature. The activities of these mutants were examined by measuring their binding affinities (Kappi) and association rates (kon) against TACE. Nearly all mutants at position Thr-2 exhibited slightly impaired affinity as well as association rate constants. On the other hand, some Ser-4 mutants displayed a remarkable increase in their binding tightness with TACE. In fact, the binding affinities of several mutants were less than 60pM, beyond the sensitivity limits of fluorimetric assays. Further studies on cell-based processing of pro-TNF-α demonstrated that wild-type N-TIMP-3 and one of its tight-binding mutants, Ser-4Met, were capable of inhibiting the proteolytic shedding of TNF-α. Furthermore, the Ser-4Met mutant was also significantly more active (P<0.05) than the wild-type N-TIMP-3 in its cellular inhibition. Comparison of N-TIMP-3 and full-length TIMP-3 revealed that, despite their identical TACE-interaction kinetics, the latter was nearly 10 times more efficient in the inhibition of TNF-α shedding, with concomitant implications for the importance of the TIMP-3 C-terminal domain in vivo.

scholarly journals Age- and Sex-Dependent Changes in Androgen Receptor Expression in the Developing Mouse Cortex and Hippocampus

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Houng-Wei Tsai ◽  
Saori Taniguchi ◽  
Jason Samoza ◽  
Aaron Ridder
Keyword(s):  
Sex Differences ◽  
Androgen Receptor ◽  
Brain Structures ◽  
Perinatal Period ◽  
Full Length ◽  
Receptor Expression ◽  
Mrna Levels ◽  
Protein Levels ◽  
Mouse Cortex ◽  
Age And Sex

During the perinatal period, male mice are exposed to higher levels of testosterone (T) than females, which promotes sexual dimorphism in their brain structures and behaviors. In addition to acting via estrogen receptors after being locally converted into estradiol by aromatase, T also acts directly through androgen receptor (AR) in the brain. Therefore, we hypothesized that AR expression in the developing mouse cortex and hippocampus was sexually dimorphic. To test our hypothesis, we measured and determined AR mRNA and protein levels in mouse cortex/hippocampus collected on the day of birth (PN0) and 7 (PN7), 14 (PN14), and 21 (PN21) days after birth. We demonstrated that, as age advanced, AR mRNA levels increased in the cortex/hippocampus of both sexes but showed no sex difference. Two AR proteins, the full-length (110 kDa) and a smaller isoform (70 kDa), were detected in the developing mouse cortex/hippocampus with an age-dependent increase in protein levels of both AR isoforms at PN21 and a transient masculine increase in expression of the full-length AR protein on PN7. Thus, we conclude that the postnatal age and sex differences in AR protein expression in combination with the sex differences in circulating T may cause sexual differentiation of the mouse cortex/hippocampus.

Alterations in mRNA and protein levels of metalloproteinases-2, -9, and -14 and tissue inhibitor of metalloproteinase-2 responses to traumatic skeletal muscle injury

2009 ◽  
Vol 297 (6) ◽  
pp. C1501-C1508 ◽  
Author(s):  
Brian R. Barnes ◽  
Eric R. Szelenyi ◽  
Gordon L. Warren ◽  
Maria L. Urso
Keyword(s):  
Skeletal Muscle ◽  
Mrna Expression ◽  
Protein Content ◽  
Muscle Injury ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Mrna Levels ◽  
Tissue Inhibitor ◽  
Skeletal Muscle Injury ◽  
Protein Levels ◽  
Sequence Of Events

This study characterizes the temporal relationship of membrane type-1 matrix metalloproteinase (MT1-MMP) and tissue inhibitor of metalloproteinase-2 (TIMP-2) expression in skeletal muscle following injury. Tibialis anterior (TA) muscles from 60 mice were exposed and injured by applying a cold steel probe (−79°C) to the muscle for 10 s. Thereafter, TA muscles from uninjured and injured legs were collected at 3, 10, 24, 48, and 72 h postinjury for analysis of local MT1-MMP, TIMP-2, and matrix metalloproteinases-2 and -9 (MMP-2 and MMP-9) mRNA and protein content via quantitative RT-PCR, immunoblotting, zymography, and immunofluorescence. All data are expressed as fold change of injured leg vs. uninjured leg. MT1-MMP mRNA levels were decreased significantly at 48 and 72 h postinjury by ∼9- and 21-fold, respectively ( P < 0.01). Both TIMP-2 and MMP-2 mRNA expression significantly decreased in the injured leg by ∼4- to 10-fold at 10–72 h postinjury ( P < 0.01). MMP-9 mRNA expression was significantly increased at 10, 24, and 48 h postinjury by 6- ( P < 0.05), 25-, and 12-fold ( P < 0.01), respectively. Protein content of latent (63 kDa) MT1-MMP was decreased at 48 and 72 h postinjury by ∼2-fold ( P < 0.01). Content of the soluble (50 kDa) fragment of MT1-MMP was significantly increased by ∼17-, 25-, and 67-fold at 24 ( P < 0.05), 48, and 72 h ( P < 0.01) postinjury, respectively. TIMP-2 protein levels diminished from 3 to 48 h postinjury by 1.5-fold to 1.8-fold ( P < 0.01), before returning to baseline levels at 72 h postinjury. Zymography revealed visual increases in gelatinase activity in molecular weight regions corresponding to MMP-9 and MMP-2. In conclusion, skeletal muscle injury initiates a sequence of events in the MT1-MMP proteolytic cascade resulting in elevated levels of the soluble (50 kDa) fragment of MT1-MMP, which could enhance pericellular extracellular matrix remodeling.

scholarly journals The N-terminal domain of ALS-linked TDP-43 assembles without misfolding

2017 ◽  
Author(s):  
Phoebe S. Tsoi ◽  
Kyoungjae J. Choi ◽  
Paul G. Leonard ◽  
Antons Sizovs ◽  
Mahdi Muhammad Moosa ◽  
...  
Keyword(s):  
Neurodegenerative Diseases ◽  
Full Length ◽  
Physiological Conditions ◽  
Terminal Domain ◽  
Terminal Domains

AbstractTDP-43 forms inclusions in several neurodegenerative diseases, and both its N- and C-terminal domains are implicated in this process. We show that the folded TDP-43 N-terminal domain oligomerizes under physiological conditions and propose that, in full-length TDP-43, association between folded N-terminal domains enhances the propensity of the intrinsically unfolded C-terminal domains to drive pathological aggregation.

Functional Characterization of Baxter's Recombinant Human ADAMTS13 Drug Candidate.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2235-2235
Author(s):  
Hanspeter Rottensteiner ◽  
Barbara Plaimauer ◽  
Gerald Schrenk ◽  
Michaela Schmidt ◽  
Bernadette Gruber ◽  
...  
Keyword(s):  
Fluid Shear Stress ◽  
Functional Characterization ◽  
Immunoblot Analysis ◽  
Full Length ◽  
Drug Candidate ◽  
Adamts13 Activity ◽  
Clinical Phase ◽  
Adhesive Surface ◽  
Production Stages ◽  
The Individual

Abstract Abstract 2235 Baxter has developed a human recombinant (r) ADAMTS13 for treatment of patients with hereditary TTP. Here we characterize preclinical and clinical lots of rADAMTS13 with respect to their functional properties. The ability of rADAMTS13 to degrade human full-length rVWF under moderate denaturing conditions was analyzed by two functional assays (VWF:CB and VWF:RCo activity) and by gel electrophoresis (multimer analysis and immunoblot analysis). In all assays, the extent of rVWF degradation served as a direct measure for ADAMTS13 activity. VWF:CB activity of rVWF was reduced upon incubation with rADAMTS13, with only minimal differences between the individual batches. VWF:RCo activity of rVWF also declined after exposure to rADAMTS13, with no relevant differences between rADAMTS13 batches. Likewise, VWF multimer analysis demonstrated the disappearance of high molecular weight multimers upon incubation with rADAMTS13. Non-reducing SDS-PAGE followed by immunostaining using a polyclonal anti-human VWF antibody clearly showed the appearance of the C-terminal and N-terminal VWF fragments, with no visible differences in the intensity of these bands between preclinical and clinical Phase I rADAMTS13 batches. Proteolytic activity of rADAMTS13 towards its multimeric VWF substrate was confirmed in two flow-based assays using full-length rVWF as substrate. One assay was based on the VenaFlux platform technology and determined the rADAMTS13 concentration-dependent decrease of the surface coverage of fluorescently-labeled platelets that had been perfused together with red blood cells and rVWF over an adhesive surface of a microcapillary. The other assay measured the extent of VWF cleavage product generation after subjecting a reaction mixture consisting of multimeric rVWF, lyophilized platelets and rADAMTS13 to fluid shear stress on a vortex mixer. Both assays revealed a high comparability between preclinical and clinical batches. All assays thus demonstrated consistency between rADAMTS13 batches over different production stages. Importantly, comparability was also shown between the ADAMTS13 activity measured by the assays using full-length VWF as substrate and FRETS-VWF73 assay used for potency assignment of rADAMTS13. Disclosures: Rottensteiner: Baxter Innovations GmbH: Employment. Plaimauer:Baxter Innovations GmbH: Employment. Schrenk:Baxter Innovations GmbH: Employment. Schmidt:Baxter Innovations GmbH: Employment. Gruber:Baxter Innovations GmbH: Employment. Schreiner:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

Ubiquitin-mediated proteasomal degradation of non-synonymous SNP variants of human ABC transporter ABCG2

2008 ◽  
Vol 411 (3) ◽  
pp. 623-631 ◽  
Author(s):  
Hiroshi Nakagawa ◽  
Ai Tamura ◽  
Kanako Wakabayashi ◽  
Kazuyuki Hoshijima ◽  
Masayuki Komada ◽  
...  
Keyword(s):  
Genetic Polymorphisms ◽  
Proteasomal Degradation ◽  
Immunoblot Analysis ◽  
Mrna Levels ◽  
Single Nucleotide ◽  
Protein Levels ◽  
Atp Binding Cassette Transporter ◽  
Novel Approach ◽  
293 Cells ◽  
The Individual

Clinical relevance is implicated between the genetic polymorphisms of the ABC (ATP-binding cassette) transporter ABCG2 (ABC subfamily G, member 2) and the individual differences in drug response. We expressed a total of seven non-synonymous SNP (single nucleotide polymorphism) variants in Flp-In-293 cells by using the Flp (flippase) recombinase system. Of these, ABCG2 F208S and S441N variants were found to be expressed at markedly low levels, whereas their mRNA levels were equal to those of the other SNP variants and ABCG2 WT (wild-type). Interestingly, protein expression levels of the ABCG2 F208S and S441N variants increased 6- to 12-fold when Flp-In-293 cells were treated with MG132, a proteasome inhibitor. Immunoprecipitation followed by immunoblot analysis showed that the ABCG2 F208S and S441N variant proteins were endogenously ubiquitinated in Flp-In-293 cells, and treatment with MG132 significantly enhanced the level of these ubiquitinated variants. Immunofluorescence microscopy demonstrated that MG132 greatly affected the ABCG2 F208S and S441N variants in terms of both protein levels and intracellular distribution. Immunoblot analysis revealed that those variants were N-glycosylated; however, their oligosaccharides were immature compared with those present on ABCG2 WT. The ABCG2 F208S and S441N variant proteins do not appear to be processed in the Golgi apparatus, but undergo ubiquitin-mediated protein degradation in proteasomes, whereas ABCG2 WT is sorted to the plasma membrane and then degraded via the lysosomal pathway. The present study provides the first evidence that certain genetic polymorphisms can affect the protein stability of ABCG2. Control of proteasomal degradation of ABCG2 would provide a novel approach in cancer chemotherapy to circumvent multidrug resistance of human cancers.

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