Apolactoferrin inhibits the catalytic domain of matrix metalloproteinase-2 by zinc chelation

2007 ◽  
Vol 85 (5) ◽  
pp. 563-572 ◽  
Author(s):  
Anthony L. Newsome ◽  
Jon Paul Johnson ◽  
Rebecca L. Seipelt ◽  
Michael W. Thompson
Keyword(s):  
Catalytic Domain ◽  
Matrix Metalloproteinase 2 ◽  
Temperature Sensitive ◽  
Mechanism Of Inhibition ◽  
Zinc Metalloproteases ◽  
Iron Binding Protein ◽  
Hemopexin Domain ◽  
Hydrolysis Of ◽  
Zinc Chelation

Lactoferrin (LTF) is a multifunctional iron-binding protein that is also capable of binding other divalent metal cations, especially Zn2+. Recent investigations indicate that lactoferrin levels are elevated in many disease conditions in which matrix metalloproteinases (MMPs), particularly MMP-2, are also elevated, suggesting that the 2 proteins may interact. This possibility was examined by determining the effect of LTF in its holo (metal-bound) and apo (metal-free) forms on the proteolytic activity of MMP-2 and other similar zinc metalloproteases. Pre-incubation with apolactoferrin, but not hololactoferrin, greatly reduced the hydrolysis of a peptide substrate by MMP-2, but not by MMP-1, -8, -9, or -13. This inhibition was specific for the 42 kDa catalytic domain fragment of MMP-2 lacking the hemopexin domain, since the 66 kDa form was poorly inhibited by apolactoferrin. The inhibition of the MMP-2 catalytic domain was strongly temperature sensitive, indicating that the conformation of one or both proteins is crucial to this interaction. To ascertain the mechanism of inhibition, increasing concentrations of ZnCl2 and FeCl2 were added to the reaction. While addition of Fe2+ did not reverse inhibition, the addition of Zn2+ resulted in a recovery of MMP-2 activity, and furthermore, zinc-saturated LTF did not inhibit MMP-2. Together, these data strongly suggest that apolactoferrin is capable of removing the catalytic zinc from the active site of MMP-2, although an exosite-based interaction between the 2 proteins cannot be fully ruled out. This inhibitory activity suggests a novel function for LTF and may represent a novel regulatory mechanism that regulates proteolysis by MMP-2 in vivo.

scholarly journals Point Mutations in Yeast CBF5 Can Abolish In Vivo Pseudouridylation of rRNA

1999 ◽  
Vol 19 (11) ◽  
pp. 7461-7472 ◽  
Author(s):  
Yeganeh Zebarjadian ◽  
Tom King ◽  
Maurille J. Fournier ◽  
Louise Clarke ◽  
John Carbon
Keyword(s):  
Catalytic Domain ◽  
Sequence Similarity ◽  
Point Mutations ◽  
Rrna Synthesis ◽  
Temperature Sensitive ◽  
In Vitro Mutagenesis ◽  
Sequence Motifs ◽  
Conserved Sequence

ABSTRACT In budding yeast (Saccharomyces cerevisiae), the majority of box H/ACA small nucleolar RNPs (snoRNPs) have been shown to direct site-specific pseudouridylation of rRNA. Among the known protein components of H/ACA snoRNPs, the essential nucleolar protein Cbf5p is the most likely pseudouridine (Ψ) synthase. Cbf5p has considerable sequence similarity to Escherichia coli TruBp, a known Ψ synthase, and shares the “KP” and “XLD” conserved sequence motifs found in the catalytic domains of three distinct families of known and putative Ψ synthases. To gain additional evidence on the role of Cbf5p in rRNA biosynthesis, we have used in vitro mutagenesis techniques to introduce various alanine substitutions into the putative Ψ synthase domain of Cbf5p. Yeast strains expressing these mutatedcbf5 genes in a cbf5Δ null background are viable at 25°C but display pronounced cold- and heat-sensitive growth phenotypes. Most of the mutants contain reduced levels of Ψ in rRNA at extreme temperatures. Substitution of alanine for an aspartic acid residue in the conserved XLD motif of Cbf5p (mutantcbf5D95A) abolishes in vivo pseudouridylation of rRNA. Some of the mutants are temperature sensitive both for growth and for formation of Ψ in the rRNA. In most cases, the impaired growth phenotypes are not relieved by transcription of the rRNA from a polymerase II-driven promoter, indicating the absence of polymerase I-related transcriptional defects. There is little or no abnormal accumulation of pre-rRNAs in these mutants, although preferential inhibition of 18S rRNA synthesis is seen in mutantcbf5D95A, which lacks Ψ in rRNA. A subset of mutations in the Ψ synthase domain impairs association of the altered Cbf5p proteins with selected box H/ACA snoRNAs, suggesting that the functional catalytic domain is essential for that interaction. Our results provide additional evidence that Cbf5p is the Ψ synthase component of box H/ACA snoRNPs and suggest that the pseudouridylation of rRNA, although not absolutely required for cell survival, is essential for the formation of fully functional ribosomes.

scholarly journals Mechanics and models of the myosin motor

2000 ◽  
Vol 355 (1396) ◽  
pp. 433-440 ◽  
Author(s):  
A. F. Huxley
Keyword(s):  
Actin Filament ◽  
Catalytic Domain ◽  
Thick Filament ◽  
Good Evidence ◽  
Temperature Sensitive ◽  
Striated Muscles ◽  
Cyclic Action ◽  
Thin Filaments ◽  
Cross Bridges ◽  
Hydrolysis Of

In striated muscles, shortening comes about by the sliding movement of thick filaments, composed mostly of myosin, relative to thin filaments, composed mostly of actin. This is brought about by cyclic action of ‘cross–bridges’ composed of the heads of myosin molecules projecting from a thick filament, which attach to an adjacent thin filament, exert force for a limited time and detach, and then repeat this cycle further along the filament. The requisite energy is provided by the hydrolysis of a molecule of adenosine triphosphate to the diphosphate and inorganic phosphate, the steps of this reaction being coupled to mechanical events within the cross–bridge. The nature of these events is discussed. There is good evidence that one of them is a change in the angle of tilt of a ‘lever arm’ relative to the ‘catalytic domain’ of the myosin head which binds to the actin filament. It is suggested here that this event is superposed on a slower, temperature–sensitive change in the orientation of the catalytic domain on the actin filament. Many uncertainties remain.

scholarly journals Conformation and Domain Movement Analysis of Human Matrix Metalloproteinase-2: Role of Associated Zn2+ and Ca2+ Ions

2019 ◽  
Author(s):  
Leah Voit-Ostricki ◽  
Sandor Lovas ◽  
Charles R. Watts
Keyword(s):  
Catalytic Domain ◽  
Conformational Stability ◽  
Movement Analysis ◽  
Binding Energies ◽  
Dynamics Simulation ◽  
Matrix Metalloproteinase 2 ◽  
Minor Role ◽  
Type I ◽  
Cross Correlation Analysis ◽  
Hemopexin Domain

Matrix Metaloproteinase-2 (MMP-2) is an extracellular Zn2+ protease specific to type I and IV collagens. Its expression is associated with several inflammatory, degenerative, and malignant diseases. Conformational properties, domain movements, and interactions between MMP-2 and its associated metal ions were characterized using a 1.0 µs molecular dynamics simulation. Dihedral principle component analysis revealed 10 families of conformations with the greatest degree of variability occurring in the link region connecting the catalytic and hemopexin domains. Dynamics cross correlation analysis indicated domain movements corresponding to opening and closing of the hemopexin domain in relation to the fibronectin and catalytic domains facilitated by the link region. Interaction energies were calculated using the MMPBSA-interaction entropy analysis method and revealed strong binding energies for the catalytic Zn2+ ion 1, Ca2+ ion 1, and Ca2+ ion 3 with significant conformational stability at the binding sites of Zn2+ ion 1 and Ca2+ ion 1. Ca2+ ion 2 diffuses freely away from its crystallographically defined binding site. Zn2+ ion 2 plays a minor role in conformational stability of the catalytic domain while Ca2+ ion 3 is strongly attracted to the highly electronegative sidechains of the Asp residues around the central β-sheet core of the hemopexin domain.

scholarly journals Conformation and Domain Movement Analysis of Human Matrix Metalloproteinase-2: Role of Associated Zn2+ and Ca2+ Ions

2019 ◽  
Vol 20 (17) ◽  
pp. 4194 ◽  
Author(s):  
Leah Voit-Ostricki ◽  
Sándor Lovas ◽  
Charles R. Watts
Keyword(s):  
Catalytic Domain ◽  
Conformational Stability ◽  
Movement Analysis ◽  
Binding Energies ◽  
Dynamics Simulation ◽  
Matrix Metalloproteinase 2 ◽  
Minor Role ◽  
Type I ◽  
Cross Correlation Analysis ◽  
Hemopexin Domain

Matrix metaloproteinase-2 (MMP-2) is an extracellular Zn2+ protease specific to type I and IV collagens. Its expression is associated with several inflammatory, degenerative, and malignant diseases. Conformational properties, domain movements, and interactions between MMP-2 and its associated metal ions were characterized using a 1.0 µs molecular dynamics simulation. Dihedral principle component analysis revealed ten families of conformations with the greatest degree of variability occurring in the link region connecting the catalytic and hemopexin domains. Dynamic cross-correlation analysis indicated domain movements corresponding to the opening and closing of the hemopexin domain in relation to the fibronectin and catalytic domains facilitated by the link region. Interaction energies were calculated using the molecular mechanics Poisson Boltzman surface area-interaction entropy (MMPBSA-IE) analysis method and revealed strong binding energies for the catalytic Zn2+ ion 1, Ca2+ ion 1, and Ca2+ ion 3 with significant conformational stability at the binding sites of Zn2+ ion 1 and Ca2+ ion 1. Ca2+ ion 2 diffuses freely away from its crystallographically defined binding site. Zn2+ ion 2 plays a minor role in conformational stability of the catalytic domain while Ca2+ ion 3 is strongly attracted to the highly electronegative sidechains of the Asp residues around the central β-sheet core of the hemopexin domain; however, the interacting residue sidechain carboxyl groups are outside of Ca2+ ion 3′s coordination sphere.

Abstract P108: Circulating Matrix Metalloproteinase-2 Invades the Heart and Causes Heart Failure

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Alejandro F Prado ◽  
Aline Azevedo ◽  
Cibele M Prado ◽  
Larissa Pernomian ◽  
Laena Pernomian ◽  
...  
Keyword(s):  
Heart Failure ◽  
Catalytic Domain ◽  
Heart Function ◽  
Matrix Metalloproteinase 2 ◽  
Saline Control ◽  
Function Loss ◽  
And Function ◽  
Tgf Β1

Background: An increase in MMP-2 levels is reported in heart failure (HF). However the role of MMP-2 in the pathogenesis of HF remains unclear. The aim of the present study was to determine the effect of increased circulating levels of MMP-2 on heart morphology and function. Methods and Results: Purified MMP-2 catalytic domain fused to GFP (catMMP-2/GFP) or saline (control) was injected into 11-wk-old male C57BL/6 mice for four weeks. The fluorescent active protein was tracked in vivo and homed in the heart. Cardiomyocyte diameter not changed between groups (catMMP-2/GFP: 11.37 ± 0.25 μm, n=7; Control: 11.38 ± 0.13 μm, n=7; P =0.97). On the other hand, fibrosis increased in the hearts of catMMP-2/GFP mice (0.82 ± 0.05% area/field, n=7 vs Control: 0.58 ± 0.02% area/field, n=7; P< 0.05). Apoptotic stained nuclei in the left ventricle (LV) of catMMP-2/GFP injected-mice amounted to 7.24%, n=4, P<0,05, whilst the LV of control animals only exhibited 0.27%, n=4. catMMP-2/GFP localized in the heart interstitium, where increased proteolytic activity (15.13 ± 1.33 U, n=5 vs Control: 9.70 ± 0.42 U, n=5; P <0.05). Hearts of catMMP-2/GFP mice showed 25% decrease in cardiac output (13 ± 1 mL/min, n=9 vs Control: 17 ± 1 mL/min, n=9), 30% decrease in ejection fraction (40 ± 2 %, n=9 vs Control: 56 ± 2 %, n=9) and stroke volume (28 ± 2 μL, n=9 vs Control: 40 ± 2 μL, n=9), and 34% decrease in fractional shortening (18 ± 1 %, n=9 vs Control: 28 ± 1 %, n=9) ( P <0.05 for all data).Western blotting showed 40% decrease in N-cadherin in animals that received catMMP-2/GFP (0.53 ± 0.08 U, n=6 vs control: 0.89 ± 0.11 U, n=6; P <0.05). Expression of signaling proteins also changed in LV of catMMP-2/GFP mice: TGF-β1 expression increased by 30% (0.60 ± 0.07 U, n=7 vs control: 0.40 ± 0.05 U, n=7, P<0,05), pAkt/Akt decreased by 40% (0.46 ± 0.05 U, n=7 vs control: 0.76 ± 0.11 U, n=7; P <0.05), pSMAD2/total SMAD2 ratio increased (1.88 ± 0.22 U, n=6 vs control: 0.93±0.12 U, n=6, P<0,05), and pSMAD3/total SMAD3 ratio increased (1.24 ± 0.22 U, n=7 vs control: 0.33 ± 0.07 U, n=7; P <0.05). Conclusions: Circulating active MMP-2 homing to the heart interstitium degrades N-cadherin and induces TGF-β1 overexpression, leading to apoptosis, and fibrosis. This mechanism may account for heart function loss when plasma levels of MMP-2 increase.

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.

Resveratrol Reduces Matrix Metalloproteinase-2 Activity Induced by Oxygen-Glucose Deprivation and Reoxygenation in Human Cerebral Microvascular Endothelial Cells

2012 ◽  
Vol 82 (4) ◽  
pp. 267-274 ◽  
Author(s):  
Zahide Cavdar ◽  
Mehtap Y. Egrilmez ◽  
Zekiye S. Altun ◽  
Nur Arslan ◽  
Nilgun Yener ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Neuroprotective Effect ◽  
Ischemia Reperfusion ◽  
Neurovascular Unit ◽  
Glucose Deprivation ◽  
Matrix Metalloproteinase 2 ◽  
Microvascular Endothelial Cells ◽  
Oxygen Glucose Deprivation ◽  
Microvascular Endothelial

The main pathophysiology in cerebral ischemia is the structural alteration in the neurovascular unit, coinciding with neurovascular matrix degradation. Among the human matrix metalloproteinases (MMPs), MMP-2 and -9, known as gelatinases, are the key enzymes for degrading type IV collagen, which is the major component of the basal membrane that surrounds the cerebral blood vessel. In the present study, we investigated the effects of resveratrol on cytotoxicity, reactive oxygen species (ROS), and gelatinases (MMP-2 and -9) in human cerebral microvascular endothelial cells exposed to 6 hours of oxygen-glucose deprivation and a subsequent 24 hours of reoxygenation with glucose (OGD/R), to mimic ischemia/reperfusion in vivo. Lactate dehydrogenase increased significantly, in comparison to that in the normoxia group. ROS was markedly increased in the OGD/R group, compared to normoxia. Correspondingly, ROS was significantly reduced with 50 μM of resveratrol. The proMMP-2 activity in the OGD/R group showed a statistically significant increase from the control cells. Resveratrol preconditioning decreased significantly the proMMP-2 in the cells exposed to OGD/R in comparison to that in the OGD/R group. Our results indicate that resveratrol regulates MMP-2 activity induced by OGD/R via its antioxidant effect, implying a possible mechanism related to the neuroprotective effect of resveratrol.

L-Tetrahydropalmatine Inhibits the Progression of Glioblastoma Tumor by Suppressing the Extracellular-Signal-Regulated Kinase/Nuclear Factor-Kappa B Signaling Pathway

2020 ◽  
Vol 19 (2) ◽  
pp. 164-171
Author(s):  
Feng Xue ◽  
Tingting Chen
Keyword(s):  
Glioblastoma Multiforme ◽  
Nuclear Factor Kappa B ◽  
Matrix Metalloproteinase 2 ◽  
Nuclear Factor ◽  
Nuclear Factor Kappa ◽  
Extracellular Signal ◽  
Apoptosis Protein ◽  
Endothelial Growth Factor

Glioblastoma multiforme is the most common malignancy of central nervous system. Herein we have evaluated the effect of L-tetrahydropalmatine, an isoquinoline alkaloid, on the tumor growth both in vivo and in vitro using C6 glioblastoma multiforme cells and BALB/c mice injected subcutaneously with C6/luc2 cells. The results of these studies show that L-tetrahydropalmatine exhibited cytotoxic effect on C6 glioblastoma multiforme cells, suppressed nuclear factor-kappa B activity, suppressed the levels of tumor-linked proteins such as matrix metalloproteinase-2/9, Cyclin-D1, vascular endothelial growth factor, and X-linked inhibitor of apoptosis protein via ERK/nuclear factor-kappa B cascade. Further, L-tetrahydropalmatine inhibited the cell migration and invasion properties of C6 cells, and also suppressed the tumor weight and volume in mice. Immunohistochemical staining of tumor tissues suggested that L-tetrahydropalmatine inhibited the extracellular-signal-regulated kinase/nuclear factor-kappa B cascade and suppressed the levels of Cyclin-D1; matrix metalloproteinase-2/9; X-linked inhibitor of apoptosis protein; and vascular endothelial growth factor, and also the progression and growth of glioblastoma multiforme in mice. In summary, L-tetrahydropalmatine inhibits the ERK/nuclear factor-kappa B cascade, decreases the tumor volume, and inhibits the proteins responsible for tumor growth both in vivo and in vitro.

Studies on the mechanism of acute inhibition of thyroglobulin hydrolysis by iodine

1985 ◽  
Vol 108 (4) ◽  
pp. 511-517 ◽  
Author(s):  
Nandalal Bagchi ◽  
Birdie Shivers ◽  
Thomas R. Brown
Keyword(s):  
Time Course ◽  
Period 2 ◽  
Iodotyrosine Deiodinase ◽  
Rate Of Hydrolysis ◽  
Underlying Mechanisms ◽  
Excess Iodide ◽  
Sites Of Action ◽  
Hydrolysis Of

Abstract. Iodine in excess is known to acutely inhibit thyroidal secretion. In the present study we have characterized the time course of the iodine effect in vitro and investigated the underlying mechanisms. Labelled thyroid glands were cultured in vitro in medium containing mononitrotyrosine, an inhibitor of iodotyrosine deiodinase. The rate of hydrolysis of labelled thyroglobulin was measured as the proportion of labelled iodotyrosines and iodothyronines recovered at the end of culture and was used as an index of thyroidal secretion. Thyrotrophin (TSH) administered in vivo acutely stimulated the rate of thyroglobulin hydrolysis. Addition of Nal to the culture medium acutely inhibited both basal and TSH-stimulated thyroglobulin hydrolysis. The effect of iodide was demonstrable after 2 h, maximal after 6 h and was not reversible upon removal of iodide. Iodide abolished the dibutyryl cAMP induced stimulation of thyroglobulin hydrolysis. Iodide required organic binding of iodine for its effect but new protein or RNA synthesis was not necessary. The inhibitory effects of iodide and lysosomotrophic agents such as NH4C1 and chloroquin on thyroglobulin hydrolysis were additive suggesting different sites of action. Iodide added in vitro altered the distribution of label in prelabelled thyroglobulin in a way that suggested increased coupling in the thyroglobulin molecule. These data indicate that 1) the iodide effect occurs progressively over a 6 h period, 2) continued presence of iodide is not necessary once the inhibition is established, 3) iodide exerts its action primarily at a post cAMP, prelysosomal site and 4) the effect requires organic binding of iodine, but not new RNA or protein synthesis. Our data are consistent with the hypothesis that excess iodide acutely inhibits thyroglobulin hydrolysis by increasing the resistance of thyroglobulin to proteolytic degradation through increased iodination and coupling.

scholarly journals Evaluation of the Safety and Immune Efficacy of Recombinant Human Respiratory Syncytial Virus Strain Long Live Attenuated Vaccine Candidates

2021 ◽  
Author(s):  
Li-Nan Wang ◽  
Xiang-Lei Peng ◽  
Min Xu ◽  
Yuan-Bo Zheng ◽  
Yue-Ying Jiao ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Gene Deletion ◽  
Human Respiratory Syncytial Virus ◽  
Temperature Sensitive ◽  
T7 Promoter ◽  
Vaccine Candidates ◽  
Rsv Infection ◽  
Syncytial Virus

AbstractHuman respiratory syncytial virus (RSV) infection is the leading cause of lower respiratory tract illness (LRTI), and no vaccine against LRTI has proven to be safe and effective in infants. Our study assessed attenuated recombinant RSVs as vaccine candidates to prevent RSV infection in mice. The constructed recombinant plasmids harbored (5′ to 3′) a T7 promoter, hammerhead ribozyme, RSV Long strain antigenomic cDNA with cold-passaged (cp) mutations or cp combined with temperature-sensitive attenuated mutations from the A2 strain (A2cpts) or further combined with SH gene deletion (A2cptsΔSH), HDV ribozyme (δ), and a T7 terminator. These vectors were subsequently co-transfected with four helper plasmids encoding N, P, L, and M2-1 viral proteins into BHK/T7-9 cells, and the recovered viruses were then passaged in Vero cells. The rescued recombinant RSVs (rRSVs) were named rRSV-Long/A2cp, rRSV-Long/A2cpts, and rRSV-Long/A2cptsΔSH, respectively, and stably passaged in vitro, without reversion to wild type (wt) at sites containing introduced mutations or deletion. Although rRSV-Long/A2cpts and rRSV-Long/A2cptsΔSH displayed  temperature-sensitive (ts) phenotype in vitro and in vivo, all rRSVs were significantly attenuated in vivo. Furthermore, BALB/c mice immunized with rRSVs produced Th1-biased immune response, resisted wtRSV infection, and were free from enhanced respiratory disease. We showed that the combination of ΔSH with attenuation (att) mutations of cpts contributed to improving att phenotype, efficacy, and gene stability of rRSV. By successfully introducing att mutations and SH gene deletion into the RSV Long parent and producing three rRSV strains, we have laid an important foundation for the development of RSV live attenuated vaccines.

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