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Article ; Online: Electrostatic effects on the folding stability of FKBP12.

Batra, Jyotica / Tjong, Harianto / Zhou, Huan-Xiang

Protein engineering, design & selection : PEDS

2016 Volume 29, Issue 8, Page(s) 301–308

Abstract: The roles of electrostatic interactions in protein folding stability have been a matter of debate, largely due to the complexity in the theoretical treatment of these interactions. We have developed computational methods for calculating electrostatic ... ...

Abstract	The roles of electrostatic interactions in protein folding stability have been a matter of debate, largely due to the complexity in the theoretical treatment of these interactions. We have developed computational methods for calculating electrostatic effects on protein folding stability. To rigorously test and further refine these methods, here we carried out experimental studies into electrostatic effects on the folding stability of the human 12-kD FK506 binding protein (FKBP12). This protein has a close homologue, FKBP12.6, with amino acid substitutions in only 18 of their 107 residues. Of the 18 substitutions, 8 involve charged residues. Upon mutating FKBP12 residues at these 8 positions individually into the counterparts in FKBP12.6, the unfolding free energy (ΔGu) of FKBP12 changed by -0.3 to 0.7 kcal/mol. Accumulating stabilizing substitutions resulted in a mutant with a 0.9 kcal/mol increase in stability. Additional charge mutations were grafted from a thermophilic homologue, MtFKBP17, which aligns to FKBP12 with 31% sequence identity over 89 positions. Eleven such charge mutations were studied, with ΔΔGu varying from -2.9 to 0.1 kcal/mol. The predicted electrostatic effects by our computational methods with refinements herein had a root-mean-square deviation of 0.9 kcal/mol from the experimental ΔΔGu values on 16 single mutations of FKBP12. The difference in ΔΔGu between mutations grafted from FKBP12.6 and those from MtFKBP17 suggests that more distant homologues are less able to provide guidance for enhancing folding stability.
MeSH term(s)	Amino Acid Substitution ; Humans ; Mutation ; Protein Folding ; Protein Stability ; Protein Unfolding ; Static Electricity ; Tacrolimus Binding Protein 1A/chemistry ; Tacrolimus Binding Protein 1A/genetics ; Thermodynamics
Chemical Substances	Tacrolimus Binding Protein 1A (EC 5.2.1.-)
Language	English
Publishing date	2016-07-05
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	1466729-0
ISSN	1741-0134 ; 1741-0126
ISSN (online)	1741-0134
ISSN	1741-0126
DOI	10.1093/protein/gzw014
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Article ; Online: Matrix metalloproteinase-10/TIMP-2 structure and analyses define conserved core interactions and diverse exosite interactions in MMP/TIMP complexes.

Jyotica Batra / Alexei S Soares / Christine Mehner / Evette S Radisky

PLoS ONE, Vol 8, Iss 9, p e

2013 Volume 75836

Abstract: Matrix metalloproteinases (MMPs) play central roles in vertebrate tissue development, remodeling, and repair. The endogenous tissue inhibitors of metalloproteinases (TIMPs) regulate proteolytic activity by binding tightly to the MMP active site. While ... ...

Abstract	Matrix metalloproteinases (MMPs) play central roles in vertebrate tissue development, remodeling, and repair. The endogenous tissue inhibitors of metalloproteinases (TIMPs) regulate proteolytic activity by binding tightly to the MMP active site. While each of the four TIMPs can inhibit most MMPs, binding data reveal tremendous heterogeneity in affinities of different TIMP/MMP pairs, and the structural features that differentiate stronger from weaker complexes are poorly understood. Here we report the crystal structure of the comparatively weakly bound human MMP-10/TIMP-2 complex at 2.1 Å resolution. Comparison with previously reported structures of MMP-3/TIMP-1, MT1-MMP/TIMP-2, MMP-13/TIMP-2, and MMP-10/TIMP-1 complexes offers insights into the structural basis of binding selectivity. Our analyses identify a group of highly conserved contacts at the heart of MMP/TIMP complexes that define the conserved mechanism of inhibition, as well as a second category of diverse adventitious contacts at the periphery of the interfaces. The AB loop of the TIMP N-terminal domain and the contact loops of the TIMP C-terminal domain form highly variable peripheral contacts that can be considered as separate exosite interactions. In some complexes these exosite contacts are extensive, while in other complexes the AB loop or C-terminal domain contacts are greatly reduced and appear to contribute little to complex stability. Our data suggest that exosite interactions can enhance MMP/TIMP binding, although in the relatively weakly bound MMP-10/TIMP-2 complex they are not well optimized to do so. Formation of highly variable exosite interactions may provide a general mechanism by which TIMPs are fine-tuned for distinct regulatory roles in biology.
Keywords	Medicine ; R ; Science ; Q
Subject code	540
Language	English
Publishing date	2013-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
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Article ; Online: Matrix metalloproteinase-10/TIMP-2 structure and analyses define conserved core interactions and diverse exosite interactions in MMP/TIMP complexes.

Batra, Jyotica / Soares, Alexei S / Mehner, Christine / Radisky, Evette S

PloS one

2013 Volume 8, Issue 9, Page(s) e75836

Abstract	Matrix metalloproteinases (MMPs) play central roles in vertebrate tissue development, remodeling, and repair. The endogenous tissue inhibitors of metalloproteinases (TIMPs) regulate proteolytic activity by binding tightly to the MMP active site. While each of the four TIMPs can inhibit most MMPs, binding data reveal tremendous heterogeneity in affinities of different TIMP/MMP pairs, and the structural features that differentiate stronger from weaker complexes are poorly understood. Here we report the crystal structure of the comparatively weakly bound human MMP-10/TIMP-2 complex at 2.1 Å resolution. Comparison with previously reported structures of MMP-3/TIMP-1, MT1-MMP/TIMP-2, MMP-13/TIMP-2, and MMP-10/TIMP-1 complexes offers insights into the structural basis of binding selectivity. Our analyses identify a group of highly conserved contacts at the heart of MMP/TIMP complexes that define the conserved mechanism of inhibition, as well as a second category of diverse adventitious contacts at the periphery of the interfaces. The AB loop of the TIMP N-terminal domain and the contact loops of the TIMP C-terminal domain form highly variable peripheral contacts that can be considered as separate exosite interactions. In some complexes these exosite contacts are extensive, while in other complexes the AB loop or C-terminal domain contacts are greatly reduced and appear to contribute little to complex stability. Our data suggest that exosite interactions can enhance MMP/TIMP binding, although in the relatively weakly bound MMP-10/TIMP-2 complex they are not well optimized to do so. Formation of highly variable exosite interactions may provide a general mechanism by which TIMPs are fine-tuned for distinct regulatory roles in biology.
MeSH term(s)	Binding Sites ; Catalytic Domain ; Crystallography, X-Ray ; Humans ; Matrix Metalloproteinase 10/chemistry ; Matrix Metalloproteinase 10/metabolism ; Matrix Metalloproteinases/chemistry ; Matrix Metalloproteinases/metabolism ; Protein Binding ; Protein Structure, Tertiary ; Substrate Specificity ; Tissue Inhibitor of Metalloproteinase-2/chemistry ; Tissue Inhibitor of Metalloproteinase-2/metabolism ; Tissue Inhibitor of Metalloproteinases/chemistry ; Tissue Inhibitor of Metalloproteinases/metabolism
Chemical Substances	TIMP2 protein, human ; Tissue Inhibitor of Metalloproteinases ; Tissue Inhibitor of Metalloproteinase-2 (127497-59-0) ; Matrix Metalloproteinases (EC 3.4.24.-) ; MMP10 protein, human (EC 3.4.24.22) ; Matrix Metalloproteinase 10 (EC 3.4.24.22)
Language	English
Publishing date	2013-09-20
Publishing country	United States
Document type	Comparative Study ; Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0075836
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Article ; Online: Nonadditive effects of mixed crowding on protein stability.

Batra, Jyotica / Xu, Ke / Zhou, Huan-Xiang

Proteins

2009 Volume 77, Issue 1, Page(s) 133–138

Abstract: The crowded environments inside cells can have significant effects on the folding stability and other biophysical properties of proteins. In this study on how macromolecular crowding affects protein folding, we took a significant step toward ... ...

Abstract	The crowded environments inside cells can have significant effects on the folding stability and other biophysical properties of proteins. In this study on how macromolecular crowding affects protein folding, we took a significant step toward realistically mimicking intracellular environments by using a mixture of two crowding agents, Ficoll and dextran. We found that the mixed crowding exerts a greater stabilizing effect than the sum of the two individual crowding agents. Therefore, the composition of crowders, not just the total concentration, has a significant influence on the effects of crowding on protein folding. Since the composition of intracellular macromolecules varies within the lifetime of a cell, our finding may provide an explanation for age being an important risk factor for protein aggregation-related diseases such as Alzheimer's disease and Parkinson's disease.
MeSH term(s)	Dextrans/chemistry ; Ficoll/chemistry ; Models, Biological ; Protein Folding ; Protein Stability ; Tacrolimus Binding Proteins/chemistry ; Thermodynamics
Chemical Substances	Dextrans ; Ficoll (25702-74-3) ; Tacrolimus Binding Proteins (EC 5.2.1.-)
Language	English
Publishing date	2009-04-30
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	806683-8
ISSN	1097-0134 ; 0887-3585
ISSN (online)	1097-0134
ISSN	0887-3585
DOI	10.1002/prot.22425
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Article ; Online: Matrix metalloproteinase-10 (MMP-10) interaction with tissue inhibitors of metalloproteinases TIMP-1 and TIMP-2: binding studies and crystal structure.

Batra, Jyotica / Robinson, Jessica / Soares, Alexei S / Fields, Alan P / Radisky, Derek C / Radisky, Evette S

The Journal of biological chemistry

2012 Volume 287, Issue 19, Page(s) 15935–15946

Abstract: Matrix metalloproteinase 10 (MMP-10, stromelysin-2) is a secreted metalloproteinase with functions in skeletal development, wound healing, and vascular remodeling; its overexpression is also implicated in lung tumorigenesis and tumor progression. To ... ...

Abstract	Matrix metalloproteinase 10 (MMP-10, stromelysin-2) is a secreted metalloproteinase with functions in skeletal development, wound healing, and vascular remodeling; its overexpression is also implicated in lung tumorigenesis and tumor progression. To understand the regulation of MMP-10 by tissue inhibitors of metalloproteinases (TIMPs), we have assessed equilibrium inhibition constants (K(i)) of putative physiological inhibitors TIMP-1 and TIMP-2 for the active catalytic domain of human MMP-10 (MMP-10cd) using multiple kinetic approaches. We find that TIMP-1 inhibits the MMP-10cd with a K(i) of 1.1 × 10(-9) M; this interaction is 10-fold weaker than the inhibition of the similar MMP-3 (stromelysin-1) catalytic domain (MMP-3cd) by TIMP-1. TIMP-2 inhibits the MMP-10cd with a K(i) of 5.8 × 10(-9) M, which is again 10-fold weaker than the inhibition of MMP-3cd by this inhibitor (K(i) = 5.5 × 10(-10) M). We solved the x-ray crystal structure of TIMP-1 bound to the MMP-10cd at 1.9 Å resolution; the structure was solved by molecular replacement and refined with an R-factor of 0.215 (R(free) = 0.266). Comparing our structure of MMP-10cd·TIMP-1 with the previously solved structure of MMP-3cd·TIMP-1 (Protein Data Bank entry 1UEA), we see substantial differences at the binding interface that provide insight into the differential binding of stromelysin family members to TIMP-1. This structural information may ultimately assist in the design of more selective TIMP-based inhibitors tailored for specificity toward individual members of the stromelysin family, with potential therapeutic applications.
MeSH term(s)	Binding Sites/genetics ; Binding, Competitive ; Catalytic Domain ; Crystallography, X-Ray ; HEK293 Cells ; Humans ; Kinetics ; Matrix Metalloproteinase 10/chemistry ; Matrix Metalloproteinase 10/genetics ; Matrix Metalloproteinase 10/metabolism ; Matrix Metalloproteinase 3/chemistry ; Matrix Metalloproteinase 3/genetics ; Matrix Metalloproteinase 3/metabolism ; Models, Molecular ; Mutation ; Protein Structure, Tertiary ; Tissue Inhibitor of Metalloproteinase-1/chemistry ; Tissue Inhibitor of Metalloproteinase-1/genetics ; Tissue Inhibitor of Metalloproteinase-1/metabolism ; Tissue Inhibitor of Metalloproteinase-2/chemistry ; Tissue Inhibitor of Metalloproteinase-2/genetics ; Tissue Inhibitor of Metalloproteinase-2/metabolism
Chemical Substances	TIMP2 protein, human ; Tissue Inhibitor of Metalloproteinase-1 ; Tissue Inhibitor of Metalloproteinase-2 (127497-59-0) ; Matrix Metalloproteinase 3 (EC 3.4.24.17) ; Matrix Metalloproteinase 10 (EC 3.4.24.22)
Language	English
Publishing date	2012-03-16
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't ; Research Support, U.S. Gov't, Non-P.H.S.
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.M112.341156
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Article ; Online: Effect of macromolecular crowding on protein binding stability: modest stabilization and significant biological consequences.

Batra, Jyotica / Xu, Ke / Qin, Sanbo / Zhou, Huan-Xiang

Biophysical journal

2009 Volume 97, Issue 3, Page(s) 906–911

Abstract: Macromolecular crowding has long been known to significantly affect protein oligomerization, and yet no direct quantitative measurements appear to have been made of its effects on the binding free energy of the elemental step of adding a single subunit. ... ...

Abstract	Macromolecular crowding has long been known to significantly affect protein oligomerization, and yet no direct quantitative measurements appear to have been made of its effects on the binding free energy of the elemental step of adding a single subunit. Here, we report the effects of two crowding agents on the binding free energy of two subunits in the Escherichia coli polymerase III holoenzyme. The crowding agents are found, paradoxically, to have only a modest stabilizing effect, of the order of 1 kcal/mol, on the binding of the two subunits. Systematic variations in the level of stabilization with crowder size are nevertheless observed. The data are consistent with theoretical predictions based on atomistic modeling of excluded-volume interactions with crowders. We reconcile the apparent paradox presented by our data by noting that the modest effects of crowding on elemental binding steps are cumulative, and thus lead to substantial stabilization of higher oligomers. Correspondingly, the effects of small variations in the level of crowding during the lifetime of a cell may be magnified, suggesting that crowding may play a role in increased susceptibility to protein aggregation-related diseases with aging.
MeSH term(s)	Amino Acid Sequence ; Computer Simulation ; DNA Polymerase III/chemistry ; DNA Polymerase III/genetics ; DNA Polymerase III/metabolism ; DNA-Directed DNA Polymerase/chemistry ; DNA-Directed DNA Polymerase/genetics ; DNA-Directed DNA Polymerase/metabolism ; Dextrans/chemistry ; Escherichia coli ; Escherichia coli Proteins/chemistry ; Escherichia coli Proteins/genetics ; Escherichia coli Proteins/metabolism ; Ficoll/chemistry ; Fluorometry ; Models, Chemical ; Models, Genetic ; Models, Molecular ; Molecular Sequence Data ; Molecular Weight ; Protein Binding ; Protein Stability ; Sequence Homology ; Thermodynamics
Chemical Substances	Dextrans ; Escherichia coli Proteins ; Ficoll (25702-74-3) ; holE protein, E coli (EC 2.7.7.-) ; DNA Polymerase III (EC 2.7.7.7) ; DNA-Directed DNA Polymerase (EC 2.7.7.7) ; dnaQ protein, E coli (EC 2.7.7.7)
Language	English
Publishing date	2009-06-22
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ZDB-ID	218078-9
ISSN	1542-0086 ; 0006-3495
ISSN (online)	1542-0086
ISSN	0006-3495
DOI	10.1016/j.bpj.2009.05.032
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Zs.A 235: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 1994: Bestellungen von Artikeln über das Online-Bestellformular Jg. 1995 - 2021: Lesesall (1.OG) ab Jg. 2022: Lesesaal (EG)
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Article ; Online: Long-range electrostatic complementarity governs substrate recognition by human chymotrypsin C, a key regulator of digestive enzyme activation.

Batra, Jyotica / Szabó, András / Caulfield, Thomas R / Soares, Alexei S / Sahin-Tóth, Miklós / Radisky, Evette S

The Journal of biological chemistry

2013 Volume 288, Issue 14, Page(s) 9848–9859

Abstract: Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which ...

Abstract	Human chymotrypsin C (CTRC) is a pancreatic serine protease that regulates activation and degradation of trypsinogens and procarboxypeptidases by targeting specific cleavage sites within their zymogen precursors. In cleaving these regulatory sites, which are characterized by multiple flanking acidic residues, CTRC shows substrate specificity that is distinct from that of other isoforms of chymotrypsin and elastase. Here, we report the first crystal structure of active CTRC, determined at 1.9-Å resolution, revealing the structural basis for binding specificity. The structure shows human CTRC bound to the small protein protease inhibitor eglin c, which binds in a substrate-like manner filling the S6-S5' subsites of the substrate binding cleft. Significant binding affinity derives from burial of preferred hydrophobic residues at the P1, P4, and P2' positions of CTRC, although acidic P2' residues can also be accommodated by formation of an interfacial salt bridge. Acidic residues may also be specifically accommodated in the P6 position. The most unique structural feature of CTRC is a ring of intense positive electrostatic surface potential surrounding the primarily hydrophobic substrate binding site. Our results indicate that long-range electrostatic attraction toward substrates of concentrated negative charge governs substrate discrimination, which explains CTRC selectivity in regulating active digestive enzyme levels.
MeSH term(s)	Binding Sites ; Biophysics/methods ; Calcium/chemistry ; Carboxypeptidases/chemistry ; Chymotrypsin/chemistry ; Crystallography, X-Ray/methods ; Enzyme Activation ; Gene Expression Regulation, Enzymologic ; HEK293 Cells ; Humans ; Kinetics ; Models, Molecular ; Molecular Conformation ; Mutation ; Pancreatic Elastase/chemistry ; Protein Isoforms ; Proteins/chemistry ; Static Electricity ; Substrate Specificity ; Surface Properties ; Trypsinogen/chemistry
Chemical Substances	Protein Isoforms ; Proteins ; eglin proteinase inhibitors ; Trypsinogen (9002-08-8) ; Carboxypeptidases (EC 3.4.-) ; Chymotrypsin (EC 3.4.21.1) ; chymotrypsin C (EC 3.4.21.2) ; Pancreatic Elastase (EC 3.4.21.36) ; Calcium (SY7Q814VUP)
Language	English
Publishing date	2013-02-19
Publishing country	United States
Document type	Journal Article ; Research Support, American Recovery and Reinvestment Act ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2997-x
ISSN	1083-351X ; 0021-9258
ISSN (online)	1083-351X
ISSN	0021-9258
DOI	10.1074/jbc.M113.457382
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Article ; Online: PEGylation extends circulation half-life while preserving in vitro and in vivo activity of tissue inhibitor of metalloproteinases-1 (TIMP-1).

Jyotica Batra / Jessica Robinson / Christine Mehner / Alexandra Hockla / Erin Miller / Derek C Radisky / Evette S Radisky

PLoS ONE, Vol 7, Iss 11, p e

2012 Volume 50028

Abstract: Excess proteolytic activity of matrix metalloproteinases (MMPs) contributes to the development of arthritis, cardiovascular diseases and cancer progression, implicating these enzymes as therapeutic targets. While many small molecule inhibitors of MMPs ... ...

Abstract	Excess proteolytic activity of matrix metalloproteinases (MMPs) contributes to the development of arthritis, cardiovascular diseases and cancer progression, implicating these enzymes as therapeutic targets. While many small molecule inhibitors of MMPs have been developed, clinical uses have been limited, in part by toxicity and off-target effects. Development of the endogenous tissue inhibitors of metalloproteinases (TIMPs) as recombinant biopharmaceuticals represents an alternative therapeutic approach; however, the short plasma half-life of recombinant TIMPs has restricted their potential in this arena. To overcome this limitation, we have modified recombinant human TIMP-1 (rhTIMP-1) by PEGylation on lysine residues. We analyzed a mixture of mono- and di-PEGylated rhTIMP-1 species modified by attachment of 20 kDa mPEG chains (PEG(20K)-TIMP-1), as confirmed by SELDI-TOF mass spectrometry. This preparation retained complete inhibitory activity toward the MMP-3 catalytic domain and partial inhibitory activity toward full length MMP-9. Pharmacokinetic evaluation showed that PEGylation extended the plasma half-life of rhTIMP-1 in mice from 1.1 h to 28 h. In biological assays, PEG(20K)-TIMP-1 inhibited both MMP-dependent cancer cell invasion and tumor cell associated gelatinase activity. Overall these results suggest that PEGylated TIMP-1 exhibits improved potential for development as an anti-cancer recombinant protein therapeutic, and additionally may offer potential for clinical applications in the treatment of other diseases.
Keywords	Medicine ; R ; Science ; Q
Subject code	610
Language	English
Publishing date	2012-01-01T00:00:00Z
Publisher	Public Library of Science (PLoS)
Document type	Article ; Online
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Article ; Online: Matrix metalloproteinase-10 is required for lung cancer stem cell maintenance, tumor initiation and metastatic potential.

Justilien, Verline / Regala, Roderick P / Tseng, I-Chu / Walsh, Michael P / Batra, Jyotica / Radisky, Evette S / Murray, Nicole R / Fields, Alan P

PloS one

2012 Volume 7, Issue 4, Page(s) e35040

Abstract: Matrix metalloproteinases (Mmps) stimulate tumor invasion and metastasis by degrading the extracellular matrix. Here we reveal an unexpected role for Mmp10 (stromelysin 2) in the maintenance and tumorigenicity of mouse lung cancer stem-like cells (CSC). ... ...

Abstract	Matrix metalloproteinases (Mmps) stimulate tumor invasion and metastasis by degrading the extracellular matrix. Here we reveal an unexpected role for Mmp10 (stromelysin 2) in the maintenance and tumorigenicity of mouse lung cancer stem-like cells (CSC). Mmp10 is highly expressed in oncosphere cultures enriched in CSCs and RNAi-mediated knockdown of Mmp10 leads to a loss of stem cell marker gene expression and inhibition of oncosphere growth, clonal expansion, and transformed growth in vitro. Interestingly, clonal expansion of Mmp10 deficient oncospheres can be restored by addition of exogenous Mmp10 protein to the culture medium, demonstrating a direct role for Mmp10 in the proliferation of these cells. Oncospheres exhibit enhanced tumor-initiating and metastatic activity when injected orthotopically into syngeneic mice, whereas Mmp10-deficient cultures show a severe defect in tumor initiation. Conversely, oncospheres implanted into syngeneic non-transgenic or Mmp10(-/-) mice show no significant difference in tumor initiation, growth or metastasis, demonstrating the importance of Mmp10 produced by cancer cells rather than the tumor microenvironment in lung tumor initiation and maintenance. Analysis of gene expression data from human cancers reveals a strong positive correlation between tumor Mmp10 expression and metastatic behavior in many human tumor types. Thus, Mmp10 is required for maintenance of a highly tumorigenic, cancer-initiating, metastatic stem-like cell population in lung cancer. Our data demonstrate for the first time that Mmp10 is a critical lung cancer stem cell gene and novel therapeutic target for lung cancer stem cells.
MeSH term(s)	Animals ; Cell Line, Tumor ; Cell Transformation, Neoplastic/genetics ; Cell Transformation, Neoplastic/metabolism ; Cell Transformation, Neoplastic/pathology ; Gene Expression Regulation, Neoplastic ; Humans ; Lung/enzymology ; Lung/metabolism ; Lung/pathology ; Lung Neoplasms/enzymology ; Lung Neoplasms/genetics ; Lung Neoplasms/pathology ; Matrix Metalloproteinase 10/genetics ; Matrix Metalloproteinase 10/metabolism ; Mice ; Mice, Inbred C57BL ; Neoplasm Metastasis/genetics ; Neoplasm Metastasis/pathology ; Neoplastic Stem Cells/enzymology ; Neoplastic Stem Cells/metabolism ; Neoplastic Stem Cells/pathology ; Tumor Microenvironment
Chemical Substances	Matrix Metalloproteinase 10 (EC 3.4.24.22)
Language	English
Publishing date	2012-04-24
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ISSN	1932-6203
ISSN (online)	1932-6203
DOI	10.1371/journal.pone.0035040
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Article ; Online: PEGylation extends circulation half-life while preserving in vitro and in vivo activity of tissue inhibitor of metalloproteinases-1 (TIMP-1).

Batra, Jyotica / Robinson, Jessica / Mehner, Christine / Hockla, Alexandra / Miller, Erin / Radisky, Derek C / Radisky, Evette S

PloS one

2012 Volume 7, Issue 11, Page(s) e50028

Abstract	Excess proteolytic activity of matrix metalloproteinases (MMPs) contributes to the development of arthritis, cardiovascular diseases and cancer progression, implicating these enzymes as therapeutic targets. While many small molecule inhibitors of MMPs have been developed, clinical uses have been limited, in part by toxicity and off-target effects. Development of the endogenous tissue inhibitors of metalloproteinases (TIMPs) as recombinant biopharmaceuticals represents an alternative therapeutic approach; however, the short plasma half-life of recombinant TIMPs has restricted their potential in this arena. To overcome this limitation, we have modified recombinant human TIMP-1 (rhTIMP-1) by PEGylation on lysine residues. We analyzed a mixture of mono- and di-PEGylated rhTIMP-1 species modified by attachment of 20 kDa mPEG chains (PEG(20K)-TIMP-1), as confirmed by SELDI-TOF mass spectrometry. This preparation retained complete inhibitory activity toward the MMP-3 catalytic domain and partial inhibitory activity toward full length MMP-9. Pharmacokinetic evaluation showed that PEGylation extended the plasma half-life of rhTIMP-1 in mice from 1.1 h to 28 h. In biological assays, PEG(20K)-TIMP-1 inhibited both MMP-dependent cancer cell invasion and tumor cell associated gelatinase activity. Overall these results suggest that PEGylated TIMP-1 exhibits improved potential for development as an anti-cancer recombinant protein therapeutic, and additionally may offer potential for clinical applications in the treatment of other diseases.
MeSH term(s)	Animals ; Cell Line, Tumor ; Gelatinases/metabolism ; Half-Life ; Humans ; Lysine/chemistry ; Matrix Metalloproteinase 3/metabolism ; Matrix Metalloproteinase 9/metabolism ; Matrix Metalloproteinase Inhibitors/administration & dosage ; Matrix Metalloproteinase Inhibitors/chemistry ; Matrix Metalloproteinase Inhibitors/metabolism ; Mice ; Neoplasm Invasiveness ; Polyethylene Glycols ; Protease Inhibitors/administration & dosage ; Protease Inhibitors/chemistry ; Protease Inhibitors/metabolism ; Recombinant Proteins/administration & dosage ; Recombinant Proteins/chemistry ; Recombinant Proteins/genetics ; Recombinant Proteins/metabolism ; Tissue Inhibitor of Metalloproteinase-1/administration & dosage ; Tissue Inhibitor of Metalloproteinase-1/chemistry ; Tissue Inhibitor of Metalloproteinase-1/genetics ; Tissue Inhibitor of Metalloproteinase-1/metabolism
Chemical Substances	Matrix Metalloproteinase Inhibitors ; Protease Inhibitors ; Recombinant Proteins ; TIMP1 protein, human ; Tissue Inhibitor of Metalloproteinase-1 ; Polyethylene Glycols (3WJQ0SDW1A) ; Gelatinases (EC 3.4.24.-) ; Matrix Metalloproteinase 3 (EC 3.4.24.17) ; Matrix Metalloproteinase 9 (EC 3.4.24.35) ; Lysine (K3Z4F929H6)
Language	English
Publishing date	2012-11-20
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, U.S. Gov't, P.H.S.
ZDB-ID	2267670-3
ISSN	1932-6203 ; 1932-6203
ISSN (online)	1932-6203
ISSN	1932-6203
DOI	10.1371/journal.pone.0050028
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