Mapping Extracellular Protein-Protein Interactions Using Extracellular Proximity Labeling (ePL).

Proximity labeling (PL) has given researchers the tools to explore protein-protein interactions (PPIs) in living systems; however, most PL studies are performed on intracellular targets. We have adapted the original PL method to investigate PPIs within the extracellular compartment, which we term extracellular PL (ePL). To demonstrate the utility of this modified technique, we investigated the interactome of the matrisome protein TIMP2. TIMPs are a family of multifunctional proteins that were initially defined by their ability to inhibit metalloproteinases, the major mediators of extracellular matrix (ECM) turnover. TIMP2 exhibits broad expression and is often abundant in both normal and diseased tissues. Understanding the functional transformation of matrisome regulators, such as TIMP2, during disease progression is essential for the development of ECM-targeted therapeutics. Using dual orientation fusion proteins of TIMP2 with BioID2/TurboID, we describe the TIMP2 proximal interactome (MassIVE MSV000095637). We also illustrate how the TIMP2 interactome changes in the presence of different stimuli, in different cell types, in unique culture conditions (2D vs 3D), and with different reaction kinetics, demonstrating the power of this technique versus classical PPI methods. We propose that screening of matrisome targets in disease models using ePL will reveal new therapeutic targets for further comprehensive studies.

Analysis of cancer cell line and tissue RNA sequencing data reveals an essential and dark matrisome.

Extracellular matrix remodeling is a hallmark of tissue development, homeostasis, and disease. The processes that mediate remodeling, and the consequences of such, are the topic of extensive focus in biomedical research. Cell culture methods represent a crucial tool utilized by those interested in matrisome function, the easiest of which are implemented with immortalized/cancer cell lines. These cell lines often form the foundations of a research proposal, or serve as vehicles of validation for other model systems. For these reasons, it is important to understand the complement of matrisome genes that are expressed when identifying appropriate cell culture models for hypothesis testing. To this end, we harvested bulk RNA sequencing data from the Cancer Cell Line Encyclopedia (CCLE) to assess matrisome gene expression in 1019 human cell lines. Our examination reveals that a large proportion of the matrisome is poorly represented in human cancer cell lines, with approximately 10% not expressed above threshold in any of the cell lines assayed. Conversely, we identify clusters of essential/common matrisome genes that are abundantly expressed in cell lines. To validate these observations against tissue data, we compared our findings with bulk RNA sequencing data from the Genotype-Tissue Expression (GTEx) portal and The Cancer Genome Atlas (TCGA) program. This comparison demonstrates general agreement between the "essential/common" and "dark/uncommon" matrisome across the three datasets, albeit with discordance observed in 59 matrisome genes between cell lines and tissues. Notably, all of the discordant genes are essential/common in tissues yet minimally expressed in cell lines, underscoring critical considerations for matrix biology researchers employing immortalized cell lines for their investigations.

Extracellular Proximity Labeling Reveals an Expanded Interactome for the Matrisome Protein TIMP2.

Classical methods of investigating protein-protein interactions (PPIs) are generally performed in non-living systems, yet in recent years new technologies utilizing proximity labeling (PL) have given researchers the tools to explore proximal PPIs in living systems. PL has distinct advantages over traditional protein interactome studies, such as the ability to identify weak and transient interactions in vitro and in vivo. Most PL studies are performed on targets within the cell or on the cell membrane. We have adapted the original PL method to investigate PPIs within the extracellular compartment, using both BioID2 and TurboID, that we term extracellular PL (ePL). To demonstrate the utility of this modified technique, we investigate the interactome of the widely expressed matrisome protein tissue inhibitor of metalloproteinases 2 (TIMP2). Tissue inhibitors of metalloproteinases (TIMPs) are a family of multi-functional proteins that were initially defined by their ability to inhibit the enzymatic activity of metalloproteinases (MPs), the major mediators of extracellular matrix (ECM) breakdown and turnover. TIMP2 exhibits a broad expression profile and is often abundant in both normal and diseased tissues. Understanding the functional transformation of matrisome regulators, like TIMP2, during the evolution of tissue microenvironments associated with disease progression is essential for the development of ECM-targeted therapeutics. Using carboxyl- and amino-terminal fusion proteins of TIMP2 with BioID2 and TurboID, we describe the TIMP2 proximal interactome. We also illustrate how the TIMP2 interactome changes in the presence of different stimuli, in different cell types, in unique culture conditions (2D vs 3D), and with different reaction kinetics (BioID2 vs. TurboID); demonstrating the power of this technique versus classical PPI methods. We propose that the screening of matrisome targets in disease models using ePL will reveal new therapeutic targets for further comprehensive studies.

The TIMP protein family: diverse roles in pathophysiology.

The tissue inhibitors of matrix metalloproteinases (TIMPs) are a family of four matrisome proteins classically defined by their roles as the primary endogenous inhibitors of metalloproteinases (MPs). Their functions however are not limited to MP inhibition, with each family member harboring numerous MP-independent biological functions that play key roles in processes such as inflammation and apoptosis. Because of these multifaceted functions, TIMPs have been cited in diverse pathophysiological contexts. Herein, we provide a comprehensive overview of the MP-dependent and -independent roles of TIMPs across a range of pathological conditions. The potential therapeutic and biomarker applications of TIMPs in these disease contexts are also considered, highlighting the biomedical promise of this complex and often misunderstood protein family.

Tissue inhibitors of metalloproteinases are proteolytic targets of matrix metalloproteinase 9.

Extracellular proteolysis and turnover are core processes of tissue homeostasis. The predominant matrix-degrading enzymes are members of the Matrix Metalloproteinase (MMP) family. MMPs extensively degrade core matrix components in addition to processing a range of other factors in the extracellular, plasma membrane, and intracellular compartments. The proteolytic activity of MMPs is modulated by the Tissue Inhibitors of Metalloproteinases (TIMPs), a family of four multi-functional matrisome proteins with extensively characterized MMP inhibitory functions. Thus, a well-regulated balance between MMP activity and TIMP levels has been described as critical for healthy tissue homeostasis, and this balance can be chronically disturbed in pathological processes. The relationship between MMPs and TIMPs is complex and lacks the constraints of a typical enzyme-inhibitor relationship due to secondary interactions between various MMPs (specifically gelatinases) and TIMP family members. We illustrate a new complexity in this system by describing how MMP9 can cleave members of the TIMP family when in molar excess. Proteolytic processing of TIMPs can generate functionally altered peptides with potentially novel attributes. We demonstrate here that all TIMPs are cleaved at their C-terminal tails by a molar excess of MMP9. This processing removes the N-glycosylation site for TIMP3 and prevents the TIMP2 interaction with latent proMMP2, a prerequisite for cell surface MMP14-mediated activation of proMMP2. TIMP2/4 are further cleaved producing ∼14 kDa N-terminal proteins linked to a smaller C-terminal domain through residual disulfide bridges. These cleaved TIMP2/4 complexes show perturbed MMP inhibitory activity, illustrating that MMP9 may bear a particularly prominent influence upon the TIMP:MMP balance in tissues.

The Continuing Saga of Tissue Inhibitor of Metalloproteinase 2: Emerging Roles in Tissue Homeostasis and Cancer Progression.

Tissue inhibitors of metalloproteinases (TIMPs) are a conserved family of proteins that were originally identified as cytokine-like erythroid growth factors. Subsequently, TIMPs were characterized as endogenous inhibitors of matrixin proteinases. These proteinases are the primary mediators of extracellular matrix turnover in pathologic conditions, such as cancer invasion and metastasis. Thus, TIMPs were immediately recognized as important regulators of tissue homeostasis. However, TIMPs also demonstrate unique biological activities that are independent of metalloproteinase regulation. Although often overlooked, these non-protease-mediated TIMP functions demonstrate a variety of direct cellular effects of potential therapeutic value. TIMP2 is the most abundantly expressed TIMP family member, and ongoing studies show that its tumor suppressor activity extends beyond protease inhibition to include direct modulation of tumor, endothelial, and fibroblast cellular responses in the tumor microenvironment. Recent data suggest that TIMP2 can suppress both primary tumor growth and metastatic niche formation. TIMP2 directly interacts with cellular receptors and matrisome elements to modulate cell signaling pathways that result in reduced proliferation and migration of neoplastic, endothelial, and fibroblast cell populations. These effects result in enhanced cell adhesion and focal contact formation while reducing tumor and endothelial proliferation, migration, and epithelial-to-mesenchymal transitions. These findings are consistent with TIMP2 homeostatic functions beyond simple inhibition of metalloprotease activity. This review examines the ongoing evolution of TIMP2 function, future perspectives in TIMP research, and the therapeutic potential of TIMP2.

Whole organism profiling of the Timp gene family.

Tissue inhibitor of metalloproteinases (TIMPs/Timps) are an endogenous family of widely expressed matrisome-associated proteins that were initially identified as inhibitors of matrix metalloproteinase activity (Metzincin family proteases). Consequently, TIMPs are often considered simply as protease inhibitors by many investigators. However, an evolving list of new metalloproteinase-independent functions for TIMP family members suggests that this concept is outdated. These novel TIMP functions include direct agonism/antagonism of multiple transmembrane receptors, as well as functional interactions with matrisome targets. While the family was fully identified over two decades ago, there has yet to be an in-depth study describing the expression of TIMPs in normal tissues of adult mammals. An understanding of the tissues and cell-types that express TIMPs 1 through 4, in both normal and disease states are important to contextualize the growing functional capabilities of TIMP proteins, which are often dismissed as non-canonical. Using publicly available single cell RNA sequencing data from the Tabula Muris Consortium, we analyzed approximately 100,000 murine cells across eighteen tissues from non-diseased organs, representing seventy-three annotated cell types, to define the diversity in Timp gene expression across healthy tissues. We describe the unique expression profiles across tissues and organ-specific cell types that all four Timp genes display. Within annotated cell-types, we identify clear and discrete cluster-specific patterns of Timp expression, particularly in cells of stromal and endothelial origins. RNA in-situ hybridization across four organs expands on the scRNA sequencing analysis, revealing novel compartments associated with individual Timp expression. These analyses emphasize a need for specific studies investigating the functional significance of Timp expression in the identified tissues and cell sub-types. This understanding of the tissues, specific cell types and microenvironment conditions in which Timp genes are expressed adds important physiological context to the growing array of novel functions for TIMP proteins.

Timp2 loss-of-function mutation and TIMP2 treatment in murine model of NSCLC: modulation of immunosuppression and oncogenic signaling.

Mounting evidence suggests that the tissue inhibitor of metalloproteinases-2 (TIMP2) can reduce tumor burden and metastasis. However, the demonstration of such anti-tumor activity and associated mechanisms using in vivo tumor models is lacking. The effects of a Timp2 functional mutation and administration of recombinant TIMP2 were examined in both orthotopic and heterotopic murine models of lung cancer using C57Bl/6 syngeneic Lewis Lung 2-luciferase 2 cells (LL2-luc2) cells. Mice harboring a functional mutation of TIMP2 (mT2) display markedly increased primary lung tumor growth, increased mortality, enriched vasculature, and enhanced infiltration of pro-tumorigenic, immunosuppressive myeloid cells. Treatment with recombinant TIMP2 reduced primary tumor growth in both mutant and wild-type (wt) mice. Comparison of transcriptional profiles of lung tissues from tumor-free, wt versus mT2 mice reveals only minor changes. However, lung tumor-bearing mice of both genotypes demonstrate significant genotype-dependent changes in gene expression following treatment with TIMP. In tumor-bearing wt mice, TIMP2 treatment reduced the expression of upstream oncogenic mediators, whereas treatment of mT2 mice resulted in an immunomodulatory phenotype. A heterotopic subcutaneous model generating metastatic pulmonary tumors demonstrated that daily administration of recombinant TIMP2 significantly downregulates the expression of heat shock proteins, suggesting a reduction of cell-stress responses. In summary, we describe how TIMP2 exerts novel, anti-tumor effects in a murine model of lung cancer and that rTIMP2 treatment supports a normalizing effect on the tumor microenvironment. Our findings show that TIMP2 treatment demonstrates significant potential as an adjuvant in the treatment of NSCLC.

Unravelling the distinct biological functions and potential therapeutic applications of TIMP2 in cancer.

Tissue inhibitors of metalloproteinases (TIMPs) are a conserved family of proteins that were originally identified as endogenous inhibitors of matrixin and adamalysin endopeptidase activity. The matrixins and adamalysins are the major mediators of extracellular matrix (ECM) turnover, thus making TIMPs important regulators of ECM structure and composition. Despite their high sequence identity and relative redundancy in inhibitory profiles, each TIMP possesses unique biological characteristics that are independent of their regulation of metalloproteinase activity. As our understanding of TIMP biology has evolved, distinct roles have been assigned to individual TIMPs in cancer progression. In this respect, data regarding TIMP2's role in cancer have borne conflicting reports of both tumor suppressor and, to a lesser extent, tumor promoter functions. TIMP2 is the most abundant TIMP family member, prevalent in normal and diseased mammalian tissues as a constitutively expressed protein. Despite its apparent stable expression, recent work highlights how TIMP2 is a cell stress-induced gene product and that its biological activity can be dictated by extracellular posttranslational modifications. Hence an understanding of TIMP2 molecular targets, and how its biological functions evolve in the progressing tumor microenvironment may reveal new therapeutic opportunities. In this review, we discuss the continually evolving functions of TIMP proteins, future perspectives in TIMP research, and the therapeutic utility of this family, with a particular focus on TIMP2.

TIMP1 and TIMP2 Downregulate TGFß Induced Decidual-like Phenotype in Natural Killer Cells.

Natural Killer (NK) cells have been found to be anergic, exhausted and pro-angiogenic in cancers. NK cell from healthy donors, exposed to TGFß, acquire the CD56brightCD9+CD49a+ decidual-like-phenotype, together with decreased levels of NKG2D activation marker, increased levels of TIM-3 exhaustion marker, similar to cancer-associated NK cells. Tissue inhibitors of metalloproteases (TIMPs) exert dual roles in cancer. The role of TIMPs in modulating immune cells is a very novel concept, and the present is the first report studying their ability to contrast TGFß action on NK cells. Here, we investigated the effects of TIMP1 and TIMP2 recombinant proteins in hindering decidual-like markers in NK cells, generated by polarizing cytolytic NK cells with TGFß. The effects of TIMP1 or TIMP2 on NK cell surface antigens were determined by multicolor flow cytometry. We found that TIMP1 and TIMP2 were effective in interfering with TGFß induced NK cell polarization towards a decidual-like-phenotype. TIMP1 and TIMP2 counteracted the effect of TGFß in increasing the percentage of CD56bright, CD16-, CD9+ and CD49a+, and restoring normal levels for TIMP 1 and 2 also inhibited decrease levels of the activation marker NKG2D induced by TGFß and decreased the TGFß upregulated exhaustion marker TIM-3. NK cell degranulation capabilities against K562 cells were also decreased by TGFß and not by TIMP1 or TIMP2. TIMP1 treatment could partially restore degranulation marker CD107a expression. Treatment with recombinant TIMP-1 or TIMP-2 showed a trend, although not statistically significant, to decrease CD49a+ and TIM-3+ expression and increase NKG2D in peripheral blood NK cells exposed to conditioned media from colon cancer cell lines. Our results suggest a potential role of TIMPs in controlling the tumor-associated cytokine TGFß-induced NK cell polarization. Given the heterogeneity of released factors within the TME, it is clear that TGFß stimulation represents a model to prove TIMP's new properties, but it cannot be envisaged as a soloist NK cell polarizing agent. Therefore, further studies from the scientific community will help defining TIMPs immunomodulatory activities of NK cells in cancer, and their possible future diagnostic-therapeutic roles.

The tumor suppressor folliculin inhibits lactate dehydrogenase A and regulates the Warburg effect.

Aerobic glycolysis in cancer cells, also known as the 'Warburg effect', is driven by hyperactivity of lactate dehydrogenase A (LDHA). LDHA is thought to be a substrate-regulated enzyme, but it is unclear whether a dedicated intracellular protein also regulates its activity. Here, we identify the human tumor suppressor folliculin (FLCN) as a binding partner and uncompetitive inhibitor of LDHA. A flexible loop within the amino terminus of FLCN controls movement of the LDHA active-site loop, tightly regulating its enzyme activity and, consequently, metabolic homeostasis in normal cells. Cancer cells that experience the Warburg effect show FLCN dissociation from LDHA. Treatment of these cells with a decapeptide derived from the FLCN loop region causes cell death. Our data suggest that the glycolytic shift of cancer cells is the result of FLCN inactivation or dissociation from LDHA. Together, FLCN-mediated inhibition of LDHA provides a new paradigm for the regulation of glycolysis.

Inhibition of Hepatic Stellate Cell Activation Suppresses Tumorigenicity of Hepatocellular Carcinoma in Mice.

Transdifferentiation (or activation) of hepatic stellate cells (HSCs) to myofibroblasts is a key event in liver fibrosis. Activated HSCs in the tumor microenvironment reportedly promote tumor progression. This study analyzed the effect of an inhibitor of HSC activation, retinol-binding protein-albumin domain III fusion protein (R-III), on protumorigenic functions of HSCs. Although conditioned medium collected from activated HSCs enhanced the migration, invasion, and proliferation of the hepatocellular carcinoma cell line Hepa-1c1c7, this effect was not observed in Hepa-1c1c7 cells treated with conditioned medium from R-III-exposed HSCs. In a subcutaneous tumor model, larger tumors with increased vascular density were formed in mice transplanted with Hepa-1c1c7+HSC than in mice transplanted with Hepa-1c1c7 cells alone. Intriguingly, when Hepa-1c1c7+HSC-transplanted mice were injected intravenously with R-III, a reduction in vascular density and extended tumor necrosis were observed. In an orthotopic tumor model, co-transplantation of HSCs enhanced tumor growth, angiogenesis, and regional metastasis accompanied by increased peritumoral lymphatic vessel density, which was abolished by R-III. In vitro study showed that R-III treatment affected the synthesis of pro-angiogenic and anti-angiogenic factors in activated HSCs, which might be the potential mechanism underlying the R-III effect. These findings suggest that the inhibition of HSC activation abrogates HSC-induced tumor angiogenesis and growth, which represents an attractive therapeutic strategy.

High-Affinity Binding of LDL Receptor-Related Protein 1 to Matrix Metalloprotease 1 Requires Protease:Inhibitor Complex Formation.

Matrix metalloprotease (MMP) activation contributes to the degradation of the extracellular matrix (ECM), resulting in a multitude of pathologies. Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifaceted endocytic and signaling receptor that is responsible for internalization and lysosomal degradation of diverse proteases, protease inhibitors, and lipoproteins along with numerous other proteins. In this study, we identified MMP-1 as a novel LRP1 ligand. Binding studies employing surface plasmon resonance revealed that both proMMP-1 and active MMP-1 bind to purified LRP1 with equilibrium dissociation constants (KD) of 19 and 25 nM, respectively. We observed that human aortic smooth muscle cells readily internalize and degrade 125I-labeled proMMP-1 in an LRP1-mediated process. Our binding data also revealed that all tissue inhibitors of metalloproteases (TIMPs) bind to LRP1 with KD values ranging from 23 to 33 nM. Interestingly, the MMP-1/TIMP-1 complex bound to LRP1 with an affinity (KD = 0.6 nM) that was 30-fold higher than that of either component alone, revealing that LRP1 prefers the protease:inhibitor complex as a ligand. Of note, modification of lysine residues on either proMMP-1 or TIMP-1 ablated the ability of the MMP-1/TIMP-1 complex to bind to LRP1. LRP1's preferential binding to enzyme:inhibitor complexes was further supported by the higher binding affinity for proMMP-9/TIMP-1 complexes than for either of these two components alone. LRP1 has four clusters of ligand-binding repeats, and MMP-1, TIMP-1, and MMP-1/TIMP-1 complexes bound to cluster III most avidly. Our results reveal an important role for LRP1 in controlling ECM homeostasis by regulating MMP-1 and MMP-9 levels.

TIMP-2 suppresses tumor growth and metastasis in murine model of triple-negative breast cancer.

Metastasis is the primary cause of treatment failures and mortality in most cancers. Triple-negative breast cancer (TNBC) is refractory to treatment and rapidly progresses to disseminated disease. We utilized an orthotopic mouse model that molecularly and phenotypically resembles human TNBC to study the effects of exogenous, daily tissue inhibitor of metalloproteinase-2 (TIMP-2) treatment on tumor growth and metastasis. Our results demonstrated that TIMP-2 treatment maximally suppressed primary tumor growth by ~36-50% and pulmonary metastasis by >92%. Immunostaining assays confirmed disruption of the epithelial to mesenchymal transition (EMT) and promotion of vascular integrity in primary tumor tissues. Immunostaining and RNA sequencing analysis of lung tissue lysates from tumor-bearing mice identified significant changes associated with metastatic colony formation. Specifically, TIMP-2 treatment disrupts periostin localization and critical cell-signaling pathways, including canonical Wnt signaling involved in EMT, as well as PI3K signaling, which modulates proliferative and metastatic behavior through p27 phosphorylation/localization. In conclusion, our study provides evidence in support of a role for TIMP-2 in suppression of triple-negative breast cancer growth and metastasis through modulation of the epithelial to mesenchymal transition, vascular normalization, and signaling pathways associated with metastatic outgrowth. Our findings suggest that TIMP-2, a constituent of the extracellular matrix in normal tissues, may have both direct and systemic antitumor and metastasis suppressor effects, suggesting potential utility in the clinical management of breast cancer progression.

Matrisome-Associated Gene Expression Patterns Correlating with TIMP2 in Cancer.

Remodeling of the extracellular matrix (ECM) to facilitate invasion and metastasis is a universal hallmark of cancer progression. However, a definitive therapeutic target remains to be identified in this tissue compartment. As major modulators of ECM structure and function, matrix metalloproteinases (MMPs) are highly expressed in cancer and have been shown to support tumor progression. MMP enzymatic activity is inhibited by the tissue inhibitor of metalloproteinase (TIMP1-4) family of proteins, suggesting that TIMPs may possess anti-tumor activity. TIMP2 is a promiscuous MMP inhibitor that is ubiquitously expressed in normal tissues. In this study, we address inconsistencies in the literature regarding the role of TIMP2 in tumor progression by analyzing co-expressed genes in tumor vs. normal tissue. Utilizing data from The Cancer Genome Atlas and Genotype-Tissue expression studies, focusing on breast and lung carcinomas, we analyzed the correlation between TIMP2 expression and the transcriptome to identify a list of genes whose expression is highly correlated with TIMP2 in tumor tissues. Bioinformatic analysis of the identified gene list highlights a core of matrix and matrix-associated genes that are of interest as potential modulators of TIMP2 function, thus ECM structure, identifying potential tumor microenvironment biomarkers and/or therapeutic targets for further study.

Co-chaperones TIMP2 and AHA1 Competitively Regulate Extracellular HSP90:Client MMP2 Activity and Matrix Proteolysis.

The extracellular molecular chaperone heat shock protein 90 (eHSP90) stabilizes protease client the matrix metalloproteinase 2 (MMP2), leading to tumor cell invasion. Although co-chaperones are critical modulators of intracellular HSP90:client function, how the eHSP90:MMP2 complex is regulated remains speculative. Here, we report that the tissue inhibitor of metalloproteinases-2 (TIMP2) is a stress-inducible extracellular co-chaperone that binds to eHSP90, increases eHSP90 binding to ATP, and inhibits its ATPase activity. In addition to disrupting the eHSP90:MMP2 complex and terminally inactivating MMP2, TIMP2 loads the client to eHSP90, keeping the protease in a transient inhibitory state. Secreted activating co-chaperone AHA1 displaces TIMP2 from the complex, providing a "reactivating" mechanism for MMP2. Gene knockout or blocking antibodies targeting TIMP2 and AHA1 released by HT1080 cancer cells modify their gelatinolytic activity. Our data suggest that TIMP2 and AHA1 co-chaperones function as a molecular switch that determines the inhibition and reactivation of the eHSP90 client protein MMP2.

Extracellular Phosphorylation of TIMP-2 by Secreted c-Src Tyrosine Kinase Controls MMP-2 Activity.

The tissue inhibitor of metalloproteinases 2 (TIMP-2) is a specific endogenous inhibitor of matrix metalloproteinase 2 (MMP-2), which is a key enzyme that degrades the extracellular matrix and promotes tumor cell invasion. Although the TIMP-2:MMP-2 complex controls proteolysis, the signaling mechanism by which the two proteins associate in the extracellular space remains unidentified. Here we report that TIMP-2 is phosphorylated outside the cell by secreted c-Src tyrosine kinase. As a consequence, phosphorylation at Y90 significantly enhances TIMP-2 potency as an MMP-2 inhibitor and weakens the catalytic action of the active enzyme. TIMP-2 phosphorylation also appears to be essential for its interaction with the latent enzyme proMMP-2 in vivo. Absence of the kinase or non-phosphorylatable Y90 abolishes TIMP-2 binding to the latent enzyme, ultimately hampering proMMP-2 activation. Together, TIMP-2 phosphorylation by secreted c-Src represents a critical extracellular regulatory mechanism that controls the proteolytic function of MMP-2.

Macromolecule-Network Electrostatics Controlling Delivery of the Biotherapeutic Cell Modulator TIMP-2.

Tissue inhibitor of metalloproteinase 2 (TIMP-2) is an endogenous 22 kDa proteinase inhibitor, demonstrating antitumorigenic, antimetastatic and antiangiogenic activities in vitro and in vivo. Recombinant TIMP-2 is currently undergoing preclinical testing in multiple, murine tumor models. Here we report the development of an inert, injectable peptide hydrogel matrix enabling encapsulation and sustained release of TIMP-2. We studied the TIMP-2 release profile from four ß-hairpin peptide gels of varying net electrostatic charge. A negatively charged peptide gel (designated AcVES3) enabling encapsulation of 4 mg/mL of TIMP-2, without effects on rheological properties, facilitated the slow sustained release (0.9%/d) of TIMP-2 over 28 d. Released TIMP-2 is structurally intact and maintains the ability to inhibit MMP activity, as well as suppress lung cancer cell proliferation in vitro. These findings suggest that the AcVES3 hydrogel will be useful as an injectable vehicle for systemic delivery of TIMP-2 in vivo for ongoing preclinical development.

Tissue Inhibitor of Metalloprotease-2 (TIMP-2): Bioprocess Development, Physicochemical, Biochemical, and Biological Characterization of Highly Expressed Recombinant Protein.

Tissue inhibitor of metalloprotease-2 (TIMP-2) is a secreted 21 kDa multifunctional protein first described as an endogenous inhibitor of matrix metalloproteinases (MMPs) that prevents breakdown of the extracellular matrix often observed in chronic diseases. TIMP-2 diminishes the level of growth factor-mediated cell proliferation in vitro, as well as neoangiogenesis and tumor growth in vivo independent of its MMP inhibitory activity. These physiological properties make TIMP-2 an excellent candidate for further preclinical development as a biologic therapy of cancer. Here we present a straightforward bioprocessing methodology that yields >35 mg/L recombinant human TIMP-2 6XHis-tagged protein (rhTIMP-2) from suspension cultures of HEK-293-F cells. Enhanced rhTIMP-2-6XHis yields were achieved by optimization of both TIMP-2 cDNA codon sequence and cell culture conditions. Using a two-step chromatographic process, we achieved >95% purity with minimal processing losses. Purified rhTIMP-2-6XHis was free of mouse antigen contamination. Circular dichroism spectroscopy indicated a well-folded rhTIMP-2-6XHis that is highly stable and refractory to pH changes. Two-dimensional heteronuclear single-quantum coherence nuclear magnetic resonance of full length rhTIMP-2-6XHis also indicated a monodisperse, well-folded protein preparation. Purified rhTIMP-2-6XHis inhibited MMP-2 enzymatic activity in a dose-dependent fashion with an IC50 of ∼1.4 nM. Pretreatment of A549 lung cancer and JygMC(A) triple-negative breast cancer cells with rhTIMP-2-6XHis in low-nanomolar amounts inhibited EGF-induced proliferation to basal (unstimulated) levels. This study therefore not only offers a robust bioprocess methodology for rhTIMP-2 production but also characterizes critical physicochemical and biological attributes that are useful for monitoring quality control of the production process.

Mps1 Mediated Phosphorylation of Hsp90 Confers Renal Cell Carcinoma Sensitivity and Selectivity to Hsp90 Inhibitors.

The molecular chaperone Hsp90 protects deregulated signaling proteins that are vital for tumor growth and survival. Tumors generally display sensitivity and selectivity toward Hsp90 inhibitors; however, the molecular mechanism underlying this phenotype remains undefined. We report that the mitotic checkpoint kinase Mps1 phosphorylates a conserved threonine residue in the amino-domain of Hsp90. This, in turn, regulates chaperone function by reducing Hsp90 ATPase activity while fostering Hsp90 association with kinase clients, including Mps1. Phosphorylation of Hsp90 is also essential for the mitotic checkpoint because it confers Mps1 stability and activity. We identified Cdc14 as the phosphatase that dephosphorylates Hsp90 and disrupts its interaction with Mps1. This causes Mps1 degradation, thus providing a mechanism for its inactivation. Finally, Hsp90 phosphorylation sensitizes cells to its inhibitors, and elevated Mps1 levels confer renal cell carcinoma selectivity to Hsp90 drugs. Mps1 expression level can potentially serve as a predictive indicator of tumor response to Hsp90 inhibitors.