Structural characterization of the human DjC20/HscB cochaperone in solution.

J-domain proteins (JDPs) form a very large molecular chaperone family involved in proteostasis processes, such as protein folding, trafficking through membranes and degradation/disaggregation. JDPs are Hsp70 co-chaperones capable of stimulating ATPase activity as well as selecting and presenting client proteins to Hsp70. In mitochondria, human DjC20/HscB (a type III JDP that possesses only the conserved J-domain in some region of the protein) is involved in [FeS] protein biogenesis and assists human mitochondrial Hsp70 (HSPA9). Human DjC20 possesses a zinc-finger domain in its N-terminus, which closely contacts the J-domain and appears to be essential for its function. Here, we investigated the hDjC20 structure in solution as well as the importance of Zn+2 for its stability. The recombinant hDjC20 was pure, folded and capable of stimulating HSPA9 ATPase activity. It behaved as a slightly elongated monomer, as attested by small-angle X-ray scattering and SEC-MALS. The presence of Zn2+ in the hDjC20 samples was verified, a stoichiometry of 1:1 was observed, and its removal by high concentrations of EDTA and DTPA was unfeasible. However, thermal and chemical denaturation in the presence of EDTA led to a reduction in protein stability, suggesting a synergistic action between the chelating agent and denaturators that facilitate protein unfolding depending on metal removal. These data suggest that the affinity of Zn+2 for the protein is very high, evidencing its importance for the hDjC20 structure.

Alternagin-C, an alpha2beta1 integrin ligand, attenuates collagen-based adhesion, stimulating the metastasis suppressor 1 expression in triple-negative breast tumor cells.

Triple-negative breast cancer has an aggressive clinical course and its treatment has been challenging due to high metastatic risk. Molecular targets have been sought to provide better strategies for this type of cancer. Integrins are cell adhesion receptors involved in tumor progression and α2ß1 integrin, a collagen receptor, has a key role in breast metastasis. Disintegrins, a family of proteins from snake venoms, selectively block the function of integrin receptors. Alternagin-C (ALT-C), a disintegrin-like protein purified from Bothrops alternatus venom, binds to α2ß1 integrin, attenuating inflammation and angiogenesis, and decreasing metalloprotease levels in the tumor microenvironment, which suggests anti-metastatic effects. However, its mechanisms of action in metastatic tumor cells have not been fully explored. Here, we investigated ALT-C effects in a triple-negative breast cancer cell line (MDA-MB-231) to elucidate how α2ß1 integrin affects cellular adhesion, migration and gene expression related to metastasis. We observed that ALT-C attenuated cell adhesion of MDA-MB-231 cells to collagen I. α2 integrin subunit silencing in MDA-MB-231 cells did not inhibit cell adhesion and migration to collagen I, indicating that other integrins play a crucial role in cell motility for this cell line. ALT-C also stimulated the metastasis suppressor 1 (MTSS1) expression and decreased metalloproteases MMP9 and MMP2. Therefore, we suggest that ALT-C contributes to impair metastasis, preventing extracellular matrix degradation and tumor attachment to collagen I, increasing MTSS1. This study is the first to elucidate the anti-metastatic mechanism involving a disintegrin-like protein from snake venom targeting α2ß1 integrin and stimulating a metastasis suppressor.

New insights on human Hsp70-escort protein 1: Chaperone activity, interaction with liposomes, cellular localizations and HSPA's self-assemblies remodeling.

The 70 kDa heat shock proteins (Hsp70) are prone to self-assembly under thermal stress conditions, forming supramolecular assemblies (SMA), what may have detrimental consequences for cellular viability. In mitochondria, the cochaperone Hsp70-escort protein 1 (Hep1) maintains mitochondrial Hsp70 (mtHsp70) in a soluble and functional state, contributing to preserving proteostasis. Here we investigated the interaction between human Hep1 (hHep1) and HSPA9 (human mtHsp70) or HSPA1A (Hsp70-1A) in monomeric and thermic SMA states to unveil further information about the involved mechanisms. hHep1 was capable of blocking the formation of HSPA SMAs under a thermic treatment and stimulated HSPA ATPase activity in both monomeric and preformed SMA. The interaction of hHep1 with both monomeric and SMA HSPAs displayed a stoichiometric ratio close to 1, suggesting that hHep1 has access to most protomers within the SMA. Interestingly, hHep1 remodeled HSPA9 and HSPA1A SMAs into smaller forms. Furthermore, hHep1 was detected in the mitochondria and nucleus of cells transfected with the respective coding DNA and interacted with liposomes resembling mitochondrial membranes. Altogether, these new features reinforce that hHep1 act as a "chaperone for a chaperone", which may play a critical role in cellular proteostasis.

Alternagin-C binding to α2ß1 integrin controls matrix metalloprotease-9 and matrix metalloprotease-2 in breast tumor cells and endothelial cells.

BACKGROUND: Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of α2ß1 integrin, one of the major collagen I receptors, in MMP activity and expression has not been studied. Alternagin-C (ALT-C), a glutamate-cysteine-aspartate-disintegrin from Bothrops alternatus venom, has high affinity for an α2ß1 integrin. Herein, we used ALT-C as a α2ß1 integrin ligand to study the effect of ALT-C on MMP-9 and MMP-2 expression as well as on tumor cells, fibroblats and endothelial cell migration. METHODS: ALT-C was purified by two steps of gel filtration followed by anion exchange chromatography. The α2ß1 integrin binding properties of ALT-C, its dissociation constant (Kd ) relative to this integrin and to collagen I (Col I) were determined by surface plasmon resonance. The effects of ALT-C (10, 40, 100 and 1000 nM) in migration assays were studied using three human cell lines: human fibroblasts, breast tumor cell line MDA-MB-231, and microvascular endothelial cells HMEC-1, considering cells found in the tumor microenvironment. ALT-C effects on MMP-9 and MMP-2 expression and activity were analyzed by quantitative PCR and gelatin zymography, respectively. Focal adhesion kinase activation was determined by western blotting. RESULTS: Our data demonstrate that ALT-C, after binding to α2ß1 integrin, acts by two distinct mechanisms against tumor progression, depending on the cell type: in tumor cells, ALT-C decreases MMP-9 and MMP-2 contents and activity, but increases focal adhesion kinase phosphorylation and transmigration; and in endothelial cells, ALT-C inhibits MMP-2, which is necessary for tumor angiogenesis. ALT-C also upregulates c-Myc mRNA level, which is related to tumor suppression. CONCLUSION: These results demonstrate that α2ß1 integrin controls MMP expression and reveal this integrin as a target for the development of antiangiogenic and antimetastatic therapies.

Alternagin-C binding to α2ß1 integrin controls matrix metalloprotease-9 and matrix metalloprotease-2 in breast tumor cells and endothelial cells

Background: Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of α2ß1 integrin, one of the major collagen I receptors, in MMP activity and expression has not been studied. Alternagin-C (ALT-C), a glutamate-cysteine-aspartate-disintegrin from Bothrops alternatus venom, has high affinity for an α2ß1 integrin. Herein, we used ALT-C as a α2ß1 integrin ligand to study the effect of ALT-C on MMP-9 and MMP-2 expression as well as on tumor cells, fibroblats and endothelial cell migration. Methods: ALT-C was purified by two steps of gel filtration followed by anion exchange chromatography. The α2ß1, integrin binding properties of ALT-C, its dissociation constant (Kd) relative to this integrin and to collagen I (Col I) were determined by surface plasmon resonance. The effects of ALT-C (10, 40, 100 and 1000 nM) in migration assays were studied using three human cell lines: human fibroblasts, breast tumor cell line MDA-MB-231, and microvascular endothelial cells HMEC-1, considering cells found in the tumor microenvironment. ALT-C effects on MMP-9 and MMP-2 expression and activity were analyzed by quantitative PCR and gelatin zymography, respectively. Focal adhesion kinase activation was determined by western blotting. Results: Our data demonstrate that ALT-C, after binding to α2ß1 integrin, acts by two distinct mechanisms against tumor progression, depending on the cell type: in tumor cells, ALT-C decreases MMP-9 and MMP-2 contents and activity, but increases focal adhesion kinase phosphorylation and transmigration; and in endothelial cells, ALT-C inhibits MMP-2, which is necessary for tumor angiogenesis. ALT-C also upregulates c-Myc mRNA level, which is related to tumor suppression. Conclusion: These results demonstrate that α2ß1 integrin controls MMP expression and reveal this integrin as a target for the development of antiangiogenic and antimetastatic therapies.(AU)

Alternagin-C binding to 21 integrin controls matrix metalloprotease-9 and matrix metalloprotease-2 in breast tumor cells and endothelial cells

Background Matrix metalloproteinases (MMPs) are key players in tumor progression, helping tumor cells to modify their microenvironment, which allows cell migration to secondary sites. The role of integrins, adhesion receptors that connect cells to the extracellular matrix, in MMP expression and activity has been previously suggested. However, the mechanisms by which integrins control MMP expression are not completely understood. Particularly, the role of 21 integrin, one of the major collagen I receptors, in MMP activity and expression has not been studied. Alternagin-C (ALT-C), a glutamate-cysteine-aspartate-disintegrin from Bothrops alternatus venom, has high affinity for an 21 integrin. Herein, we used ALT-C as a 21 integrin ligand to study the effect of ALT-C on MMP-9 and MMP-2 expression as well as on tumor cells, fibroblats and endothelial cell migration. Methods ALT-C was purified by two steps of gel filtration followed by anion exchange chromatography. The 21, integrin binding properties of ALT-C, its dissociation constant (Kd) relative to this integrin and to collagen I (Col I) were determined by surface plasmon resonance. The effects of ALT-C (10, 40, 100 and 1000 nM) in migration assays were studied using three human cell lines: human fibroblasts, breast tumor cell line MDA-MB-231, and microvascular endothelial cells HMEC-1, considering cells found in the tumor microenvironment. ALT-C effects on MMP-9 and MMP-2 expression and activity were analyzed by quantitative PCR and gelatin zymography, respectively. Focal adhesion kinase activation was determined by western blotting. Results Our data demonstrate that ALT-C, after binding to 21 integrin...

ADAM9 silencing inhibits breast tumor cells transmigration through blood and lymphatic endothelial cells.

ADAMs are transmembrane multifunctional proteins that contain disintegrin and metalloprotease domains. ADAMs act in a diverse set of biological processes, including fertilization, inflammatory responses, myogenesis, cell migration, cell proliferation and ectodomain cleavage of membrane proteins. These proteins also have additional functions in pathological processes as cancer and metastasis development. ADAM9 is a member of ADAM protein family that is overexpressed in several types of human carcinomas. The aim of this study was to investigate the role of ADAM9 in hematogenous and lymphatic tumor cell dissemination assisting the development of new therapeutic tools. The role of ADAM9 in the interaction of breast tumor cells (MDA-MB-231) and endothelial cells was studied through RNA silencing. ADAM9 silencing in MDA-MB-231 cells had no influence in expression of several genes related to the metastatic process such as ADAM10, ADAM12, ADAM17, cMYC, MMP9, VEGF-A, VEGF-C, osteopontin and collagen XVII. However, there was a minor decrease in ADAM15 expression but an increase in that of MMP2. Moreover, ADAM9 silencing had no effect in the adhesion of MDA-MB-231 cells to vascular (HMEC-1 and HUVEC) and lymphatic cells (HMVEC-dLyNeo) under flow condition. Nevertheless, siADAM9 in MDA-MB-231 decreased transendothelial cell migration in vitro through HUVEC, HMEC-1 and HMVEC-dLyNeo (50%, 40% and 32% respectively). These results suggest a role for ADAM9 on the extravasation step of the metastatic cascade through both blood and lymph vessels.