Biochemical evidence of the interactions of membrane type-1 matrix metalloproteinase (MT1-MMP) with adenine nucleotide translocator (ANT): potential implications linking proteolysis with energy metabolism in cancer cells.

Burnham Institute for Medical Research, La Jolla, CA 92037, USA.
Biochemical Journal (Impact Factor: 4.78). 03/2009; 420(1):37-47. DOI: 10.1042/BJ20090082
Source: PubMed

ABSTRACT Invasion-promoting MT1-MMP (membrane type-1 matrix metalloproteinase) is a key element in cell migration processes. To identify the proteins that interact and therefore co-precipitate with this proteinase from cancer cells, we used the proteolytically active WT (wild-type), the catalytically inert E240A and the C-end truncated (tailless; DeltaCT) MT1-MMP-FLAG constructs as baits. The identity of the pulled-down proteins was determined by LC-MS/MS (liquid chromatography tandem MS) and then confirmed by Western blotting using specific antibodies. We determined that, in breast carcinoma MCF cells (MCF-7 cells), ANT (adenine nucleotide translocator) efficiently interacted with the WT, E240A and DeltaCT constructs. The WT and E240A constructs also interacted with alpha-tubulin, an essential component of clathrin-mediated endocytosis. In turn, tubulin did not co-precipitate with the DeltaCT construct because of the inefficient endocytosis of the latter, thus suggesting a high level of selectivity of our test system. To corroborate these results, we then successfully used the ANT2-FLAG construct as a bait to pull-down MT1-MMP, which was naturally produced by fibrosarcoma HT1080 cells. We determined that the presence of the functionally inert catalytic domain alone was sufficient to cause the proteinase to interact with ANT2, thus indicating that there is a non-proteolytic mode of these interactions. Overall, it is tempting to hypothesize that by interacting with pro-invasive MT1-MMP, ANT plays a yet to be identified role in a coupling mechanism between energy metabolism and pericellular proteolysis in migrating cancer cells.

  • [Show abstract] [Hide abstract]
    ABSTRACT: We have identified the adenine nucleotide translocator (ANT) isoforms ANT1 and ANT2 that are present in the plasma membrane of mouse cerebellar neurons as novel binding partners of the cell adhesion molecule L1. The direct interaction between ANT and L1 is mediated by sites within the fibronectin type III domains of L1 and the first and third extracellular loops of the ANT proteins. We also show that L1 interacts with the ANT binding partner matrix metalloprotease 14 (MMP14) and that the ANT proteins bind directly to the L1 interaction partner glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Moreover, we provide evidence that the functional interplay between L1, ANT proteins, MMP14, and GAPDH at the plasma membrane mediates L1-induced neurite outgrowth of cerebellar neurons. Disruption of this interplay by ANT inhibitors, ANT-derived synthetic peptides, and/or function-blocking MMP14 and ANT antibodies leads to alterations in L1-dependent neurite outgrowth. Stimulation of L1-mediated signaling in cerebellar neurons triggers transient ATP secretion via ANT proteins and leads to transient src family-dependent tyrosine phosphorylation of L1, ANT1, ANT2, and MMP14. Thus, our results indicate that plasma membrane-localized ANT1 and ANT2 regulate L1-mediated neurite outgrowth in conjunction with MMP14.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 03/2012; 32(11):3917-30. DOI:10.1523/JNEUROSCI.6165-11.2012 · 6.75 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Connexins are the structural subunits of gap junctions and act as protein platforms for signaling complexes. Little is known about tissue-specific connexin signaling nexuses given significant challenges associated with affinity-purifying endogenous channel complexes to the level required for interaction analyses. Here, we used multiple subcellular fractionation techniques to isolate connexin32-enriched membrane microdomains from murine liver. We show, for the first time, that connexin32 localizes to the plasma membrane and inner mitochondrial membrane of hepatocytes. Using a combination of immunoprecipitation-high throughput mass spectrometry, reciprocal co-IP, and subcellular fractionation methodologies, we report a novel interactome validated using null-mutant controls. Eighteen connexin32 interacting proteins were identified. The majority represent resident mitochondrial proteins, a minority plasma membrane, endoplasmic reticulum, or cytoplasmic partners. In particular, connexin32 interacts with connexin26 and with the mitochondrial protein, sideroflexin-1 at the plasma membrane. Connexin32 interaction enhances connexin26 stability. Converging bioinformatics, biochemical, and confocal analyses support a role for connexin-32 in transiently tethering mitochondria to connexin32-enriched membrane microdomains through interaction with proteins in the outer mitochondrial membrane, including sideroflexin-1. Complex formation increases the pool of sideroflexin-1 that is present at the plasma membrane. Together, these data identify a novel plasma membrane/mitochondrial signaling nexus in the connexin32 interactome.
    Journal of Proteome Research 04/2013; 12(6). DOI:10.1021/pr301166p · 5.00 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Keywords: Matrix metalloproteinase MT1-MMP (MMP-14) Phage display peptide library Near-infrared fluorescence optical imaging Membrane type-1 matrix metalloproteinase (MT1-MMP) is a key member of the matrix metalloproteinase (MMP) family. It participates in pericellular proteolysis of extracellular matrix (ECM) macromolecules and is essential for many biological and pathological processes, such as tumor development, angiogenesis and metastasis. A ligand that specifically binds to MT1-MMP may facilitate the labeling of this molecule, allow imaging at the cellular and organism levels, and provide a means for targeted drug delivery specific to MT1-MMP. A non-substrate MT1-MMP binding peptide was identified by screening a Ph.D.-12™ phage display peptide library and conjugated with near-infrared fluorescent (NIRF) dye Cy5.5 for tumor imaging. Peptide HWKHLHNTKTFL (denoted as MT1-AF7p) showed high MT1-MMP binding affinity. Computer modeling verified that MT1-AF7p binds to the MT-loop region of MT1-MMP and interacts with MT1-MMP through hydrogen bonding and hydrophobic interactions. MDA-MB-435 xenografts with high MT1-MMP expression had significantly higher tumor accumulation and better tumor contrast than the low MT1-MMP expressing A549 xenografts after intravenous injection of Cy5.5-MT1-AF7p. Using NIRF imaging, we have demonstrated specific targeting of MT1-AF7p to MT1-MMP-expressing tumors. Thus, MT1-AF7p is an important tool for noninvasive monitoring of MT1-MMP expression in tumors, and it shows great potential as an imaging agent for MT1-MMP-positive tumors. Published by Elsevier B.V.
    Journal of Controlled Release 04/2013; 150(3):248. · 7.26 Impact Factor

Full-text (2 Sources)

Available from
May 31, 2014