ABSTRACT
The adaptability of proteins to their work environments is fundamental for cellular life. Here, we describe how the hemopexin-like domain of the multifunctional blood glycoprotein vitronectin binds Ca2+ to adapt to excursions of temperature and shear stress. Using X-ray crystallography, molecular dynamics simulations, NMR, and differential scanning fluorimetry, we describe how Ca2+ and its flexible hydration shell enable the protein to perform conformational changes that relay beyond the calcium-binding site and alter the number of polar contacts to enhance conformational stability. By means of mutagenesis, we identify key residues that cooperate with Ca2+ to promote protein stability, and we show that calcium association confers protection against shear stress, a property that is advantageous for proteins that circulate in the vasculature, like vitronectin.
Subject(s)
Calcium , Vitronectin , Calcium/metabolism , Vitronectin/chemistry , Vitronectin/metabolism , Protein Binding , Hemopexin/metabolism , Binding Sites , Crystallography, X-Ray , Protein ConformationABSTRACT
Iron is essential for growth of Mycobacterium tuberculosis, the causative agent of tuberculosis. To acquire iron from the host, M. tuberculosis uses the siderophores called mycobactins and carboxymycobactins. Here, we show that the rv0455c gene is essential for M. tuberculosis to grow in low-iron medium and that secretion of both mycobactins and carboxymycobactins is drastically reduced in the rv0455c deletion mutant. Both water-soluble and membrane-anchored Rv0455c are functional in siderophore secretion, supporting an intracellular role. Lack of Rv0455c results in siderophore toxicity, a phenotype observed for other siderophore secretion mutants, and severely impairs replication of M. tuberculosis in mice, demonstrating the importance of Rv0455c and siderophore secretion during disease. The crystal structure of a Rv0455c homolog reveals a novel protein fold consisting of a helical bundle with a 'cinch' formed by an essential intramolecular disulfide bond. These findings advance our understanding of the distinct M. tuberculosis siderophore secretion system.
Subject(s)
Mycobacterium tuberculosis , Tuberculosis , Animals , Iron/metabolism , Mice , Mycobacterium tuberculosis/metabolism , Siderophores/metabolism , Tuberculosis/microbiology , VirulenceABSTRACT
Plekha7 (Pleckstrin homology [PH] domain containing, family A member 7) regulates the assembly of proteins of the cytoplasmic apical zonula adherens junction (AJ), thus ensuring cell-cell adhesion and tight-junction barrier integrity. Little is known of Plekha7 function in cancer. In colorectal cancer (CRC) Plekha7 expression is elevated compared to adjacent normal tissue levels, increasing with clinical stage. Plekha7 was present at plasma membrane AJ with wild-type KRas (wt-KRas) but was dispersed in cells expressing mutant KRas (mut-KRas). Fluorescence lifetime imaging microscopy (FLIM) indicated a direct Plekha7 interaction with wt-KRas but scantily with mut-KRas. Inhibiting Plekha7 specifically decreased mut-KRas cell signaling, proliferation, attachment, migration, and retarded mut-KRAS CRC tumor growth. Binding of diC8-phosphoinositides (PI) to the PH domain of Plekha7 was relatively low affinity. This may be because a D175 amino acid residue plays a "sentry" role preventing PI(3,4)P2 and PI(3,4,5)P3 binding. Molecular or pharmacological inhibition of the Plekha7 PH domain prevented the growth of mut-KRas but not wt-KRas cells. Taken together the studies suggest that Plekha7, in addition to maintaining AJ structure plays a role in mut-KRas signaling and phenotype through interaction of its PH domain with membrane mut-KRas, but not wt-KRas, to increase the efficiency of mut-KRas downstream signaling.
Subject(s)
Biomarkers, Tumor/metabolism , Carrier Proteins/metabolism , Colorectal Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Mutation , Proto-Oncogene Proteins p21(ras)/genetics , Apoptosis , Biomarkers, Tumor/genetics , Carrier Proteins/genetics , Cell Adhesion , Cell Proliferation , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Humans , Intercellular Junctions , Signal Transduction , Tight Junctions , Tumor Cells, CulturedABSTRACT
PLEKHA7 (pleckstrin homology domain containing family A member 7) plays key roles in intracellular signaling, cytoskeletal organization, and cell adhesion, and is associated with multiple human cancers. The interactions of its pleckstrin homology (PH) domain with membrane phosphatidyl-inositol-phosphate (PIP) lipids are critical for proper cellular localization and function, but little is known about how PLEKHA7 and other PH domains interact with membrane-embedded PIPs. Here we describe the structural basis for recognition of membrane-bound PIPs by PLEHA7. Using X-ray crystallography, nuclear magnetic resonance, molecular dynamics simulations, and isothermal titration calorimetry, we show that the interaction of PLEKHA7 with PIPs is multivalent, distinct from a discrete one-to-one interaction, and induces PIP clustering. Our findings reveal a central role of the membrane assembly in mediating protein-PIP association and provide a roadmap for understanding how the PH domain contributes to the signaling, adhesion, and nanoclustering functions of PLEKHA7.
Subject(s)
Carrier Proteins/chemistry , Binding Sites , Carrier Proteins/metabolism , Cell Membrane/chemistry , Cell Membrane/metabolism , Humans , Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Phosphatidylinositols/chemistry , Phosphatidylinositols/metabolism , Protein BindingABSTRACT
Retinoid X receptor-alpha (RXRα) binds to DNA either as homodimers or heterodimers, but it also forms homotetramers whose function is poorly defined. We previously discovered that an N-terminally-cleaved form of RXRα (tRXRα), produced in tumour cells, activates phosphoinositide 3-kinase (PI3K) signalling by binding to the p85α subunit of PI3K and that K-80003, an anti-cancer agent, inhibits this process. Here, we report through crystallographic and biochemical studies that K-80003 binds to and stabilizes tRXRα tetramers via a 'three-pronged' combination of canonical and non-canonical mechanisms. K-80003 binding has no effect on tetramerization of RXRα, owing to the head-tail interaction that is absent in tRXRα. We also identify an LxxLL motif in p85α, which binds to the coactivator-binding groove on tRXRα and dissociates from tRXRα upon tRXRα tetramerization. These results identify conformational selection as the mechanism for inhibiting the nongenomic action of tRXRα and provide molecular insights into the development of RXRα cancer therapeutics.
Subject(s)
Antineoplastic Agents/pharmacology , Phosphatidylinositol 3-Kinases/drug effects , Protein Multimerization/drug effects , Retinoid X Receptor alpha/antagonists & inhibitors , Sulindac/analogs & derivatives , A549 Cells , Animals , Class Ia Phosphatidylinositol 3-Kinase , Crystallography, X-Ray , HEK293 Cells , Hep G2 Cells , Humans , MCF-7 Cells , Mice , Mice, Nude , Neoplasm Transplantation , Phosphatidylinositol 3-Kinases/metabolism , Retinoid X Receptor alpha/metabolism , Signal Transduction , Sulindac/pharmacologyABSTRACT
Obesity-associated insulin resistance plays a central role in type 2 diabetes. As such, tyrosine phosphatases that dephosphorylate the insulin receptor (IR) are potential therapeutic targets. The low-molecular-weight protein tyrosine phosphatase (LMPTP) is a proposed IR phosphatase, yet its role in insulin signaling in vivo has not been defined. Here we show that global and liver-specific LMPTP deletion protects mice from high-fat diet-induced diabetes without affecting body weight. To examine the role of the catalytic activity of LMPTP, we developed a small-molecule inhibitor with a novel uncompetitive mechanism, a unique binding site at the opening of the catalytic pocket, and an exquisite selectivity over other phosphatases. This inhibitor is orally bioavailable, and it increases liver IR phosphorylation in vivo and reverses high-fat diet-induced diabetes. Our findings suggest that LMPTP is a key promoter of insulin resistance and that LMPTP inhibitors would be beneficial for treating type 2 diabetes.
Subject(s)
Diabetes Mellitus, Type 2/drug therapy , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Protein Tyrosine Phosphatases/antagonists & inhibitors , Protein Tyrosine Phosphatases/genetics , Small Molecule Libraries , Animals , Binding Sites , Diabetes Mellitus, Type 2/enzymology , Diabetes Mellitus, Type 2/genetics , Drug Evaluation, Preclinical , Enzyme Activation/drug effects , Gene Deletion , Inhibitory Concentration 50 , Mice , Mice, Knockout , Mice, Obese , Models, Biological , Molecular Structure , Molecular Weight , Small Molecule Libraries/pharmacology , Structure-Activity RelationshipABSTRACT
The hypoxia-inducible transcription factor HIF1α drives expression of many glycolytic enzymes. Here, we show that hypoxic glycolysis, in turn, increases HIF1α transcriptional activity and stimulates tumor growth, revealing a novel feed-forward mechanism of glycolysis-HIF1α signaling. Negative regulation of HIF1α by AMPK1 is bypassed in hypoxic cells, due to ATP elevation by increased glycolysis, thereby preventing phosphorylation and inactivation of the HIF1α transcriptional coactivator p300. Notably, of the HIF1α-activated glycolytic enzymes we evaluated by gene silencing, aldolase A (ALDOA) blockade produced the most robust decrease in glycolysis, HIF-1 activity, and cancer cell proliferation. Furthermore, either RNAi-mediated silencing of ALDOA or systemic treatment with a specific small-molecule inhibitor of aldolase A was sufficient to increase overall survival in a xenograft model of metastatic breast cancer. In establishing a novel glycolysis-HIF-1α feed-forward mechanism in hypoxic tumor cells, our results also provide a preclinical rationale to develop aldolase A inhibitors as a generalized strategy to treat intractable hypoxic cancer cells found widely in most solid tumors. Cancer Res; 76(14); 4259-69. ©2016 AACR.
Subject(s)
Fructose-Bisphosphate Aldolase/antagonists & inhibitors , Glycolysis , Hypoxia-Inducible Factor 1, alpha Subunit/physiology , Neoplasms/drug therapy , Signal Transduction/physiology , AMP-Activated Protein Kinases/physiology , Animals , Cell Hypoxia , Cell Line, Tumor , E1A-Associated p300 Protein/physiology , Humans , Mice , Neoplasms/metabolism , Xenograft Model Antitumor AssaysABSTRACT
A high throughput screen for compounds that induce TRAIL-mediated apoptosis identified ML100 as an active chemical probe, which potentiated TRAIL activity in prostate carcinoma PPC-1 and melanoma MDA-MB-435 cells. Follow-up in silico modeling and profiling in cell-based assays allowed us to identify NSC130362, pharmacophore analog of ML100 that induced 65-95% cytotoxicity in cancer cells and did not affect the viability of human primary hepatocytes. In agreement with the activation of the apoptotic pathway, both ML100 and NSC130362 synergistically with TRAIL induced caspase-3/7 activity in MDA-MB-435 cells. Subsequent affinity chromatography and inhibition studies convincingly demonstrated that glutathione reductase (GSR), a key component of the oxidative stress response, is a target of NSC130362. In accordance with the role of GSR in the TRAIL pathway, GSR gene silencing potentiated TRAIL activity in MDA-MB-435 cells but not in human hepatocytes. Inhibition of GSR activity resulted in the induction of oxidative stress, as was evidenced by an increase in intracellular reactive oxygen species (ROS) and peroxidation of mitochondrial membrane after NSC130362 treatment in MDA-MB-435 cells but not in human hepatocytes. The antioxidant reduced glutathione (GSH) fully protected MDA-MB-435 cells from cell lysis induced by NSC130362 and TRAIL, thereby further confirming the interplay between GSR and TRAIL. As a consequence of activation of oxidative stress, combined treatment of different oxidative stress inducers and NSC130362 promoted cell death in a variety of cancer cells but not in hepatocytes in cell-based assays and in in vivo, in a mouse tumor xenograft model.
Subject(s)
Apoptosis/drug effects , Glutathione Reductase/metabolism , High-Throughput Screening Assays , Oxidative Stress , TNF-Related Apoptosis-Inducing Ligand/metabolism , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Animals , Antineoplastic Agents/pharmacology , Cell Line, Tumor , Dose-Response Relationship, Drug , Doxorubicin/pharmacology , Drug Discovery , Glutathione/metabolism , Glutathione Reductase/antagonists & inhibitors , Humans , Mice , Reactive Oxygen Species , Small Molecule LibrariesABSTRACT
Nicotinate mononucleotide adenylyltransferase NadD is an essential enzyme in the biosynthesis of the NAD cofactor, which has been implicated as a target for developing new antimycobacterial therapies. Here we report the crystal structure of Mycobacterium tuberculosis NadD (MtNadD) at a resolution of 2.4 Å. A remarkable new feature of the MtNadD structure, compared with other members of this enzyme family, is a 310 helix that locks the active site in an over-closed conformation. As a result, MtNadD is rendered inactive as it is topologically incompatible with substrate binding and catalysis. Directed mutagenesis was also used to further dissect the structural elements that contribute to the interactions of the two MtNadD substrates, i.e. ATP and nicotinic acid mononucleotide (NaMN). For inhibitory profiling of partially active mutants and wild type MtNadD, we used a small molecule inhibitor of MtNadD with moderate affinity (Ki â¼ 25 µM) and antimycobacterial activity (MIC80) â¼ 40-80 µM). This analysis revealed interferences with some of the residues in the NaMN binding subsite consistent with the competitive inhibition observed for the NaMN substrate (but not ATP). A detailed steady-state kinetic analysis of MtNadD suggests that ATP must first bind to allow efficient NaMN binding and catalysis. This sequential mechanism is consistent with the requirement of transition to catalytically competent (open) conformation hypothesized from structural modeling. A possible physiological significance of this mechanism is to enable the down-regulation of NAD synthesis under ATP-limiting dormancy conditions. These findings point to a possible new strategy for designing inhibitors that lock the enzyme in the inactive over-closed conformation.
Subject(s)
Antitubercular Agents/pharmacology , Drug Discovery , Enzyme Inhibitors/pharmacology , Mycobacterium tuberculosis/enzymology , Nicotinamide-Nucleotide Adenylyltransferase/metabolism , Amino Acid Sequence , Antitubercular Agents/chemistry , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Kinetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mycobacterium tuberculosis/drug effects , Nicotinamide-Nucleotide Adenylyltransferase/antagonists & inhibitors , Nicotinamide-Nucleotide Adenylyltransferase/chemistry , Protein Conformation , Sequence Homology, Amino Acid , Structure-Activity RelationshipABSTRACT
Retinoid X receptor-alpha (RXRα), an intriguing and unique drug target, can serve as an intracellular target mediating the anticancer effects of certain nonsteroidal anti-inflammatory drugs (NSAIDs), including sulindac. We report the synthesis and characterization of two sulindac analogs, K-8008 and K-8012, which exert improved anticancer activities over sulindac in a RXRα-dependent manner. The analogs inhibit the interaction of the N-terminally truncated RXRα (tRXRα) with the p85α subunit of PI3K, leading to suppression of AKT activation and induction of apoptosis. Crystal structures of the RXRα ligand-binding domain (LBD) with K-8008 or K-8012 reveal that both compounds bind to tetrameric RXRα LBD at a site different from the classical ligand-binding pocket. Thus, these results identify K-8008 and K-8012 as tRXRα modulators and define a binding mechanism for regulating the nongenomic action of tRXRα.
Subject(s)
Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Neoplasms/drug therapy , Retinoid X Receptor alpha/antagonists & inhibitors , Retinoid X Receptor alpha/chemistry , Sulindac/analogs & derivatives , Sulindac/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Binding Sites/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , HeLa Cells , Hep G2 Cells , Humans , Mice , Mice, Inbred BALB C , Mice, Nude , Models, Molecular , Molecular Structure , Neoplasms/metabolism , Neoplasms/pathology , Retinoid X Receptor alpha/metabolism , Structure-Activity Relationship , Sulindac/chemistry , Tumor Cells, CulturedABSTRACT
Bacteroides fragilis causes the majority of anaerobic infections in humans. The presence of a pathogenicity island in the genome discriminates pathogenic and commensal B. fragilis strains. The island encodes metalloproteinase II (MPII), a potential virulence protein, and one of three homologous fragilysin isozymes (FRA; also termed B. fragilis toxin or BFT). Here, we report biochemical data on the structural-functional characteristics of the B. fragilis pathogenicity island proteases by reporting the crystal structure of MPII at 2.13 Å resolution, combined with detailed characterization of the cleavage preferences of MPII and FRA3 (as a representative of the FRA isoforms), identified using a high-throughput peptide cleavage assay with 18 583 substrate peptides. We suggest that the evolution of the MPII catalytic domain can be traced to human and archaebacterial proteinases, whereas the prodomain fold is a feature specific to MPII and FRA. We conclude that the catalytic domain of both MPII and FRA3 evolved differently relative to the prodomain, and that the prodomain evolved specifically to fit the B. fragilis pathogenicity. Overall, our data provide insights into the evolution of cleavage specificity and activation mechanisms in the virulent metalloproteinases.
Subject(s)
Bacteroides fragilis/enzymology , Genomic Islands/genetics , Metalloproteases/genetics , Bacteroides fragilis/genetics , Metalloproteases/chemistryABSTRACT
Glucose transporters are required to bring glucose into cells, where it is an essential energy source and precursor in protein and lipid synthesis. These transporters are involved in important common diseases such as cancer and diabetes. Here, we report the crystal structure of the Staphylococcus epidermidis glucose/H(+) symporter in an inward-facing conformation at 3.2-Å resolution. The Staphylococcus epidermidis glucose/H(+) symporter is homologous to human glucose transporters, is very specific and has high avidity for glucose, and is inhibited by the human glucose transport inhibitors cytochalasin B, phloretin, and forskolin. On the basis of the crystal structure in conjunction with mutagenesis and functional studies, we propose a mechanism for glucose/H(+) symport and discuss the symport mechanism versus facilitated diffusion.
Subject(s)
Glucose Transport Proteins, Facilitative/antagonists & inhibitors , Glucose Transport Proteins, Facilitative/chemistry , Models, Molecular , Protein Conformation , Staphylococcus epidermidis/chemistry , Biological Transport/physiology , Cloning, Molecular , Colforsin/pharmacology , Crystallization , Cytochalasin B/pharmacology , Escherichia coli , Glucose Transport Proteins, Facilitative/genetics , Humans , Mutagenesis , Mutagenesis, Site-Directed , Phloretin/pharmacology , Sequence Homology , Staphylococcus epidermidis/geneticsABSTRACT
The K7L gene product of the smallpox virus is a protease implicated in the maturation of viral proteins. K7L belongs to protease Clan CE, which includes distantly related cysteine proteases from eukaryotes, pathogenic bacteria, and viruses. Here, we describe its recombinant high level expression, biochemical mechanism, substrate preference, and regulation. Earlier studies inferred that the orthologous I7L vaccinia protease cleaves at an AG-X motif in six viral proteins. Our data for K7L suggest that the AG-X motif is necessary but not sufficient for optimal cleavage activity. Thus, K7L requires peptides extended into the P7 and P8 positions for efficient substrate cleavage. Catalytic activity of K7L is substantially enhanced by homodimerization, by the substrate protein P25K as well as by glycerol. RNA and DNA also enhance cleavage of the P25K protein but not of synthetic peptides, suggesting that nucleic acids augment the interaction of K7L with its protein substrate. Library-based peptide preference analyses enabled us to design an activity-based probe that covalently and selectively labels K7L in lysates of transfected and infected cells. Our study thus provides proof-of-concept for the design of inhibitors and probes that may contribute both to a better understanding of the role of K7L in the virus life cycle and the design of novel anti-virals.
Subject(s)
Antiviral Agents/chemistry , Molecular Probes/chemistry , Peptide Hydrolases/chemistry , Peptide Library , Protease Inhibitors/chemistry , Variola virus/enzymology , Viral Proteins/antagonists & inhibitors , Amino Acid Motifs , Animals , Cell Line , Cricetinae , Drug Design , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Smallpox/drug therapy , Smallpox/enzymology , Smallpox/genetics , Variola virus/genetics , Viral Proteins/chemistry , Viral Proteins/genetics , Viral Proteins/metabolismABSTRACT
The complement membrane attack complex (MAC) forms transmembrane pores in pathogen membranes. The first step in MAC assembly is cleavage of C5 to generate metastable C5b, which forms a stable complex with C6, termed C5b-6. C5b-6 initiates pore formation via the sequential recruitment of homologous proteins: C7, C8, and 12-18 copies of C9, each of which comprises a central MAC-perforin domain flanked by auxiliary domains. We recently proposed a model of pore assembly, in which the auxiliary domains play key roles, both in stabilizing the closed conformation of the protomers and in driving the sequential opening of the MAC-perforin ß-sheet of each new recruit to the growing pore. Here, we describe an atomic model of C5b-6 at 4.2 Å resolution. We show that C5b provides four interfaces for the auxiliary domains of C6. The largest interface is created by the insertion of an interdomain linker from C6 into a hydrophobic groove created by a major reorganization of the α-helical domain of C5b. In combination with the rigid body docking of N-terminal elements of both proteins, C5b becomes locked into a stable conformation. Both C6 auxiliary domains flanking the linker pack tightly against C5b. The net effect is to induce the clockwise rigid body rotation of four auxiliary domains, as well as the opening/twisting of the central ß-sheet of C6, in the directions predicted by our model to activate or prime C6 for the subsequent steps in MAC assembly. The complex also suggests novel small molecule strategies for modulating pathological MAC assembly.
Subject(s)
Complement Membrane Attack Complex/chemistry , Complement System Proteins/chemistry , Complement Membrane Attack Complex/metabolism , Complement System Proteins/metabolism , Crystallography, X-Ray , Humans , Hydrophobic and Hydrophilic Interactions , Protein Structure, Quaternary , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
The complement membrane attack complex (MAC) is formed by the sequential assembly of C5b with four homologous proteins as follows: one copy each of C6, C7, and C8 and 12-14 copies of C9. Together these form a lytic pore in bacterial membranes. C6 through C9 comprise a MAC-perforin domain flanked by 4-9 "auxiliary" domains. Here, we report the crystal structure of C6, the first and longest of the pore proteins to be recruited by C5b. Comparisons with the structures of the C8αßγ heterodimer and perforin show that the central domain of C6 adopts a "closed" (perforin-like) state that is distinct from the "open" conformations in C8. We further show that C6, C8α, and C8ß contain three homologous subdomains ("upper," "lower," and "regulatory") related by rotations about two hinge points. In C6, the regulatory segment includes four auxiliary domains that stabilize the closed conformation, inhibiting release of membrane-inserting elements. In C8ß, rotation of the regulatory segment is linked to an opening of the central ß-sheet of its clockwise partner, C8α. Based on these observations, we propose a model for initiation and unidirectional propagation of the MAC in which the auxiliary domains play key roles: in the assembly of the C5b-8 initiation complex; in driving and regulating the opening of the ß-sheet of the MAC-performin domain of each new recruit as it adds to the growing pore; and in stabilizing the final pore. Our model of the assembled pore resembles those of the cholesterol-dependent cytolysins but is distinct from that recently proposed for perforin.
Subject(s)
Complement C6/chemistry , Complement Membrane Attack Complex , Models, Biological , Models, Molecular , Complement C6/metabolism , Complement System Proteins/chemistry , Complement System Proteins/metabolism , Crystallography, X-Ray , Humans , Protein Structure, Secondary , Protein Structure, Tertiary , Structure-Activity RelationshipABSTRACT
Mycobacterium tuberculosis is the causative agent of human tuberculosis (TB). Mycobacterial secretory protein ESAT-6 induces matrix metalloproteinase (MMP)-9 in epithelial cells neighboring infected macrophages. MMP-9 then enhances recruitment of uninfected macrophages, which contribute to nascent granuloma maturation and bacterial growth. Disruption of MMP-9 function attenuates granuloma formation and bacterial growth. The abundant mycobacterial 65 kDa heat shock protein (HSP65) chaperone is the major target for the immune response and a critical component in M. tuberculosis adhesion to macrophages. We hypothesized that HSP65 is susceptible to MMP-9 proteolysis and that the resulting HSP65 immunogenic peptides affect host adaptive immunity. To identify MMPs that cleave HSP65, we used MMP-2 and MMP-9 gelatinases, the simple hemopexin domain MMP-8, membrane-associated MMP-14, MMP-15, MMP-16 and MMP-24, and glycosylphosphatidylinositol-linked MMP-17 and MMP-25. We determined both the relative cleavage efficiency of MMPs against the HSP65 substrate and the peptide sequence of the cleavage sites. Cleavage of the unstructured PAGHG474L C-terminal region initiates the degradation of HSP65 by MMPs. This initial cleavage destroys the substrate-binding capacity of the HSP65 chaperone. Multiple additional cleavages of the unfolded HSP65 then follow. MMP-2, MMP-8, MMP-14, MMP-15 and MMP-16, in addition to MMP-9, generate the known highly immunogenic N-terminal peptide of HSP65. Based on our biochemical data, we now suspect that MMP proteolysis of HSP65 in vivo, including MMP-9 proteolysis, also results in the abundant generation of the N-terminal immunogenic peptide and that this peptide, in addition to intact HSP65, contributes to the complex immunomodulatory interplay in the course of TB infection.
Subject(s)
Antigens, Bacterial/metabolism , Bacterial Proteins/metabolism , Chaperonin 60/metabolism , Matrix Metalloproteinases/metabolism , Mycobacterium tuberculosis/immunology , Peptide Fragments/metabolism , Tuberculosis/immunology , Adaptive Immunity , Amino Acid Sequence , Antigens, Bacterial/chemistry , Bacterial Proteins/chemistry , Catalytic Domain , Chaperonin 60/chemistry , Epitopes/chemistry , Epitopes/metabolism , Humans , Hydrolysis/drug effects , Immunomodulation , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Matrix Metalloproteinase Inhibitors , Matrix Metalloproteinases/genetics , Molecular Sequence Data , Peptide Fragments/antagonists & inhibitors , Peptide Fragments/chemistry , Peptide Fragments/genetics , Protease Inhibitors/pharmacology , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Substrate Specificity , Tuberculosis/prevention & controlABSTRACT
Membrane PTK7 pseudo-kinase plays an essential role in planar cell polarity and the non-canonical Wnt pathway in vertebrates. Recently, a new N-ethyl-N-nitrosourea-induced mutant named chuzhoi (chz) was isolated in mice. chz embryos have severe birth defects, including a defective neural tube, defective heart and lung development, and a shortened anterior-posterior body axis. The chz mutation was mapped to the Ala-Asn-Pro tripeptide insertion into the junction region between the fifth and the sixth Ig-like domains of PTK7. Unexpectedly, chz reduced membrane localization of the PTK7 protein. We hypothesized and then proved that the chz mutation caused an insertion of an additional membrane type 1 matrix metalloproteinase cleavage site in PTK7 and that the resulting aberrant proteolysis of chz affected the migratory parameters of the cells. It is likely that aberrations in the membrane type 1 matrix metalloproteinase/PTK7 axis are detrimental to cell movements that shape the body plan and that chz represents a novel model system for increasing our understanding of the role of proteolysis in developmental pathologies, including congenital defects.
Subject(s)
Abnormalities, Drug-Induced/enzymology , Cell Adhesion Molecules/metabolism , Matrix Metalloproteinase 14/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Abnormalities, Drug-Induced/genetics , Alkylating Agents/adverse effects , Alkylating Agents/pharmacology , Animals , Cell Adhesion Molecules/genetics , Cell Line, Tumor , Cell Movement/drug effects , Cell Movement/genetics , Cell Shape/drug effects , Cell Shape/genetics , Ethylnitrosourea/adverse effects , Ethylnitrosourea/pharmacology , Humans , Matrix Metalloproteinase 14/genetics , Mice , Mutation , Protein Structure, Tertiary , Receptor Protein-Tyrosine Kinases/geneticsABSTRACT
In flaviviruses and hepatitis C virus (HCV), the NS3 gene encodes the N-terminal protease (NS3pro) and the C-terminal helicase (NS3hel). In HCV, the downstream NS4A is required for the NS3pro activity and exhibits a conserved EFDEMEE motif. To identify the role of this motif, we compared the ATPase and helicase activities of NS3 alone with those of the NS3-NS4A constructs. Our results suggest that the EFDEMEE motif is essential for regulating the ATPase activity of NS3hel. It is likely that this motif interferes with the ATP-binding site of NS3hel. It is becoming clear that NS4A functions as a cofactor of both proteinase and helicase in HCV.
Subject(s)
Carrier Proteins/genetics , Carrier Proteins/metabolism , Hepacivirus/genetics , Hepacivirus/metabolism , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Motifs , Amino Acid Sequence , Amino Acid Substitution , Base Sequence , Binding Sites/genetics , Carrier Proteins/chemistry , DNA Primers/genetics , Genes, Viral , Humans , Hydrolysis , Intracellular Signaling Peptides and Proteins , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Conformation , RNA Helicases/chemistry , RNA Helicases/genetics , RNA Helicases/metabolism , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Viral Nonstructural Proteins/chemistryABSTRACT
PTK7 is an essential component of the Wnt/planar cell polarity (PCP) pathway. We provide evidence that the Wnt/PCP pathway converges with pericellular proteolysis in both normal development and cancer. Here, we demonstrate that membrane type-1 matrix metalloproteinase (MT1-MMP), a key proinvasive proteinase, functions as a principal sheddase of PTK7. MT1-MMP directly cleaves the exposed PKP(621)↓LI sequence of the seventh Ig-like domain of the full-length membrane PTK7 and generates, as a result, an N-terminal, soluble PTK7 fragment (sPTK7). The enforced expression of membrane PTK7 in cancer cells leads to the actin cytoskeleton reorganization and the inhibition of cell invasion. MT1-MMP silencing and the analysis of the uncleavable L622D PTK7 mutant confirm the significance of MT1-MMP proteolysis of PTK7 in cell functions. Our data also demonstrate that a fine balance between the metalloproteinase activity and PTK7 levels is required for normal development of zebrafish (Danio rerio). Aberration of this balance by the proteinase inhibition or PTK7 silencing results in the PCP-dependent convergent extension defects in the zebrafish. Overall, our data suggest that the MT1-MMP-PTK7 axis plays an important role in both cancer cell invasion and normal embryogenesis in vertebrates. Further insight into these novel mechanisms may promote understanding of directional cell motility and lead to the identification of therapeutics to treat PCP-related developmental disorders and malignancy.
Subject(s)
Cell Adhesion Molecules/metabolism , Embryo, Nonmammalian/embryology , Matrix Metalloproteinase 14/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Animals , Binding Sites/genetics , Cell Adhesion Molecules/chemistry , Cell Adhesion Molecules/genetics , Cell Line , Cell Line, Tumor , Cell Movement , Cell Polarity , Cytoskeleton/metabolism , Embryo, Nonmammalian/metabolism , Gene Expression Regulation, Developmental , Gene Knockdown Techniques , Humans , In Situ Hybridization , Matrix Metalloproteinase 14/genetics , Molecular Sequence Data , Mutation , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Protein Binding , Protein Structure, Tertiary , Receptor Protein-Tyrosine Kinases/chemistry , Receptor Protein-Tyrosine Kinases/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Transfection , Wnt Proteins/metabolism , Zebrafish/embryology , Zebrafish/genetics , Zebrafish/metabolism , Zebrafish Proteins/chemistry , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
The functional activity of invasion-promoting membrane type 1 matrix metalloproteinase (MT1-MMP) is elevated in cancer. This elevated activity promotes cancer cell migration, invasion, and metastasis. MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its prodomain. Excision by furin was considered sufficient for the prodomain release and MT1-MMP activation. We determined, however, that the full-length intact prodomain released by furin alone is a potent autoinhibitor of MT1-MMP. Additional MMP cleavages within the prodomain sequence are required to release the MT1-MMP enzyme activity. Using mutagenesis of the prodomain sequence and mass spectrometry analysis of the prodomain fragments, we demonstrated that the intradomain cleavage of the PGD/L(50) site initiates the MT1-MMP activation, whereas the (108)RRKR(111)/Y(112) cleavage by furin completes the removal and the degradation of the autoinhibitory prodomain and the liberation of the functional activity of the emerging enzyme of MT1-MMP.