RÉSUMÉ
Lysyl hydroxylase 2 (LH2) catalyzes the formation of highly stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs), thus promoting lung cancer metastasis through its capacity to modulate specific types of collagen cross-links within the tumor stroma. Using 1 and 2 from our previous high-throughput screening (HTS) as lead probes, we prepared a series of 1,3-diketone analogues, 1-18, and identified 12 and 13 that inhibit LH2 with IC50's of approximately 300 and 500 nM, respectively. Compounds 12 and 13 demonstrate selectivity for LH2 over LH1 and LH3. Quantum mechanics/molecular mechanics (QM/MM) modeling indicates that the selectivity of 12 and 13 may stem from noncovalent interactions like hydrogen bonding between the morpholine/piperazine rings with the LH2-specific Arg661. Treatment of 344SQ WT cells with 13 resulted in a dose-dependent reduction in their migration potential, whereas the compound did not impede the migration of the same cell line with an LH2 knockout (LH2KO).
RÉSUMÉ
The catalytic function of lysyl hydroxylase-2 (LH2), a member of the Fe(II)/αKG-dependent oxygenase superfamily, is to catalyze the hydroxylation of lysine to hydroxylysine in collagen, resulting in stable hydroxylysine aldehyde-derived collagen cross-links (HLCCs). Reports show that high amounts of LH2 lead to the accumulation of HLCCs, causing fibrosis and specific types of cancer metastasis. Some members of the Fe(II)/αKG-dependent family have also been reported to have intramolecular O2 tunnels, which aid in transporting one of the required cosubstrates into the active site. While LH2 can be a promising target to combat these diseases, efficacious inhibitors are still lacking. We have used computational simulations to investigate a series of 44 small molecules as lead compounds for LH2 inhibition. Tunneling analyses indicate the existence of several intramolecular tunnels. The lengths of the calculated O2-transporting tunnels in holoenzymes are relatively longer than those in the apoenzyme, suggesting that the ligands may affect the enzyme's structure and possibly block (at least partially) the tunnels. The sequence alignment analysis between LH enzymes from different organisms shows that all of the amino acid residues with the highest occurrence rate in the oxygen tunnels are conserved. Our results suggest that the enolate form of diketone compounds establishes stronger interactions with the Fe(II) in the active site. Branching the enolate compounds with functional groups such as phenyl and pyridinyl enhances the interaction with various residues around the active site. Our results provide information about possible leads for further LH2 inhibition design and development.
Sujet(s)
Hydroxylysine , Procollagen-lysine, 2-oxoglutarate 5-dioxygenase , Collagène/composition chimique , Collagène/métabolisme , Composés du fer II , Lysine/métabolisme , Procollagen-lysine, 2-oxoglutarate 5-dioxygenase/antagonistes et inhibiteurs , Procollagen-lysine, 2-oxoglutarate 5-dioxygenase/composition chimiqueRÉSUMÉ
Lysyl hydroxylase 2 (LH2) is a member of LH family that catalyzes the hydroxylation of lysine (Lys) residues on collagen, and this particular isozyme has been implicated in various diseases. While its function as a telopeptidyl LH is generally accepted, several fundamental questions remain unanswered: 1. Does LH2 catalyze the hydroxylation of all telopeptidyl Lys residues of collagen? 2. Is LH2 involved in the helical Lys hydroxylation? 3. What are the functional consequences when LH2 is completely absent? To answer these questions, we generated LH2-null MC3T3 cells (LH2KO), and extensively characterized the type I collagen phenotypes in comparison with controls. Cross-link analysis demonstrated that the hydroxylysine-aldehyde (Hylald)-derived cross-links were completely absent from LH2KO collagen with concomitant increases in the Lysald-derived cross-links. Mass spectrometric analysis revealed that, in LH2KO type I collagen, telopeptidyl Lys hydroxylation was completely abolished at all sites while helical Lys hydroxylation was slightly diminished in a site-specific manner. Moreover, di-glycosylated Hyl was diminished at the expense of mono-glycosylated Hyl. LH2KO collagen was highly soluble and digestible, fibril diameters were diminished, and mineralization impaired when compared to controls. Together, these data underscore the critical role of LH2-catalyzed collagen modifications in collagen stability, organization and mineralization in MC3T3 cells.
Sujet(s)
Collagène de type I , Procollagen-lysine, 2-oxoglutarate 5-dioxygenase , Collagène/métabolisme , Collagène de type I/métabolisme , Hydroxylation , Lysine/métabolisme , Procollagen-lysine, 2-oxoglutarate 5-dioxygenase/génétique , Procollagen-lysine, 2-oxoglutarate 5-dioxygenase/métabolisme , Maturation post-traductionnelle des protéinesRÉSUMÉ
Epithelial-to-mesenchymal transition (EMT) is a transcriptionally governed process by which cancer cells establish a front-rear polarity axis that facilitates motility and invasion. Dynamic assembly of focal adhesions and other actin-based cytoskeletal structures on the leading edge of motile cells requires precise spatial and temporal control of protein trafficking. Yet, the way in which EMT-activating transcriptional programs interface with vesicular trafficking networks that effect cell polarity change remains unclear. Here, by utilizing multiple approaches to assess vesicular transport dynamics through endocytic recycling and retrograde trafficking pathways in lung adenocarcinoma cells at distinct positions on the EMT spectrum, we find that the EMT-activating transcription factor ZEB1 accelerates endocytosis and intracellular trafficking of plasma membrane-bound proteins. ZEB1 drives turnover of the MET receptor tyrosine kinase by hastening receptor endocytosis and transport to the lysosomal compartment for degradation. ZEB1 relieves a plus-end-directed microtubule-dependent kinesin motor protein (KIF13A) and a clathrin-associated adaptor protein complex subunit (AP1S2) from microRNA-dependent silencing, thereby accelerating cargo transport through the endocytic recycling and retrograde vesicular pathways, respectively. Depletion of KIF13A or AP1S2 mitigates ZEB1-dependent focal adhesion dynamics, front-rear axis polarization, and cancer cell motility. Thus, ZEB1-dependent transcriptional networks govern vesicular trafficking dynamics to effect cell polarity change.
Sujet(s)
Endosomes/métabolisme , Transition épithélio-mésenchymateuse/génétique , Transition épithélio-mésenchymateuse/physiologie , Tumeurs du poumon/métabolisme , Facteur de transcription Zeb1/génétique , Facteur de transcription Zeb1/métabolisme , Actines/métabolisme , Complexe protéique adaptateur, sous-unités sigma , Adénocarcinome pulmonaire/métabolisme , Lignée cellulaire tumorale , Polarité de la cellule , Cytosquelette/métabolisme , Endocytose , Contacts focaux/métabolisme , Régulation de l'expression des gènes tumoraux , Humains , Kinésine , Tumeurs du poumon/génétique , Protéines membranaires/métabolisme , microARN/métabolisme , Métastase tumoraleRÉSUMÉ
Cancer cells are a major source of enzymes that modify collagen to create a stiff, fibrotic tumor stroma. High collagen lysyl hydroxylase 2 (LH2) expression promotes metastasis and is correlated with shorter survival in lung adenocarcinoma (LUAD) and other tumor types. LH2 hydroxylates lysine (Lys) residues on fibrillar collagen's amino- and carboxy-terminal telopeptides to create stable collagen cross-links. Here, we show that electrostatic interactions between the LH domain active site and collagen determine the unique telopeptidyl lysyl hydroxylase (tLH) activity of LH2. However, CRISPR/Cas-9-mediated inactivation of tLH activity does not fully recapitulate the inhibitory effect of LH2 knock out on LUAD growth and metastasis in mice, suggesting that LH2 drives LUAD progression, in part, through a tLH-independent mechanism. Protein homology modeling and biochemical studies identify an LH2 isoform (LH2b) that has previously undetected collagen galactosylhydroxylysyl glucosyltransferase (GGT) activity determined by a loop that enhances UDP-glucose-binding in the GLT active site and is encoded by alternatively spliced exon 13 A. CRISPR/Cas-9-mediated deletion of exon 13 A sharply reduces the growth and metastasis of LH2b-expressing LUADs in mice. These findings identify a previously unrecognized collagen GGT activity that drives LUAD progression.
Sujet(s)
Adénocarcinome pulmonaire/physiopathologie , Évolution de la maladie , Glucosyltransferases/métabolisme , Tumeurs du poumon/physiopathologie , Animaux , SourisRÉSUMÉ
Cancer cells function as primary architects of the tumor microenvironment. However, the molecular features of cancer cells that govern stromal cell phenotypes remain unclear. Here, we show that cancer-associated fibroblast (CAF) heterogeneity is driven by lung adenocarcinoma (LUAD) cells at either end of the epithelial-to-mesenchymal transition (EMT) spectrum. LUAD cells that have high expression of the EMT-activating transcription factor ZEB1 reprogram CAFs through a ZEB1-dependent secretory program and direct CAFs to the tips of invasive projections through a ZEB1-driven CAF repulsion process. The EMT, in turn, sensitizes LUAD cells to pro-metastatic signals from CAFs. Thus, CAFs respond to contextual cues from LUAD cells to promote metastasis.
Sujet(s)
Adénocarcinome pulmonaire/génétique , Fibroblastes associés au cancer/métabolisme , Cellules épithéliales/métabolisme , Tumeurs du rein/génétique , Tumeurs du poumon/génétique , Cellules souches mésenchymateuses/métabolisme , Facteur de transcription Zeb1/génétique , Adénocarcinome pulmonaire/métabolisme , Adénocarcinome pulmonaire/secondaire , alpha-Globulines/génétique , alpha-Globulines/métabolisme , Animaux , Fibroblastes associés au cancer/anatomopathologie , Communication cellulaire , Lignée cellulaire tumorale , Mouvement cellulaire , Prolifération cellulaire , Récepteur-2 à domaine discoïdine/génétique , Récepteur-2 à domaine discoïdine/métabolisme , Cellules épithéliales/anatomopathologie , Transition épithélio-mésenchymateuse/génétique , Analyse de profil d'expression de gènes , Régulation de l'expression des gènes tumoraux , Humains , Tumeurs du rein/métabolisme , Tumeurs du rein/secondaire , Tumeurs du poumon/métabolisme , Tumeurs du poumon/anatomopathologie , Mâle , Cellules souches mésenchymateuses/anatomopathologie , Souris , Souris transgéniques , Transduction du signal , Microenvironnement tumoral/génétique , Facteur de transcription Zeb1/métabolismeRÉSUMÉ
Therapeutic strategies designed to target TP53-deficient cancer cells remain elusive. Here, we showed that TP53 loss initiated a pharmacologically actionable secretory process that drove lung adenocarcinoma (LUAD) progression. Molecular, biochemical, and cell biological studies showed that TP53 loss increased the expression of Golgi reassembly and stacking protein 55 kDa (G55), a Golgi stacking protein that maintains Golgi organelle integrity and is part of a GOLGIN45 (G45)-myosin IIA-containing protein complex that activates secretory vesicle biogenesis in the Golgi. TP53 loss activated G55-dependent secretion by relieving G55 and myosin IIA from miR-34a-dependent silencing. G55-dependent secreted proteins enhanced the proliferative and invasive activities of TP53-deficient LUAD cells and promoted angiogenesis and CD8+ T cell exhaustion in the tumor microenvironment. A small molecule that blocks G55-G45 interactions impaired secretion and reduced TP53-deficient LUAD growth and metastasis. These results identified a targetable secretory vulnerability in TP53-deficient LUAD cells.
Sujet(s)
Adénocarcinome pulmonaire/métabolisme , Appareil de Golgi/métabolisme , Tumeurs du poumon/métabolisme , Protéine p53 suppresseur de tumeur/déficit , Protéines du transport vésiculaire/métabolisme , Adénocarcinome pulmonaire/génétique , Adénocarcinome pulmonaire/anatomopathologie , Animaux , Lignée cellulaire tumorale , Appareil de Golgi/génétique , Appareil de Golgi/anatomopathologie , Humains , Tumeurs du poumon/génétique , Tumeurs du poumon/anatomopathologie , Souris , Métastase tumorale , Protéine p53 suppresseur de tumeur/métabolisme , Protéines du transport vésiculaire/génétiqueRÉSUMÉ
Heightened secretion of protumorigenic effector proteins is a feature of malignant cells. Yet, the molecular underpinnings and therapeutic implications of this feature remain unclear. Here, we identify a chromosome 1q region that is frequently amplified in diverse cancer types and encodes multiple regulators of secretory vesicle biogenesis and trafficking, including the Golgi-dedicated enzyme phosphatidylinositol (PI)-4-kinase IIIß (PI4KIIIß). Molecular, biochemical, and cell biological studies show that PI4KIIIß-derived PI-4-phosphate (PI4P) synthesis enhances secretion and accelerates lung adenocarcinoma progression by activating Golgi phosphoprotein 3 (GOLPH3)-dependent vesicular release from the Golgi. PI4KIIIß-dependent secreted factors maintain 1q-amplified cancer cell survival and influence prometastatic processes in the tumor microenvironment. Disruption of this functional circuitry in 1q-amplified cancer cells with selective PI4KIIIß antagonists induces apoptosis and suppresses tumor growth and metastasis. These results support a model in which chromosome 1q amplifications create a dependency on PI4KIIIß-dependent secretion for cancer cell survival and tumor progression.
Sujet(s)
Adénocarcinome pulmonaire/métabolisme , Chromosomes humains de la paire 1/métabolisme , Phosphotransferases (Alcohol Group Acceptor)/métabolisme , Adénocarcinome pulmonaire/génétique , Animaux , Chromosomes humains de la paire 1/génétique , Test ELISA , Appareil de Golgi/métabolisme , Humains , Techniques in vitro , Protéines membranaires/génétique , Protéines membranaires/métabolisme , Souris , Phosphotransferases (Alcohol Group Acceptor)/antagonistes et inhibiteurs , Microtomographie aux rayons XRÉSUMÉ
Collagenous stromal accumulations predict a worse clinical outcome in a variety of malignancies. Better tools are needed to elucidate the way in which collagen influences cancer cells. Here, we report a method to generate collagenous matrices that are deficient in key post-translational modifications and evaluate cancer cell behaviors on those matrices. We utilized genetic and biochemical approaches to inhibit lysine hydroxylation and glucosylation on collagen produced by MC-3T3-E1 murine osteoblasts (MC cells). Seeded onto MC cell-derived matrix surface, multicellular aggregates containing lung adenocarcinoma cells alone or in combination with cancer-associated fibroblasts dissociated with temporal and spatial patterns that were influenced by collagen modifications. These findings demonstrate the feasibility of generating defined collagen matrices that are suitable for cell culture studies.
RÉSUMÉ
Lysyl hydroxylase-2 (LH2) catalyzes the hydroxylation of telopeptidyl lysine residues on collagen, leading to the formation of stable collagen cross-links that connect collagen molecules and stabilize the extracellular matrix. High levels of LH2 have been reported in the formation and stabilization of hydroxylysine aldehyde-derived collagen cross-links (HLCCs), leading to fibrosis and cancer metastasis in certain tissues. Identification of small-molecule inhibitors targeting LH2 activity requires a robust and suitable assay system, which is currently lacking. Thus, despite being a promising target for these diseases, small-molecule inhibitors for LH2 have yet to be reported. Therefore, we developed a luminescence-based strategy to monitor LH activity and validated its ability to identify new inhibitors in a screen of approximately 65,000 compounds against LH2. Primary hits were confirmed using the same LH assay against mimiviral L230. This newly developed LH assay is robust, suitable for high-throughput screening, and able to identify potent specific inhibitors of LH2.
Sujet(s)
Antienzymes/pharmacologie , Procollagen-lysine, 2-oxoglutarate 5-dioxygenase/antagonistes et inhibiteurs , Procollagen-lysine, 2-oxoglutarate 5-dioxygenase/métabolisme , Bibliothèques de petites molécules/pharmacologie , Tests de criblage à haut débit/méthodesRÉSUMÉ
In the originally published version of this Article, financial support was not fully acknowledged. The PDF and HTML versions of the Article have now been corrected to also include support from the National Institutes of Health grant T32GM008280 to Sarah Alvarado.
RÉSUMÉ
Collagen lysyl hydroxylases (LH1-3) are Fe2+- and 2-oxoglutarate (2-OG)-dependent oxygenases that maintain extracellular matrix homeostasis. High LH2 levels cause stable collagen cross-link accumulations that promote fibrosis and cancer progression. However, developing LH antagonists will require structural insights. Here, we report a 2 Å crystal structure and X-ray scattering on dimer assemblies for the LH domain of L230 in Acanthamoeba polyphaga mimivirus. Loop residues in the double-stranded ß-helix core generate a tail-to-tail dimer. A stabilizing hydrophobic leucine locks into an aromatic tyrosine-pocket on the opposite subunit. An active site triad coordinates Fe2+. The two active sites flank a deep surface cleft that suggest dimerization creates a collagen-binding site. Loss of Fe2+-binding disrupts the dimer. Dimer disruption and charge reversal in the cleft increase Km and reduce LH activity. Ectopic L230 expression in tumors promotes collagen cross-linking and metastasis. These insights suggest inhibitor targets for fibrosis and cancer.
Sujet(s)
Fer/composition chimique , Mimiviridae/enzymologie , Procollagen-lysine, 2-oxoglutarate 5-dioxygenase/composition chimique , Multimérisation de protéines , Protéines virales/composition chimique , Séquence d'acides aminés , Animaux , Domaine catalytique , Lignée cellulaire tumorale , Collagène/composition chimique , Collagène/métabolisme , Cristallographie aux rayons X , Stabilité enzymatique , Humains , Fer/métabolisme , Tumeurs du poumon/génétique , Tumeurs du poumon/métabolisme , Tumeurs du poumon/anatomopathologie , Souris nude , Mimiviridae/génétique , Mutation , Métastase tumorale , Procollagen-lysine, 2-oxoglutarate 5-dioxygenase/génétique , Procollagen-lysine, 2-oxoglutarate 5-dioxygenase/métabolisme , Liaison aux protéines , Diffusion aux petits angles , Similitude de séquences d'acides aminés , Transplantation hétérologue , Protéines virales/génétique , Protéines virales/métabolisme , Diffraction des rayons XRÉSUMÉ
Insulin-degrading enzyme (IDE) hydrolyzes bioactive peptides, including insulin, amylin, and the amyloid ß peptides. Polyanions activate IDE toward some substrates, yet an endogenous polyanion activator has not yet been identified. Here we report that inositol phosphates (InsPs) and phosphatdidylinositol phosphates (PtdInsPs) serve as activators of IDE. InsPs and PtdInsPs interact with the polyanion-binding site located on an inner chamber wall of the enzyme. InsPs activate IDE by up to â¼95-fold, affecting primarily Vmax The extent of activation and binding affinity correlate with the number of phosphate groups on the inositol ring, with phosphate positional effects observed. IDE binds PtdInsPs from solution, immobilized on membranes, or presented in liposomes. Interaction with PtdInsPs, likely PtdIns(3)P, plays a role in localizing IDE to endosomes, where the enzyme reportedly encounters physiological substrates. Thus, InsPs and PtdInsPs can serve as endogenous modulators of IDE activity, as well as regulators of its intracellular spatial distribution.
Sujet(s)
Endosomes/métabolisme , Inositol phosphates/métabolisme , Insulinase/métabolisme , Phosphatidyl inositols/métabolisme , Androstadiènes/pharmacologie , Animaux , Sites de fixation , Cellules COS , Chlorocebus aethiops , Endosomes/effets des médicaments et des substances chimiques , Activation enzymatique , Enzymes immobilisées/métabolisme , Concentration en ions d'hydrogène , Insulinase/composition chimique , Insulinase/génétique , Liposomes/composition chimique , Liposomes/métabolisme , Mutation , WortmannineRÉSUMÉ
Hydroxylysine aldehyde-derived collagen cross-links (HLCCs) accumulate in fibrotic tissues and certain types of cancer and are thought to drive the progression of these diseases. HLCC formation is initiated by lysyl hydroxylase 2 (LH2), an Fe(II) and α-ketoglutarate (αKG)-dependent oxygenase that hydroxylates telopeptidyl lysine residues on collagen. Development of LH2 antagonists for the treatment of these diseases will require a reliable source of recombinant LH2 protein and a non-radioactive LH2 enzymatic activity assay that is amenable to high throughput screens of small molecule libraries. However, LH2 protein generated using E coli- or insect-based expression systems is either insoluble or enzymatically unstable, and the LH2 enzymatic activity assays that are currently available measure radioactive CO2 released from 14C-labeled αKG during its conversion to succinate. To address these deficiencies, we have developed a scalable process to purify human LH2 protein from Chinese hamster ovary cell-derived conditioned media samples and a luciferase-based assay that quantifies LH2-dependent conversion of αKG to succinate. These methodologies may be applicable to other Fe(II) and αKG-dependent oxygenase systems.
Sujet(s)
Acides cétoglutariques/composition chimique , Procollagen-lysine, 2-oxoglutarate 5-dioxygenase/biosynthèse , Animaux , Cellules CHO , Carbone/composition chimique , Dioxyde de carbone/composition chimique , Chromatographie en phase liquide , Collagène/composition chimique , Cricetulus , Milieux de culture conditionnés/composition chimique , Glycosylation , Humains , Luciferases/composition chimique , Lysine/composition chimique , Protéines recombinantes/biosynthèse , Acide succinique/composition chimique , Spectrométrie de masse en tandemRÉSUMÉ
Tumor cells gain metastatic capacity through a Golgi phosphoprotein 3-dependent (GOLPH3-dependent) Golgi membrane dispersal process that drives the budding and transport of secretory vesicles. Whether Golgi dispersal underlies the pro-metastatic vesicular trafficking that is associated with epithelial-to-mesenchymal transition (EMT) remains unclear. Here, we have shown that, rather than causing Golgi dispersal, EMT led to the formation of compact Golgi organelles with improved ribbon linking and cisternal stacking. Ectopic expression of the EMT-activating transcription factor ZEB1 stimulated Golgi compaction and relieved microRNA-mediated repression of the Golgi scaffolding protein PAQR11. Depletion of PAQR11 dispersed Golgi organelles and impaired anterograde vesicle transport to the plasma membrane as well as retrograde vesicle tethering to the Golgi. The N-terminal scaffolding domain of PAQR11 was associated with key regulators of Golgi compaction and vesicle transport in pull-down assays and was required to reconstitute Golgi compaction in PAQR11-deficient tumor cells. Finally, high PAQR11 levels were correlated with EMT and shorter survival in human cancers, and PAQR11 was found to be essential for tumor cell migration and metastasis in EMT-driven lung adenocarcinoma models. We conclude that EMT initiates a PAQR11-mediated Golgi compaction process that drives metastasis.
Sujet(s)
Adénocarcinome/métabolisme , Mouvement cellulaire , Transition épithélio-mésenchymateuse , Appareil de Golgi/métabolisme , Tumeurs du poumon/métabolisme , Protéines tumorales/métabolisme , Récepteurs à la progestérone/métabolisme , Adénocarcinome/génétique , Adénocarcinome/anatomopathologie , Lignée cellulaire tumorale , Délétion de gène , Appareil de Golgi/génétique , Appareil de Golgi/anatomopathologie , Humains , Tumeurs du poumon/génétique , Tumeurs du poumon/anatomopathologie , Protéines membranaires/génétique , Protéines membranaires/métabolisme , microARN/génétique , microARN/métabolisme , Métastase tumorale , Protéines tumorales/génétique , Domaines protéiques , ARN tumoral/génétique , ARN tumoral/métabolisme , Récepteurs à la progestérone/génétique , Facteur de transcription Zeb1/génétique , Facteur de transcription Zeb1/métabolismeRÉSUMÉ
The Neuropilins (Nrps) are a family of essential cell surface receptors involved in multiple fundamental cellular signaling cascades. Nrp family members have key functions in VEGF-dependent angiogenesis and semaphorin-dependent axon guidance, controlling signaling and cross-talk between these fundamental physiological processes. More recently, Nrp function has been found in diverse signaling and adhesive functions, emphasizing their role as pleiotropic co-receptors. Pathological Nrp function has been shown to be important in aberrant activation of both canonical and alternative pathways. Here we review key recent insights into Nrp function in human health and disease.
Sujet(s)
Membrane cellulaire/métabolisme , Neuropilines/métabolisme , Motifs d'acides aminés , Animaux , Axones/métabolisme , Adhérence cellulaire , Humains , Ligands , Souris , Phénotype , Liaison aux protéines , Structure tertiaire des protéines , Transport des protéines , Sémaphorines/métabolisme , Transduction du signal , Facteur de croissance endothéliale vasculaire de type A/métabolismeRÉSUMÉ
Calcineurin is an essential serine/threonine phosphatase that plays vital roles in neuronal development and function, heart growth, and immune system activation. Calcineurin is unique in that it is the only phosphatase known to be activated by calmodulin in response to increasing intracellular calcium concentrations. Calcium-loaded calmodulin binds to the regulatory domain of calcineurin, resulting in a conformational change that removes an autoinhibitory domain from the active site of the phosphatase. We have determined a 1.95 Å crystal structure of calmodulin bound to a peptide corresponding to its binding region from calcineurin. In contrast to previous structures of this complex, our structure has a stoichiometry of 1:1 and has the canonical collapsed, wraparound conformation observed for many calmodulin-substrate complexes. In addition, we have used size-exclusion chromatography and time-resolved fluorescence to probe the stoichiometry of binding of calmodulin to a construct corresponding to almost the entire regulatory domain from calcineurin, again finding a 1:1 complex. Taken in sum, our data strongly suggest that a single calmodulin protein is necessary and sufficient to bind to and activate each calcineurin enzyme.
Sujet(s)
Calcineurine/métabolisme , Calmoduline/métabolisme , Séquence nucléotidique , Calcineurine/composition chimique , Calmoduline/composition chimique , Chromatographie sur gel , Amorces ADN , Électrophorèse sur gel de polyacrylamide , Humains , Conformation moléculaire , Spectrométrie de fluorescenceRÉSUMÉ
Plants use the insoluble polyglucan starch as their primary glucose storage molecule. Reversible phosphorylation, at the C6 and C3 positions of glucose moieties, is the only known natural modification of starch and is the key regulatory mechanism controlling its diurnal breakdown in plant leaves. The glucan phosphatase Starch Excess4 (SEX4) is a position-specific starch phosphatase that is essential for reversible starch phosphorylation; its absence leads to a dramatic accumulation of starch in Arabidopsis, but the basis for its function is unknown. Here we describe the crystal structure of SEX4 bound to maltoheptaose and phosphate to a resolution of 1.65 Å. SEX4 binds maltoheptaose via a continuous binding pocket and active site that spans both the carbohydrate-binding module (CBM) and the dual-specificity phosphatase (DSP) domain. This extended interface is composed of aromatic and hydrophilic residues that form a specific glucan-interacting platform. SEX4 contains a uniquely adapted DSP active site that accommodates a glucan polymer and is responsible for positioning maltoheptaose in a C6-specific orientation. We identified two DSP domain residues that are responsible for SEX4 site-specific activity and, using these insights, we engineered a SEX4 double mutant that completely reversed specificity from the C6 to the C3 position. Our data demonstrate that the two domains act in consort, with the CBM primarily responsible for engaging glucan chains, whereas the DSP integrates them in the catalytic site for position-specific dephosphorylation. These data provide important insights into the structural basis of glucan phosphatase site-specific activity and open new avenues for their biotechnological utilization.
Sujet(s)
Protéines d'Arabidopsis/composition chimique , Dual-specificity phosphatases/composition chimique , Glucanes/composition chimique , Glucose/composition chimique , Amidon/composition chimique , Arabidopsis/enzymologie , Protéines d'Arabidopsis/métabolisme , Glucides/composition chimique , Domaine catalytique , Clonage moléculaire , Dual-specificity phosphatases/métabolisme , Phosphates/composition chimique , Phosphorylation , Feuilles de plante/métabolisme , Liaison aux protéines , Conformation des protéinesRÉSUMÉ
Neuropilin-1 (Nrp1), an essential type I transmembrane receptor, binds two secreted ligand families, vascular endothelial growth factor (VEGF) and class III Semaphorin (Sema3). VEGF-A and Sema3F have opposing roles in regulating Nrp1 vascular function in angiogenesis. VEGF-A functions as one of the most potent pro-angiogenic cytokines, while Sema3F is a uniquely potent endogenous angiogenesis inhibitor. Sema3 family members require proteolytic processing by furin to allow competitive binding to Nrp1. We demonstrate that the furin-processed C-terminal domain of Sema3F (C-furSema) potently inhibits VEGF-A-dependent activation of endothelial cells. We find that this potent activity is due to unique heterobivalent engagement of Nrp1 by two distinct sites in the C-terminal domain of Sema3F. One of the sites is the C-terminal arginine, liberated by furin cleavage, and the other is a novel upstream helical motif centered on the intermolecular disulfide. Using a novel chimeric C-furSema, we demonstrate that combining a single C-terminal arginine with the helical motif is necessary and sufficient for potent inhibition of binding of VEGF-A to Nrp1. We further demonstrate that the multiple furin-processed variants of Sema3A, with the altered proximity of the two binding motifs, have dramatically different potencies. This suggests that furin processing not only switches Sema3 to an activated form but also, depending on the site processed, can also tune potency. These data establish the basis for potent competitive binding of Sema3 to Nrp1 and provide a basis for the design of bivalent Nrp inhibitors.