RESUMEN
Calmodulin-like (CML) proteins are major EF-hand-containing, calcium (Ca2+)-binding proteins with crucial roles in plant development and in coordinating plant stress tolerance. Given their abundance in plants, the properties of Ca2+ sensors and identification of novel target proteins of CMLs deserve special attention. To this end, we recombinantly produced and biochemically characterized CML36 from Arabidopsis thaliana We analyzed Ca2+ and Mg2+ binding to the individual EF-hands, observed metal-induced conformational changes, and identified a physiologically relevant target. CML36 possesses two high-affinity Ca2+/Mg2+ mixed binding sites and two low-affinity Ca2+-specific sites. Binding of Ca2+ induced an increase in the α-helical content and a conformational change that lead to the exposure of hydrophobic regions responsible for target protein recognition. Cation binding, either Ca2+ or Mg2+, stabilized the secondary and tertiary structures of CML36, guiding a large structural transition from a molten globule apo-state to a compact holoconformation. Importantly, through in vitro binding and activity assays, we showed that CML36 interacts directly with the regulative N terminus of the Arabidopsis plasma membrane Ca2+-ATPase isoform 8 (ACA8) and that this interaction stimulates ACA8 activity. Gene expression analysis revealed that CML36 and ACA8 are co-expressed mainly in inflorescences. Collectively, our results support a role for CML36 as a Ca2+ sensor that binds to and modulates ACA8, uncovering a possible involvement of the CML protein family in the modulation of plant-autoinhibited Ca2+ pumps.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/química , ATPasas Transportadoras de Calcio/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Membrana Celular/enzimología , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Calmodulina/genética , Activación EnzimáticaRESUMEN
Sulfur-containing amino acids play essential roles in many organisms. The protozoan parasite Toxoplasma gondii includes the genes for cystathionine ß-synthase and cystathionine γ-lyase (TgCGL), as well as for cysteine synthase, which are crucial enzymes of the transsulfuration and de novo pathways for cysteine biosynthesis, respectively. These enzymes are specifically expressed in the oocyst stage of T. gondii. However, their functionality has not been investigated. Herein, we expressed and characterized the putative CGL from T. gondii. Recombinant TgCGL almost exclusively catalyses the α,γ-hydrolysis of l-cystathionine to form l-cysteine and displays marginal reactivity toward l-cysteine. Structure-guided homology modelling revealed two striking amino acid differences between the human and parasite CGL active-sites (Glu59 and Ser340 in human to Ser77 and Asn360 in toxoplasma). Mutation of Asn360 to Ser demonstrated the importance of this residue in modulating the specificity for the catalysis of α,ß- versus α,γ-elimination of l-cystathionine. Replacement of Ser77 by Glu completely abolished activity towards l-cystathionine. Our results suggest that CGL is an important functional enzyme in T. gondii, likely implying that the reverse transsulfuration pathway is operative in the parasite; we also probed the roles of active-site architecture and substrate binding conformations as determinants of reaction specificity in transsulfuration enzymes.
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Cistationina gamma-Liasa/genética , Cistationina gamma-Liasa/metabolismo , Análisis Mutacional de ADN/métodos , Mutación/genética , Toxoplasma/enzimología , Cistationina , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Toxoplasma/genética , Toxoplasma/metabolismoRESUMEN
BACKGROUND: Lysine demethylase enzymes (KDMs) are an emerging class of therapeutic targets, that catalyse the removal of methyl marks from histone lysine residues regulating chromatin structure and gene expression. KDM4A isoform plays an important role in the epigenetic dysregulation in various cancers and is linked to aggressive disease and poor clinical outcomes. Despite several efforts, the KDM4 family lacks successful specific molecular inhibitors. RESULTS: Herein, starting from a structure-based fragments virtual screening campaign we developed a synergic framework as a guide to rationally design efficient KDM4A inhibitors. Commercial libraries were used to create a fragments collection and perform a virtual screening campaign combining docking and pharmacophore approaches. The most promising compounds were tested in-vitro by a Homogeneous Time-Resolved Fluorescence-based assay developed for identifying selective substrate-competitive inhibitors by means of inhibition of H3K9me3 peptide demethylation. 2-(methylcarbamoyl)isonicotinic acid was identified as a preliminary active fragment, displaying inhibition of KDM4A enzymatic activity. Its chemical exploration was deeply investigated by computational and experimental approaches which allowed a rational fragment growing process. The in-silico studies guided the development of derivatives designed as expansion of the primary fragment hit and provided further knowledge on the structure-activity relationship. CONCLUSIONS: Our study describes useful insights into key ligand-KDM4A protein interaction and provides structural features for the development of successful selective KDM4A inhibitors.
Asunto(s)
Histona Demetilasas con Dominio de Jumonji , Lisina , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Lisina/metabolismo , Metilación de ADN , Histonas/metabolismo , Relación Estructura-ActividadRESUMEN
Calcium- and Integrin-Binding protein 2 (CIB2) is a small and ubiquitously expressed protein with largely unknown biological function but ascertained role in hearing physiology and disease. Recent studies found that CIB2 binds Ca2+ with moderate affinity and dimerizes under conditions mimicking the physiological ones. Here we provided new lines of evidence on CIB2 oligomeric state and the mechanism of interaction with the α7B integrin target. Based on a combination of native mass spectrometry, chemical cross-linking/mass spectrometry, analytical gel filtration, dynamic light scattering and molecular dynamics simulations we conclude that CIB2 is monomeric under all tested conditions and presents uncommon hydrodynamic properties, most likely due to the high content of hydrophobic solvent accessible surface. Surface plasmon resonance shows that the interaction with α7B occurs with relatively low affinity and is limited to the cytosolic region proximal to the membrane, being kinetically favored in the presence of physiological Mg2+ and in the absence of Ca2+. Although CIB2 binds to an α7B peptide in a 1:1 stoichiometry, the formation of the complex might induce binding of another CIB2 molecule.
Asunto(s)
Proteínas de Unión al Calcio/metabolismo , Hidrodinámica , Multimerización de Proteína , Animales , Calcio/metabolismo , Proteínas de Unión al Calcio/química , Calmodulina/metabolismo , Bovinos , Membrana Celular/metabolismo , Dispersión Dinámica de Luz , Humanos , Integrinas , Magnesio/metabolismo , Modelos Moleculares , Peso Molecular , Péptidos/metabolismo , Unión Proteica , Estabilidad Proteica , Estructura Terciaria de Proteína , Proteolisis , Homología Estructural de ProteínaRESUMEN
Calcium and integrin binding protein 2 (CIB2) shares with the other members of the CIB family the ability to bind Ca2+ and Mg2+ via two functional EF-hand motifs, namely EF3 and EF4. As a cation sensor, CIB2 is able to switch to a conformation likely associated with specific biological functions yet to be clarified. Recent findings demonstrate the involvement of CIB2 in hearing physiology and a single, conservative point mutation (p.E64D) has been related to Usher Syndrome type 1J (USH1J) and non-syndromic hearing loss. We present an exhaustive biochemical and biophysical characterization of human wild type (WT) and E64D CIB2. We found that CIB2 does not possibly work as a calcium sensor under physiological conditions, its affinity for Ca2+ (Kdapp = 0.5 mM) being too low for detecting normal intracellular levels. Instead, CIB2 displays a significantly high affinity for Mg2+ (Kdapp = 290 µM), and it is probably Mg2+ -bound under physiological conditions. At odds with the homologous protein CIB1, CIB2 forms a non-covalent dimer under conditions that mimic the physiological ones, and as such it interacts with its physiological target α7B integrin. NMR spectroscopy revealed a long-range allosteric communication between the residue E64, located at the N-terminal domain, and the metal cation binding site EF3, located at the C-terminal domain. The conservative E64D mutation breaks up such inter-domain communication resulting in the impaired ability of CIB2 to switch to its Mg2+-bound form. The ability to bind the target integrin peptide was substantially conserved for E64D CIB2, thus suggesting that the molecular defect associated with USH1J resides in its inability to sense Mg2+ and adopt the required conformation.
RESUMEN
In addition to the well-known Ca(2+) sensor calmodulin, plants possess many calmodulin-like proteins (CMLs) that are predicted to have specific roles in the cell. Herein, we described the biochemical and biophysical characterization of recombinant Arabidopsis thaliana CML14. We applied isothermal titration calorimetry to analyze the energetics of Ca(2+) and Mg(2+) binding to CML14, and nuclear magnetic resonance spectroscopy, together with intrinsic and ANS-based fluorescence, to evaluate the structural effects of metal binding and metal-induced conformational changes. Furthermore, differential scanning calorimetry and limited proteolysis were used to characterize protein thermal and local stability. Our data demonstrate that CML14 binds one Ca(2+) ion with micromolar affinity (Kd â¼ 12 µM) and the presence of 10 mM Mg(2+) decreases the Ca(2+) affinity by â¼5-fold. Although binding of Ca(2+) to CML14 increases protein stability, it does not result in a more hydrophobic protein surface and does not induce the large conformational rearrangement typical of Ca(2+) sensors, but causes only localized structural changes in the unique functional EF-hand. Our data, together with a molecular modelling prediction, provide interesting insights into the biochemical properties of Arabidopsis CML14 and may be useful to direct additional studies aimed at understanding its physiological role.
Asunto(s)
Proteínas de Arabidopsis/química , Arabidopsis/química , Calcio/química , Calmodulina/química , Magnesio/química , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sitios de Unión , Calcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Cationes Bivalentes , Clonación Molecular , Secuencia Conservada , Motivos EF Hand , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Interacciones Hidrofóbicas e Hidrofílicas , Magnesio/metabolismo , Modelos Moleculares , Mutación , Unión Proteica , Estabilidad Proteica , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de SecuenciaRESUMEN
To investigate novel colorectal cancer (CRC)-associated antigens that could be targets of humoral or cellular responses, we analyzed the reactivity of serum from a long-surviving CRC patient (for more than 100 months of follow-up) in clinical remission, by serologic proteome analysis. Two-dimensional Western blotting (2D-WB) and mass spectrometry analysis revealed a strong reactivity of this serum against protein disulfide isomerase A3 (PDIA3). Anti-PDIA3 antibodies are not a diagnostic marker of CRC, 2D-WB and Luminex analysis revealed that they were equally present in about 10% of sera from healthy subjects and CRC patients. Kaplan-Meier analysis of survival in CRC patient cohort, after 48 months of follow-up, showed a trend of higher survival in patients with increased levels of autoantibodies to PDIA3. Therefore, the interplay between the presence of these antibodies and T-cell response was investigated. Peripheral blood T cells from CRC patients with high immunoglobulin G (IgG) reactivity to PDIA3 also secreted interferon gamma (IFN-γ) when stimulated in vitro with recombinant PDIA3, whereas those from CRC with low IgG reactivity to PDIA3 did not. PDIA3-pulsed dendritic cells efficiently induced proliferation and IFN-γ production of autologous CD4(+) and CD8(+) T cells. Finally, ex vivo analysis of tumor-infiltrating T lymphocytes from CRC patients with autoantibodies to PDIA3 revealed that PDIA3-specific Th1 effector cells accumulated in tumor tissue. These data indicate that the presence of autoantibodies to PDIA3 favors the development of an efficient and specific T-cell response against PDIA3 in CRC patients. These results may be relevant for the design of novel immunotherapeutic strategies in CRC patients.