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1.
Comput Struct Biotechnol J ; 23: 2001-2010, 2024 Dec.
Article in English | MEDLINE | ID: mdl-38770160

ABSTRACT

In a recent study, we have identified BPH03 as a promising scaffold for the development of compounds aimed at modulating the interaction between PED/PEA15 (Phosphoprotein Enriched in Diabetes/Phosphoprotein Enriched in Astrocytes 15) and PLD1 (phospholipase D1), with potential applications in type II diabetes therapy. PED/PEA15 is known to be overexpressed in certain forms of diabetes, where it binds to PLD1, thereby reducing insulin-stimulated glucose transport. The inhibition of this interaction reestablishes basal glucose transport, indicating PED as a potential target of ligands capable to recover glucose tolerance and insulin sensitivity. In this study, we employ computational methods to provide a detailed description of BPH03 interaction with PED, evidencing the presence of a hidden druggable pocket within its PLD1 binding surface. We also elucidate the conformational changes that occur during PED interaction with BPH03. Moreover, we report new NMR data supporting the in-silico findings and indicating that BPH03 disrupts the PED/PLD1 interface displacing PLD1 from its interaction with PED. Our study represents a significant advancement toward the development of potential therapeutics for the treatment of type II diabetes.

2.
Int J Mol Sci ; 25(7)2024 Mar 26.
Article in English | MEDLINE | ID: mdl-38612485

ABSTRACT

Galectins, ß-galactoside-binding proteins, play relevant roles in different biological processes; therefore, they are becoming emerging targets for diagnostic and therapeutic approaches [...].


Subject(s)
Galectins
3.
Int J Mol Sci ; 24(19)2023 Sep 29.
Article in English | MEDLINE | ID: mdl-37834166

ABSTRACT

Proteins of the MucR/Ros family play a crucial role in bacterial infection or symbiosis with eukaryotic hosts. MucR from Sinorhizobium meliloti plays a regulatory role in establishing symbiosis with the host plant, both dependent and independent of Quorum Sensing. Here, we report the first characterization of MucR isolated from Sinorhizobium meliloti by mass spectrometry and demonstrate that this protein forms higher-order oligomers in its native condition of expression by SEC-MALS. We show that MucR purified from Sinorhizobium meliloti can bind DNA and recognize the region upstream of the ndvA gene in EMSA, revealing that this gene is a direct target of MucR. Although MucR DNA binding activity was already described, a detailed characterization of Sinorhizobium meliloti DNA targets has never been reported. We, thus, analyze sequences recognized by MucR in the rem gene promoter, showing that this protein recognizes AT-rich sequences and does not require a consensus sequence to bind DNA. Furthermore, we investigate the dependence of MucR DNA binding on the length of DNA targets. Taken together, our studies establish MucR from Sinorhizobium meliloti as a member of a new family of Histone-like Nucleoid Structuring (H-NS) proteins, thus explaining the multifaceted role of this protein in many species of alpha-proteobacteria.


Subject(s)
Repressor Proteins , Sinorhizobium meliloti , Repressor Proteins/genetics , Sinorhizobium meliloti/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Transcription Factors/metabolism , DNA/genetics , DNA/metabolism , Symbiosis , Gene Expression Regulation, Bacterial
4.
Brief Funct Genomics ; 22(2): 227-240, 2023 04 13.
Article in English | MEDLINE | ID: mdl-36827221

ABSTRACT

SARS-CoV-2 encodes eight accessory proteins, one of which, ORF8, has a poorly conserved sequence with SARS-CoV and its role in viral pathogenicity has recently been identified. ORF8 in SARS-CoV-2 has a unique functional feature that allows it to form a dimer structure linked by a disulfide bridge between Cys20 and Cys20 (S-S). This study provides structural characterization of natural mutant variants as well as the identification of potential drug candidates capable of binding directly to the interchain disulfide bridge. The lead compounds reported in this work have a tendency to settle in the dimeric interfaces by direct interaction with the disulfide bridge. These molecules may disturb the dimer formation and may have an inhibition impact on its potential functional role in host immune evasion and virulence pathogenicity. This work provides detailed insights on the sequence and structural variability through computational mutational studies, as well as potent drug candidates with the ability to interrupt the intermolecular disulfide bridge formed between Cys20 and Cys20. Furthermore, the interactions of ORF8 peptides complexed with MHC-1 is studied, and the binding mode reveals that certain ORF8 peptides bind to MHC-1 in a manner similar to other viral peptides. Overall, this study is a narrative of various computational approaches used to provide detailed structural insights into SARS-CoV-2 ORF8 interchain disulfide bond disruptors.


Subject(s)
COVID-19 , SARS-CoV-2 , Humans , Dimerization
5.
Sci Rep ; 10(1): 9283, 2020 06 09.
Article in English | MEDLINE | ID: mdl-32518326

ABSTRACT

Ros/MucR is a widespread family of bacterial zinc-finger (ZF) containing proteins that integrate multiple functions such as virulence, symbiosis and/or cell cycle transcription. NMR solution structure of Ros DNA-binding domain (region 56-142, i.e. Ros87) has been solved by our group and shows that the prokaryotic ZF domain shows interesting structural and functional features that differentiate it from its eukaryotic counterpart as it folds in a significantly larger zinc-binding globular domain. We have recently proposed a novel functional model for this family of proteins suggesting that they may act as H-NS-'like' gene silencers. Indeed, the N-terminal region of this family of proteins appears to be responsible for the formation of functional oligomers. No structural characterization of the Ros N-terminal domain (region 1-55) is available to date, mainly because of serious solubility problems of the full-length protein. Here we report the first structural characterization of the N-terminal domain of the prokaryotic ZF family examining by means of MD and NMR the structural preferences of the full-length Ros protein from Agrobacterium tumefaciens.


Subject(s)
Agrobacterium tumefaciens/metabolism , DNA-Binding Proteins/genetics , Protein Domains , Protein Structure, Secondary/genetics , Zinc Fingers/genetics , Agrobacterium tumefaciens/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , DNA-Binding Proteins/metabolism , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular
6.
Front Mol Neurosci ; 12: 195, 2019.
Article in English | MEDLINE | ID: mdl-31467503

ABSTRACT

Cystatin B (CSTB) is a ubiquitous protein belonging to a superfamily of protease inhibitors. CSTB may play a critical role in brain physiology because its mutations cause progressive myoclonic epilepsy-1A (EPM1A), the most common form of progressive myoclonic epilepsy. However, the molecular mechanisms underlying the role of CSTB in the central nervous system (CNS) are largely unknown. To investigate the possible involvement of CSTB in the synaptic plasticity, we analyzed its expression in synaptosomes as a model system in studying the physiology of the synaptic regions of the CNS. We found that CSTB is not only present in the synaptosomes isolated from rat and mouse brain cortex, but also secreted into the medium in a depolarization-controlled manner. In addition, using biorthogonal noncanonical amino acid tagging (BONCAT) procedure, we demonstrated, for the first time, that CSTB is locally synthesized in the synaptosomes. The synaptic localization of CSTB was confirmed in a human 3D model of cortical development, namely cerebral organoids. Altogether, these results suggest that CSTB may play a role in the brain plasticity and open a new perspective in studying the involvement of CSTB deregulation in neurodegenerative and neuropsychiatric diseases.

7.
Sci Rep ; 9(1): 10519, 2019 07 19.
Article in English | MEDLINE | ID: mdl-31324836

ABSTRACT

The scalp-ear-nipple (SEN) syndrome is an autosomal-dominant disorder characterized by cutis aplasia of the scalp and malformations of breast, external ears, digits, and nails. Genetic analyses have shown that the disease is caused by missense mutations of the KCTD1 protein, although the functional/structural basis of SEN insurgence is hitherto unknown. With the aim of unravelling the molecular basis of the SEN syndrome associated with KCTD1 mutations we here expressed and characterized several disease causing mutants. A preliminary dissection of the protein provides insights into the role that individual domains play in KCTD1 stability. The characterization of SEN-causing mutants indicates that, although the mutation sites are located in distant regions of the BTB domain or of the pre-BTB region, all of them are unable to interact with the transcription factor AP-2α, a well-known KCTD1 biological partner. Notably, all mutations, including the one located in the pre-BTB region, produce a significant destabilization of the protein. The structural role of the pre-BTB region in KCTD1 and other proteins of the family is corroborated by its sequence conservation in orthologs and paralogs. Interestingly, SEN-causing mutations also favor the tendency of KCTD1 to adopt structural states that are characterized by the ability to bind the ß-amyloid fluorescent dye thioflavin T. The formation of aggregation-prone species may have important implications for the disease etiology. Collectively, these findings provide an intriguing picture of the functional and structural alterations induced by KCTD1 mutations that ultimately lead to disease.


Subject(s)
Abnormalities, Multiple/genetics , Co-Repressor Proteins/genetics , Ear, External/abnormalities , Hypospadias/genetics , Muscle Hypotonia/genetics , Mutation, Missense , Nipples/abnormalities , Point Mutation , Scalp/abnormalities , Amino Acid Substitution , Benzothiazoles/metabolism , Co-Repressor Proteins/metabolism , Genes, Dominant , Humans , Models, Molecular , Protein Conformation , Protein Denaturation , Protein Domains , Protein Stability , Recombinant Proteins/metabolism , Transcription Factor AP-2/metabolism
8.
Bioorg Chem ; 84: 434-443, 2019 03.
Article in English | MEDLINE | ID: mdl-30576907

ABSTRACT

EphA2 receptor plays a critical and debatable function in cancer and is considered a target in drug discovery. Lately, there has been a growing interest in its cytosolic C-terminal SAM domain (EphA2-SAM) as it engages protein modulators of receptor endocytosis and stability. Interestingly, EphA2-SAM binds the SAM domain from the lipid phosphatase Ship2 (Ship2-SAM) mainly producing pro-oncogenic outcomes. In an attempt to discover novel inhibitors of the EphA2-SAM/Ship2-SAM complex with possible anticancer properties, we focused on the central region of Ship2-SAM (known as Mid-Loop interface) responsible for its binding to EphA2-SAM. Starting from the amino acid sequence of the Mid-Loop interface virtual peptide libraries were built through ad hoc inserted mutations with either l- or d- amino acids and screened against EphA2-SAM by docking techniques. A few virtual hits were synthesized and experimentally tested by a variety of direct and competition-type interaction assays relying on NMR (Nuclear Magnetic Resonance), SPR (Surface Plasmon Resonance), MST (Microscale Thermophoresis) techniques. These studies guided the discovery of an original EphA2-SAM ligand antagonist of its interaction with Ship2-SAM.


Subject(s)
Drug Design , Molecular Docking Simulation , Peptides/chemistry , Receptor, EphA2/chemistry , Amino Acid Sequence , Binding Sites , Humans , Ligands , Nuclear Magnetic Resonance, Biomolecular , Peptide Library , Peptides/blood , Peptides/metabolism , Protein Stability , Receptor, EphA2/metabolism , Sterile Alpha Motif
9.
Sci Rep ; 8(1): 17238, 2018 11 22.
Article in English | MEDLINE | ID: mdl-30467359

ABSTRACT

MucR is a member of the Ros/MucR family of prokaryotic zinc-finger proteins found in the α-proteobacteria which regulate the expression of genes required for the successful pathogenic and symbiotic interactions of these bacteria with the eukaryotic hosts. The structure and function of their distinctive zinc-finger domain has been well-studied, but only recently the quaternary structure of the full length proteins was investigated demonstrating their ability to form higher-order oligomers. The aim of this study was to identify the region of MucR involved in higher-order oligomer formation by analysing deletion and point mutants of this protein by Light Scattering, and to determine the role that MucR oligomerization plays in the regulatory function of this protein. Here we demonstrate that a conserved hydrophobic region at the N-terminus of MucR is responsible for higher-order oligomer formation and that MucR oligomerization is essential for its regulatory function in Brucella. All these features of MucR are shared by the histone-like nucleoid structuring protein, (H-NS), leading us to propose that the prokaryotic zinc-finger proteins in the MucR/Ros family control gene expression employing a mechanism similar to that used by the H-NS proteins, rather than working as classical transcriptional regulators.


Subject(s)
Bacterial Proteins/genetics , Brucella abortus/genetics , Gene Expression Regulation, Bacterial/genetics , DNA, Bacterial/genetics , Gene Deletion , Point Mutation/genetics , Prokaryotic Cells/physiology , Zinc Fingers/genetics
10.
Bioorg Chem ; 80: 602-610, 2018 10.
Article in English | MEDLINE | ID: mdl-30036816

ABSTRACT

Sam (Sterile alpha motif) domains represent small helical protein-protein interaction modules which play versatile functions in different cellular processes. The Sam domain from the EphA2 receptor binds the Sam domain of the lipid phosphatase Ship2 and this interaction modulates receptor endocytosis and degradation primarily generating pro-oncogenic effects in cell. To identify molecule antagonists of the EphA2-Sam/Ship2-Sam complex with anti-cancer activity, we focused on hydrocarbon helical stapled peptides. EphA2-Sam and one of its interactors (i.e., the first Sam domain of the adaptor protein Odin) were used as model systems for peptide design. Increase in helicity in the stapled peptides, with respect to the corresponding linear/native-like regions, was proved by structural studies conducted through CD (Circular Dichroism) and NMR (Nuclear Magnetic Resonance). Interestingly, interaction assays by means of NMR, SPR (Surface Plasmon Resonance) and MST (MicroScale Thermophoresis) techniques led to the discovery of a novel ligand of Ship2-Sam.


Subject(s)
Peptides/chemistry , Peptides/pharmacology , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases/metabolism , Protein Interaction Maps/drug effects , Receptor, EphA2/metabolism , Amino Acid Sequence , Drug Discovery , Humans , Models, Molecular , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases/chemistry , Protein Binding/drug effects , Receptor, EphA2/chemistry , Sterile Alpha Motif/drug effects
11.
Chem Sci ; 9(13): 3290-3298, 2018 Apr 07.
Article in English | MEDLINE | ID: mdl-29780459

ABSTRACT

Significant advances in the understanding of the molecular determinants of fibrillogenesis can be expected from comparative studies of the aggregation propensities of proteins with highly homologous structures but different folding pathways. Here, we fully characterize, by means of stopped-flow, T-jump, CD and DSC experiments, the unfolding mechanisms of three highly homologous proteins, zinc binding Ros87 and Ml153-149 and zinc-lacking Ml452-151. The results indicate that the three proteins significantly differ in terms of stability and (un)folding mechanisms. Particularly, Ros87 and Ml153-149 appear to be much more stable to guanidine denaturation and are characterized by folding mechanisms including the presence of an intermediate. On the other hand, metal lacking Ml452-151 folds according to a classic two-state model. Successively, we have monitored the capabilities of Ros87, Ml452-151 and Ml153-149 to form amyloid fibrils under native conditions. Particularly, we show, by CD, fluorescence, DLS, TEM and SEM experiments, that after 168 hours, amyloid formation of Ros87 has started, while Ml153-149 has formed only amorphous aggregates and Ml452-151 is still monomeric in solution. This study shows how metal binding can influence protein folding pathways and thereby control conformational accessibility to aggregation-prone states, which in turn changes aggregation kinetics, shedding light on the role of metal ions in the development of protein deposition diseases.

12.
FEBS Open Bio ; 8(4): 711-718, 2018 04.
Article in English | MEDLINE | ID: mdl-29632823

ABSTRACT

The protein MucR from Brucella spp. is involved in the expression regulation of genes necessary for host interaction and infection. MucR is a member of the Ros/MucR family, which comprises prokaryotic zinc-finger proteins and includes Ros from Agrobacterium tumefaciens and the Ml proteins from Mesorhizobium loti. MucR from Brucella spp. can regulate the expression of virulence genes and repress its own gene expression. Despite the well-known role played by MucR in the repression of its own gene, no target sequence has yet been identified in the mucR promoter gene. In this study, we provide the first evidence that MucR from Brucella abortus binds more than one target site in the promoter region of its own gene, suggesting a molecular mechanism by which this protein represses its own expression. Furthermore, a circular dichroism analysis reveals that MucR is a heat-stable protein. Overall, the results of this study suggest that MucR might resemble a H-NS protein.

13.
Sci Rep ; 7(1): 17474, 2017 12 12.
Article in English | MEDLINE | ID: mdl-29234063

ABSTRACT

The lipid phosphatase Ship2 represents a drug discovery target for the treatment of different diseases, including cancer. Its C-terminal sterile alpha motif domain (Ship2-Sam) associates with the Sam domain from the EphA2 receptor (EphA2-Sam). This interaction is expected to mainly induce pro-oncogenic effects in cells therefore, inhibition of the Ship2-Sam/EphA2-Sam complex may represent an innovative route to discover anti-cancer therapeutics. In the present work, we designed and analyzed several peptide sequences encompassing the interaction interface of EphA2-Sam for Ship2-Sam. Peptide conformational analyses and interaction assays with Ship2-Sam conducted through diverse techniques (CD, NMR, SPR and MST), identified a positively charged penta-amino acid native motif in EphA2-Sam, that once repeated three times in tandem, binds Ship2-Sam. NMR experiments show that the peptide targets the negatively charged binding site of Ship2-Sam for EphA2-Sam. Preliminary in vitro cell-based assays indicate that -at 50 µM concentration- it induces necrosis of PC-3 prostate cancer cells with more cytotoxic effect on cancer cells than on normal dermal fibroblasts. This work represents a pioneering study that opens further opportunities for the development of inhibitors of the Ship2-Sam/EphA2-Sam complex for therapeutic applications.


Subject(s)
Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases/antagonists & inhibitors , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases/metabolism , Receptor, EphA2/antagonists & inhibitors , Receptor, EphA2/metabolism , Sterile Alpha Motif , Antineoplastic Agents/pharmacology , Binding Sites , Cell Line, Tumor , Drug Design , Escherichia coli , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Male , Membrane Proteins , Models, Molecular , Necrosis/chemically induced , Necrosis/metabolism , Nuclear Magnetic Resonance, Biomolecular , Peptides/chemistry , Peptides/pharmacology , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases/chemistry , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases/genetics , Preliminary Data , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Protein Binding , Receptor, EphA2/chemistry , Receptor, EphA2/genetics , Saccharomyces cerevisiae Proteins , Sterile Alpha Motif/drug effects
14.
Sci Rep ; 7(1): 15805, 2017 Nov 17.
Article in English | MEDLINE | ID: mdl-29150637

ABSTRACT

Mesorhizobium loti contains ten genes coding for proteins sharing high amino acid sequence identity with members of the Ros/MucR transcription factor family. Five of these Ros/MucR family members from Mesorhizobium loti (Ml proteins) have been recently structurally and functionally characterized demonstrating that Ml proteins are DNA-binding proteins. However, the DNA-binding studies were performed using the Ros DNA-binding site with the Ml proteins. Currently, there is no evidence as to when the Ml proteins are expressed during the Mesorhizobium lo ti life cycle as well as no information concerning their natural DNA-binding site. In this study, we examine the ml genes expression profile in Mesorhizobium loti and show that ml1, ml2, ml3 and ml5 are expressed during planktonic growth and in biofilms. DNA-binding experiments show that the Ml proteins studied bind a conserved AT-rich site in the promoter region of the exoY gene from Mesorhizobium loti and that the proteins make important contacts with the minor groove of DNA. Moreover, we demonstrate that the Ml proteins studied form higher-order oligomers through their N-terminal region and that the same AT-rich site is recognized by MucR from Brucella abortus using a similar mechanism involving contacts with the minor groove of DNA and oligomerization.


Subject(s)
AT Rich Sequence/genetics , Bacterial Proteins/metabolism , Brucella abortus/metabolism , DNA, Bacterial/genetics , Mesorhizobium/metabolism , Protein Multimerization , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Base Sequence , Biofilms/growth & development , Brucella abortus/genetics , Colony Count, Microbial , Gene Expression Regulation, Bacterial , Genes, Bacterial , Mesorhizobium/genetics , Mutation/genetics , Netropsin/metabolism , Phenotype , Plankton/growth & development , Protein Binding
15.
Biochimie ; 131: 106-114, 2016 Dec.
Article in English | MEDLINE | ID: mdl-27678190

ABSTRACT

The insomniac protein of Drosophila melanogaster (INC) has a crucial role in sleep homeostasis as flies lacking the inc gene exhibit strikingly reduced and poorly consolidated sleep. Nevertheless, in vitro characterizations of INC biophysical properties and partnerships have not been yet reported. Here we report the heterologous expression of the protein and its characterization using a number of different techniques. Present data indicate that INC is endowed with a remarkable stability, which results from the cooperation of the two protein domains. Moreover, we also demonstrated and quantified the ability of INC to recognize its potential partners Cul3 and dGRASP. Taking into account the molecular organization of the protein, these two partners may be anchored simultaneously. Although there is no evident relationship between the reported INC functions and dGRASP binding, our data suggest that INC may cooperate as ligase adaptor to dGRASP ubiquitination. SAXS data collected on the complex between INC and Cul3, which represent the first structural characterization of this type of assemblies, clearly highlight the highly dynamic nature of these complexes. This strongly suggests that the functional behavior of these proteins cannot be understood if dynamic effects are not considered. Finally, the strict analogy of the biochemical/biophysical properties of INC and of its human homolog KCTD5 may reliably indicate that this latter protein and/or the closely related proteins KCTD2/KCTD17 may play important roles in human sleep regulation.


Subject(s)
Cullin Proteins/metabolism , Drosophila Proteins/metabolism , Homeostasis , Membrane Proteins/metabolism , Sleep/physiology , Amino Acid Sequence , Animals , Biophysical Phenomena , Circular Dichroism , Cullin Proteins/chemistry , Cullin Proteins/genetics , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Drosophila melanogaster/physiology , Golgi Matrix Proteins , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Protein Binding , Protein Denaturation , Scattering, Small Angle , Sequence Homology, Amino Acid , Temperature , X-Ray Diffraction
16.
PLoS One ; 10(5): e0126808, 2015.
Article in English | MEDLINE | ID: mdl-25974686

ABSTRACT

Cullin 3 (Cul3) recognition by BTB domains is a key process in protein ubiquitination. Among Cul3 binders, a great attention is currently devoted to KCTD proteins, which are implicated in fundamental biological processes. On the basis of the high similarity of BTB domains of these proteins, it has been suggested that the ability to bind Cul3 could be a general property among all KCTDs. In order to gain new insights into KCTD functionality, we here evaluated and/or quantified the binding of Cul3 to the BTB of KCTD proteins, which are known to be involved either in cullin-independent (KCTD12 and KCTD15) or in cullin-mediated (KCTD6 and KCTD11) activities. Our data indicate that KCTD6(BTB) and KCTD11(BTB) bind Cul3 with high affinity forming stable complexes with 4:4 stoichiometries. Conversely, KCTD12(BTB) and KCTD15(BTB) do not interact with Cul3, despite the high level of sequence identity with the BTB domains of cullin binding KCTDs. Intriguingly, comparative sequence analyses indicate that the capability of KCTD proteins to recognize Cul3 has been lost more than once in distinct events along the evolution. Present findings also provide interesting clues on the structural determinants of Cul3-KCTD recognition. Indeed, the characterization of a chimeric variant of KCTD11 demonstrates that the swapping of α2ß3 loop between KCTD11(BTB) and KCTD12(BTB) is sufficient to abolish the ability of KCTD11(BTB) to bind Cul3. Finally, present findings, along with previous literature data, provide a virtually complete coverage of Cul3 binding ability of the members of the entire KCTD family.


Subject(s)
Cullin Proteins/metabolism , Repressor Proteins/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Calorimetry , Cell Cycle Proteins , Chromatography, Gel , Circular Dichroism , Cullin Proteins/chemistry , Cullin Proteins/genetics , Humans , Molecular Dynamics Simulation , Molecular Sequence Data , Potassium Channels/chemistry , Potassium Channels/genetics , Potassium Channels/metabolism , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Repressor Proteins/chemistry , Repressor Proteins/genetics , Sequence Alignment , Transferases
17.
Biochim Biophys Acta ; 1844(7): 1289-98, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24747150

ABSTRACT

Recent investigations have highlighted a key role of the proteins of the KCTD (K-potassium channel tetramerization domain containing proteins) family in several fundamental biological processes. Despite the growing importance of KCTDs, our current understanding of their biophysical and structural properties is very limited. Biochemical characterizations of these proteins have shown that most of them act as substrate adaptor in E3 ligases during protein ubiquitination. Here we present a characterization of the KCTD5-Cullin3 interactions which are mediated by the KCTD5 BTB domain. Isothermal titration calorimetry experiments reveal that KCTD5 avidly binds the Cullin3 (Cul3). The complex presents a 5:5 stoichiometry and a dissociation constant of 59 nM. Molecular modeling and molecular dynamics simulations clearly indicate that the two proteins form a stable (KCTD5-Cul3)(5) pinwheel-shaped heterodecamer in which two distinct KCTD5 subunits cooperate in the binding of each cullin chain. Molecular dynamics simulations indicate that different types of interactions contribute to the stability of the assembly. Interestingly, residues involved in Cul3 recognitions are conserved in the KCTD5 orthologs and paralogs implicated in important biological processes. These residues are also rather well preserved in most of the other KCTD proteins. By using molecular modeling techniques, the entire ubiquitination system including the E3 ligase, the E2 conjugating enzyme and ubiquitin was generated. The analysis of the molecular architecture of this complex machinery provides insights into the ubiquitination processes which involve E3 ligases with a high structural complexity.


Subject(s)
Cullin Proteins/metabolism , Molecular Dynamics Simulation , Potassium Channels/metabolism , Ubiquitin/metabolism , Amino Acid Sequence , Cullin Proteins/chemistry , Humans , Models, Molecular , Molecular Sequence Data , Potassium Channels/chemistry , Protein Binding , Protein Conformation , Sequence Homology, Amino Acid , Substrate Specificity , Ubiquitination
18.
Biochemistry ; 51(10): 2136-45, 2012 Mar 13.
Article in English | MEDLINE | ID: mdl-22332920

ABSTRACT

The EphA2 receptor plays key roles in many physiological and pathological events, including cancer. The process of receptor endocytosis and the consequent degradation have attracted attention as possible means of overcoming the negative outcomes of EphA2 in cancer cells and decreasing tumor malignancy. A recent study indicates that Sam (sterile alpha motif) domains of Odin, a member of the ANKS (ankyrin repeat and sterile alpha motif domain-containing) family of proteins, are important for the regulation of EphA2 endocytosis. Odin contains two tandem Sam domains (Odin-Sam1 and -Sam2). Herein, we report on the nuclear magnetic resonance (NMR) solution structure of Odin-Sam1; through a variety of assays (employing NMR, surface plasmon resonance, and isothermal titration calorimetry techniques), we clearly demonstrate that Odin-Sam1 binds to the Sam domain of EphA2 in the low micromolar range. NMR chemical shift perturbation experiments and molecular modeling studies point out that the two Sam domains interact with a head-to-tail topology characteristic of several Sam-Sam complexes. This binding mode is similar to that we have previously proposed for the association between the Sam domains of the lipid phosphatase Ship2 and EphA2. This work further validates structural elements relevant for the heterotypic Sam-Sam interactions of EphA2 and provides novel insights for the design of potential therapeutic compounds that can modulate receptor endocytosis.


Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Receptor, EphA2/chemistry , Receptor, EphA2/metabolism , Adaptor Proteins, Signal Transducing/genetics , Endocytosis/physiology , Humans , In Vitro Techniques , Models, Molecular , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Nuclear Magnetic Resonance, Biomolecular , Protein Interaction Domains and Motifs , Receptor, EphA2/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Solutions , Surface Plasmon Resonance , Thermodynamics
19.
Biochimie ; 93(4): 715-24, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21237243

ABSTRACT

The family of human proteins containing a potassium channel tetramerization domain (KCTD) includes 21 members whose function is largely unknown. Recent reports have however suggested that these proteins are implicated in very important biological processes. KCTD11/REN, the best-characterized member of the family to date, plays a crucial role in the ubiquitination of HDAC1 by acting, in complex with Cullin3, as an E3 ubiquitin ligase. By combining bioinformatics and mutagenesis analyses, here we show that the protein is expressed in two alternative variants: a short previously characterized form (sKCTD11) composed by 232 amino acids and a longer variant (lKCTD11) which contains an N-terminal extension of 39 residues. Interestingly, we demonstrate that lKCTD11 starts with a non-canonical AUU codon. Although both sKCTD11 and lKCTD11 bear a POZ/BTB domain in their N-terminal region, this domain is complete only in the long form. Indeed, sKCTD11 presents an incomplete POZ/BTB domain. Nonetheless, sKCTD11 is still able to bind Cul3, although to much lesser extent than lKCTD11, and to perform its biological activity. The heterologous expression of sKCTD11 and lKCTD11 and their individual domains in Escherichia coli yielded soluble products as fusion proteins only for the longer form. In contrast to the closely related KCTD5 which is pentameric, the characterization of both lKCTD11 and its POZ/BTB domain by gel filtration and light scattering indicates that the protein likely forms stable tetramers. In line with this result, experiments conducted in cells show that the active protein is not monomeric. Based on these findings, homology-based models were built for lKCTD11 BTB and for its complex with Cul3. These analyses indicate that a stable lKCTD11 BTB-Cul3 three-dimensional model with a 4:4 stoichiometry can be generated. Moreover, these models provide insights into the determinants of the tetramer stability and into the regions involved in lKCTD11-Cul3 recognition.


Subject(s)
Potassium Channels/chemistry , Potassium Channels/genetics , Ubiquitin-Protein Ligases/chemistry , Amino Acid Sequence , Base Sequence , Cell Cycle Proteins , Cullin Proteins/chemistry , Cullin Proteins/genetics , HEK293 Cells , Humans , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed/methods , Protein Binding/genetics , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Transferases , Ubiquitin/chemistry
20.
Nat Cell Biol ; 12(2): 132-42, 2010 Feb.
Article in English | MEDLINE | ID: mdl-20081843

ABSTRACT

Hedgehog signalling is crucial for development and is deregulated in several tumours, including medulloblastoma. Regulation of the transcriptional activity of Gli (glioma-associated oncogene) proteins, effectors of the Hedgehog pathway, is poorly understood. We show here that Gli1 and Gli2 are acetylated proteins and that their HDAC-mediated deacetylation promotes transcriptional activation and sustains a positive autoregulatory loop through Hedgehog-induced upregulation of HDAC1. This mechanism is turned off by HDAC1 degradation through an E3 ubiquitin ligase complex formed by Cullin3 and REN, a Gli antagonist lost in human medulloblastoma. Whereas high HDAC1 and low REN expression in neural progenitors and medulloblastomas correlates with active Hedgehog signalling, loss of HDAC activity suppresses Hedgehog-dependent growth of neural progenitors and tumour cells. Consistent with this, abrogation of Gli1 acetylation enhances cellular proliferation and transformation. These data identify an integrated HDAC- and ubiquitin-mediated circuitry, where acetylation of Gli proteins functions as an unexpected key transcriptional checkpoint of Hedgehog signalling.


Subject(s)
Cullin Proteins/metabolism , Hedgehog Proteins/metabolism , Histone Deacetylases/metabolism , Nerve Tissue Proteins/metabolism , Oncogene Proteins/metabolism , Signal Transduction/physiology , Trans-Activators/metabolism , Acetylation , Animals , Cell Cycle Proteins , Cell Line , Cell Line, Tumor , Cells, Cultured , Chromatin Immunoprecipitation , Cullin Proteins/genetics , Electrophoresis, Polyacrylamide Gel , Hedgehog Proteins/genetics , Histone Deacetylase 1/genetics , Histone Deacetylase 1/metabolism , Histone Deacetylase 2/genetics , Histone Deacetylase 2/metabolism , Histone Deacetylases/genetics , Humans , Immunoblotting , Immunohistochemistry , Kruppel-Like Transcription Factors/genetics , Kruppel-Like Transcription Factors/metabolism , Medulloblastoma/genetics , Medulloblastoma/metabolism , Mice , NIH 3T3 Cells , Nerve Tissue Proteins/genetics , Oncogene Proteins/genetics , Protein Binding , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Spectrometry, Mass, Electrospray Ionization , Trans-Activators/genetics , Transferases , Zinc Finger Protein GLI1 , Zinc Finger Protein Gli2
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