Important Role In Protein-protein Interactions Research Articles

In Silico-Based Structural Evaluation to Categorize the Pathogenicity of Mutations Identified in the RAD Class of Proteins.

RAD genes, known as double-strand break repair proteins, play a major role in maintaining the genomic integrity of a cell by carrying out essential DNA repair functions via double-strand break repair pathways. Mutations in the RAD class of proteins show high susceptibility to breast and ovarian cancers; however, adequate research on the mutations identified in these genes has not been extensively reported for their deleterious effects. Changes in the folding pattern of RAD proteins play an important role in protein-protein interactions and also functions. Missense mutations identified from four cancer databases, cBioPortal, COSMIC, ClinVar, and gnomAD, cause aberrant conformations, which may lead to faulty DNA repair mechanisms. It is therefore necessary to evaluate the effects of pathogenic mutations of RAD proteins and their subsequent role in breast and ovarian cancers. In this study, we have used eight computational prediction servers to analyze pathogenic mutations and understand their effects on the protein structure and function. A total of 5122 missense mutations were identified from four different cancer databases, of which 1165 were predicted to be pathogenic using at least five pathogenicity prediction servers. These mutations were characterized as high-risk mutations based on their location in the conserved domains and subsequently subjected to structural stability characterization. The mutations included in the present study were selected from clinically relevant mutants in breast cancer pedigrees. Comparative folding patterns and intra-atomic interaction results showed alterations in the structural behavior of RAD proteins, specifically RAD51C triggered by mutations G125V and L138F and RAD51D triggered by mutations S207L and E233G.

Heat Shock Proteins in Oxidative Stress and Ischemia/Reperfusion Injury and Benefits from Physical Exercises: A Review to the Current Knowledge.

Heat shock proteins (HSPs) are molecular chaperones produced in response to oxidative stress (OS). These proteins are involved in the folding of newly synthesized proteins and refolding of damaged or misfolded proteins. Recent studies have been focused on the regulatory role of HSPs in OS and ischemia/reperfusion injury (I/R) where reactive oxygen species (ROS) play a major role. ROS perform many functions, including cell signaling. Unfortunately, they are also the cause of pathological processes leading to various diseases. Biological pathways such as p38 MAPK, HSP70 and Akt/GSK-3β/eNOS, HSP70, JAK2/STAT3 or PI3K/Akt/HSP70, and HSF1/Nrf2-Keap1 are considered in the relationship between HSP and OS. New pathophysiological mechanisms involving ROS are being discovered and described the protein network of HSP interactions. Understanding of the mechanisms involved, e.g., in I/R, is important to the development of treatment methods. HSPs are multifunctional proteins because they closely interact with the antioxidant and the nitric oxide generation systems, such as HSP70/HSP90/NOS. A deficiency or excess of antioxidants modulates the activation of HSF and subsequent HSP biosynthesis. It is well known that HSPs are involved in the regulation of several redox processes and play an important role in protein-protein interactions. The latest research focuses on determining the role of HSPs in OS, their antioxidant activity, and the possibility of using HSPs in the treatment of I/R consequences. Physical exercises are important in patients with cardiovascular diseases, as they affect the expression of HSPs and the development of OS.

Structural Insights Into PfARO and Characterization of its Interaction With PfAIP

Apicomplexan parasites contain rhoptries, which are specialized secretory organelles that coordinate host cell invasion. During the process of invasion, rhoptries secrete their contents to facilitate interaction with, and entry into, the host cell. Here, we report the crystal structure of the rhoptry protein Armadillo Repeats-Only (ARO) from the human malaria parasite, Plasmodium falciparum (PfARO). The structure of PfARO comprises five tandem Armadillo-like (ARM) repeats, with adjacent ARM repeats stacked in a head-to-tail orientation resulting in PfARO adopting an elongated curved shape. Interestingly, the concave face of PfARO contains two distinct patches of highly conserved residues that appear to play an important role in protein-protein interaction. We functionally characterized the P. falciparum homolog of ARO interacting protein (PfAIP) and demonstrate that it localizes to the rhoptries. We show that conditional mislocalization of PfAIP leads to deficient red blood cell invasion. Guided by the structure, we identified mutations of PfARO that lead to mislocalization of PfAIP. Using proximity-based biotinylation we probe into PfAIP interacting proteins.

Studies of protein–protein interactions in Fanconi anemia pathway to unravel the DNA interstrand crosslink repair mechanism

Fanconi anemia (FA), a cancer predisposition syndrome exhibits hallmark feature of radial chromosome formation, and hypersensitivity to DNA crosslinking agents. A set of FA pathway proteins mainly FANCI, FANCD2 and BRCA2 are expressed to repair the covalent crosslink between the dsDNA. However, FA, BRCA pathways play an important role in DNA ICL repair as well as in homologous recombination repair, but the presumptive role of FA-BRCA proteins has not clearly explored particularly in context to function associated protein–protein interactions (PPIs). Here, in-vivo, in-vitro and in-silico studies have been performed for functionally relevant domains of FANCI, FANCD2 and BRCA2. To our conclusion, FANCI ARM repeat interacts with FANCD2 CUE domain and BRCA2 C-terminal region. Interestingly, FANCD2 CUE domain also interacts strongly with BRCA2 C-terminal region. Interactions between BRCA2 CTR and functionally relevant mutations Ser222Ala (cell cycle checkpoint mutant) and Leu231Arg (DNA ICL repair mutant) present in FANCD2 CUE domain have been analysed. To our finding, these mutations abrogate the binding between FANCD2 CUE domain and BRCA2 CTR. Furthermore, (1) different domain of FANCI, FANCD2 and BRCA2 are playing important role in PPIs, (2) mutations cause the impairment in the PPIs which in turn may disrupt the DNA ICL repair mechanism.

Minimalistic Predictor of Protein Binding Energy: Contribution of Solvation Factor to Protein Binding

It has long been known that solvation plays an important role in protein-protein interactions. Here, we use a minimalistic solvation-based model for predicting protein binding energy to estimate quantitatively the contribution of the solvation factor in protein binding. The factor is described by a simple linear combination of buried surface areas according to amino-acid types. Even without structural optimization, our minimalistic model demonstrates a predictive power comparable to more complex methods, making the proposed approach the basis for high throughput applications. Application of the model to a proteomic database shows that receptor-substrate complexes involved in signaling have lower affinities than enzyme-inhibitor and antibody-antigen complexes, and they differ by chemical compositions on interfaces. Also, we found that protein complexes with components that come from the same genes generally have lower affinities than complexes formed by proteins from different genes, but in this case the difference originates from different interface areas. The model was implemented in the software PYTHON, and the source code can be found on the Shakhnovich group webpage: http://faculty.chemistry.harvard.edu/shakhnovich/software.

A potential role of UBC28 interacting RING finger protein TaRF1 in spike development of wheat

RING finger proteins are the most abundant proteins in plants and may be essential for diverse aspects of cellular regulation in plant growth and development. Many RING finger proteins are E3 ubiquitin ligases, which play important roles in protein-protein interactions and ubiquitin-dependent protein degradation. The TaRF1 gene encodes a novel RING finger protein. In this study, we characterized the wheat (Triticum aestivum) RING finger domain as a hexapoid wheat ubiquitin ligase. To study the role of TaRF1 in wheat, we isolated TaRF1 from wheat spike cDNA. TaRF1 was 756 bp and encoded a putative 251-amino-acid with a predicted molecular mass of 28.57 kDa and isoelectric point of 5.75. A typical C3HC4-type RING finger domain was found at the C-terminal region of the TaRF1 protein. TaRF1 expression was investigated in the developmental stages and under various stresses by using reverse transcription polymerase chain reaction and was confirmed by subcellular localization of TaRF1 labeled with green fluorescent protein. Using the yeast 2-hybrid screen, we identified potential TaRF1-interacting proteins in a wheat spike library. Among the 8 clones that were identified as potential interacting partners of TaRF1 using yeast 2-hybrid screening, we found the strongest interaction between TaRF1 and the ubiquitin E2 enzyme TaUBC28 in tobacco leaves through biomolecular fluorescence complementation. The selectivity of interactions between E2 enzymes and RING E3 ligases represents a central and crucial part of the ubiquitin-conjugation pathways in organisms. These results indicate that the TaRF1 protein can interact with TaUBC28 in vitro and in vivo.

Involvement of the catalytically important Asp54 residue ofMycobacterium smegmatisDevR in protein-protein interactions between DevR and DevS

The DevSR two-component system in Mycobacterium smegmatis consists of the DevS histidine kinase and the DevR response regulator. It is a regulatory system that is involved in the adaptation of mycobacteria to hypoxic and NO stresses. Using the yeast two-hybrid assay and pull-down assay, it was demonstrated that the phosphoaccepting Asp (Asp54) of DevR is important for protein-protein interactions between DevR and DevS. The negative charge of Asp54 of DevR was shown to play an important role in protein-protein interactions between DevR and DevS. When the Lys104 residue, which is involved in transmission of conformational changes induced by phosphorylation of the response regulator, was replaced with Ala, the mutant form of DevR was not phosphorylated by DevS and functionally inactive in vivo. However, the K104A mutation in DevR only slightly affected protein-protein interactions between DevR and DevS.

Automated computational framework for the analysis of electrostatic similarities of proteins

Charge plays an important role in protein-protein interactions. In the case of excessively charged proteins, their electrostatic potentials contribute to the processes of recognition and binding with other proteins or ligands. We present an automated computational framework for determining the contribution of each charged amino acid to the electrostatic properties of proteins, at atomic resolution level. This framework involves computational alanine scans, calculation of Poisson-Boltzmann electrostatic potentials, calculation of electrostatic similarity distances (ESDs), hierarchical clustering analysis of ESDs, calculation of solvation free energies of association, and visualization of the spatial distributions of electrostatic potentials. The framework is useful to classify families of mutants with similar electrostatic properties and to compare them with the parent proteins in the complex. The alanine scan mutants introduce perturbations in the local electrostatic properties of the proteins and aim in delineating the contribution of each mutated amino acid in the spatial distribution of electrostatic potential, and in biological function when electrostatics is a dominant contributing factor in protein-protein interactions. The framework can be used to design new proteins with tailored electrostatic properties, such as immune system regulators, inhibitors, and vaccines, and in guiding experimental studies. We present an example for the interaction of the immune system protein C3d (the d-fragment of complement protein C3) with its receptor CR2, and we discuss our data in view of a binding site controversy.

Requirement of Calcium Binding, Myristoylation, and Protein-Protein Interaction for the Copine BON1 Function in Arabidopsis

Copines are highly conserved proteins with lipid-binding activities found in animals, plants, and protists. They contain two calcium-dependent phospholipid binding C2 domains at the amino terminus and a VWA domain at the carboxyl terminus. The biological roles of most copines are not understood and the biochemical properties required for their functions are largely unknown. The Arabidopsis copine gene BON1/CPN1 is a negative regulator of cell death and defense responses. Here we probed the potential biochemical activities of BON1 through mutagenic studies. We found that mutations of aspartates in the C2 domains did not alter plasma membrane localization but compromised BON1 activity. Mutation at putative myristoylation residue glycine 2 altered plasma membrane localization of BON1 and rendered BON1 inactive. Mass spectrometry analysis of BON1 further suggests that the N-peptide of BON1 is modified. Furthermore, mutations that affect the interaction between BON1 and its functional partner BAP1 abolished BON1 function. This analysis reveals an unanticipated regulation of copine protein localization and function by calcium and lipid modification and suggests an important role in protein-protein interaction for the VWA domain of copines.

Molecular characterization and expression of the MYND-ZF gene from Clonorchis sinensis

The MYND-type zinc finger protein (MYND-ZF) is a large group of proteins containing the MYND domain which play an important role in protein-protein interactions. A cDNA clone encoding a novel MYND-ZF was isolated and identified from a Clonorchis sinensis (C. sinensis) adult cDNA library. The open reading frame of this novel cDNA sequence contains 1,440 base pairs with a putative protein of 479 amino acids showing a high homology with the MYND-ZF identified from other species. Recombinant CsMYND-ZF was expressed and purified from Escherichia coli BL21 (DE3). CsMYND-ZF transcripts were detected in the cDNA of adult worms and metacercariae but not in eggs of C. sinensis. Immunohistochemistry results revealed that CsMYND-ZF was deposited at the tegument of adult worms and metacercariae C. sinensis using anti-recombinant CsMYND-ZF serum. These findings may contribute to the development of a reliable diagnostic method.

Unique nucleotide polymorphism of ankyrin gene cluster in Arabidopsis

The ankyrin (ANK) gene cluster is a part of a multigene family encoding ANK transmembrane proteins in Arabidopsis thaliana, and plays an important role in protein-protein interactions and in signal pathways. In contrast to other regions of a genome, the ANK gene cluster exhibits an extremely high level of DNA polymorphism in an approximately 5-kb region, without apparent decay. Phylogenetic analysis detects two clear, deeply differentiated haplotypes (dimorphism). The divergence between haplotypes of accession Col-0 and Ler-0 (Hap-C and Hap-L) is estimated to be 10.7%, approximately equal to the 10.5% average divergence between A. thaliana and A. lyrata. Sequence comparisons for the ANK gene cluster homologues in Col-0 indicate that the members evolve independently, and that the similarity among paralogues is lower than between alleles. Very little intralocus recombination or gene conversion is detected in ANK regions. All these characteristics of the ANK gene cluster are consistent with a tandem gene duplication and birth-and-death process. The possible mechanisms for and implications of this elevated nucleotide variation are also discussed, including the suggestion of balancing selection.

Molecular and functional characterization of a novel splice variant of ANKHD1 that lacks the KH domain and its role in cell survival and apoptosisc

Multiple ankyrin repeat motif-containing proteins play an important role in protein-protein interactions. ANKHD1 proteins are known to possess multiple ankyrin repeat domains and a single KH domain with no known function. Using yeast two-hybrid system analysis, we identified a novel splice variant of ANKHD1. This splice variant of ANKHD1, which we designated as HIV-1 Vpr-binding ankyrin repeat protein (VBARP), does not contain the signature KH domain, and codes for only a single ankyrin repeat motif. We characterized VBARP by molecular and functional analysis, revealing that VBARP is ubiquitously expressed in different tissues as well as cell lines of different lineage. In addition, blast searches indicated that orthologs and homologs to VBARP exist in different phyla, suggesting that VBARP might be evolutionarily conserved, and thus may be involved in basic cellular function(s). Furthermore, biochemical analysis revealed the presence of two VBARP isoforms coding for 69 and 49 kDa polypeptides, respectively, that are primarily localized in the cytoplasm. Functional analysis using short interfering RNA approaches indicate that this gene product is essential for cell survival through its regulation of caspases. Taken together, these results indicate that VBARP is a novel splice variant of ANKHD1 and may play a role in cellular apoptosis (antiapoptotic) and cell survival pathway(s).

Apolipoprotein L6, a Novel Proapoptotic Bcl-2 Homology 3–Only Protein, Induces Mitochondria-Mediated Apoptosis in Cancer Cells

Cancer cells frequently possess defects in the genetic and biochemical pathways of apoptosis. Members of the Bcl-2 family play pivotal roles in regulating apoptosis and possess at least one of four Bcl-2 homology (BH) domains, designated BH1 to BH4. The BH3 domain is the only one conserved in proapoptotic BH3-only proteins and plays an important role in protein-protein interactions in apoptosis by regulating homodimerization and heterodimerization of the Bcl-2 family members. To date, 10 BH3-only proapoptotic proteins have been identified and characterized in the human genome. The completion of the Human Genome Project and the availability of various public databases and sequence analysis algorithms allowed us to use the bioinformatic database-mining approach to identify one novel BH3-only protein, apolipoprotein L6 (ApoL6). The full-length cDNA of ApoL6 was identified, cloned, and functionally expressed in p53-null colorectal cancer cells (DLD-1). We found that overexpression of wild-type ApoL6 induced mitochondria-mediated apoptosis in DLD-1 cells characterized by release of cytochrome c and Smac/DIABLO from mitochondria and activation of caspase-9, whereas ApoL6 BH3 domain deletion allele did not. In addition, overexpression of ApoL6 also induced activation of caspase-8. Furthermore, we showed that adenovirus harboring the full-length cDNA of ApoL6 induced marked apoptosis in a variety of cancer cell types, and ApoL6 recruited and interacted with lipid/fatty acid components during the induction of apoptosis. To our knowledge, this is the first example that intracellular overproduction of an apolipoprotein induces marked apoptosis.

The Biology and Enzymology of Protein Tyrosine O-Sulfation

The Biology and Enzymology of Protein Tyrosine O-Sulfation

Ubiquitin superfolds: intrinsic and attachable regulators of cellular activities?

Ubiquitinylation, the post-translational covalent conjugation of ubiquitin to other proteins, mediates diverse cellular processes in addition to the proteasome-catalysed degradation signalled by multiple ubiquitinylation. Ubiquitin superfolds have also been found in other proteins. The amino acid sequences of these superfolds are unrelated to ubiquitin, but they have an almost identical three-dimensional shape to that of ubiquitin. Additionally, a number of 'ubiquitin-like' proteins, some of which can be conjugated to other proteins, may also contain the ubiquitin superfold. Intrinsic and attachable ubiquitin superfolds can act as powerful ligands and probably have important roles in protein-protein interactions in the cell.

Equilibrium and kinetic binding analysis of the N-terminal domain of the Pf1 gene 5 protein and its interaction with single-stranded DNA

The Pf1 gene 5 protein is a single-stranded DNA-binding protein that binds cooperatively to the viral strand of Pf1 DNA during replication. A variety of N-terminal fragments of the Pf1 gene 5 protein have been expressed and purified. We have identified an N-terminal single-stranded DNA-binding domain (residues 1 to 105) that is much more globular than the intact protein (1 to 144). Larger fragments (1 to 115) as well as smaller fragments (1 to 91) were unable to bind DNA effectively. Analysis of the truncated proteins by gel retardation and fluorescence anisotropy indicates that the N-terminal domain binds DNA with a reduced affinity, due principally to a reduction in cooperativity, and that binding is highly concentration-dependent. Kinetic analysis shows that the rates of association and dissociation of the N-terminal domain from the complex with DNA are faster than those observed for the intact protein. The results suggest that the flexible C-terminal domain of the Pf1 gene 5 protein plays an important role in protein-protein interactions that stabilise adjacent protein dimers in the DNA-protein complex.

Characterization of mechanisms of interleukin-6 gene repression by estrogen receptor.

Estrogens are the most effective agents available for preventing osteoporosis, and their principal role in bone metabolism is the inhibition of interleukin-6 (IL-6) production in osteoblasts and bone marrow stromal cells. We examined the mechanism of inhibitory effect of estrogens on the 190 bp proximal promoter of the IL-6 gene. Promoter activity induced by transfection of both NF-kappaB p65 subunit and NF-IL6 was decreased by 45% by estradiol (E2)-estrogen receptor (ER) complexes. The inhibitory effect of E2 was also observed on a mutant IL-6 promoter in which the NF-IL6 binding site was disrupted. E2 repressed the wild-type promoter activity induced by NF-kappaB p65 subunit alone, but had no effect on that induced by NF-IL6 alone. These findings suggested that estrogens inhibit IL-6 production by interfering with the function of NF-kappaB rather than that of NF-IL6. The ER mutant, HE19, which does not contain the A/B domain, repressed the induction by NF-kappaB to the same extent as wild-type ER HE0, whereas the effect of C-terminal deletion mutant, HE21, was only marginal. The antiestrogen, 4-hydroxytamoxifen (OHT), had no effect on IL-6 promoter activity, suggesting that E2-induced conformational change of the hormone binding domain plays an important role in protein-protein interaction between ER and NF-kappaB. E2 had no effect on the nuclear translocation of NF-kappaB, and electrophoretic mobility shift assay showed that the presence of E2-ER complexes did not affect the ability of NF-kappaB to bind to specific DNA sequences.

Interactions between the Cytosolic Components p47phox and p67phox of the Human Neutrophil NADPH Oxidase That Are Not Required for Activation in the Cell-free System

Activation of the human NADPH oxidase requires the interaction of at least four cytosolic proteins and one membrane-bound heterodimeric protein. Src homology 3 (SH3) domains and their proline-rich counterstructures have been shown to play an important role in protein-protein interactions. Because it was found that the cytosolic oxidase components p67phox, p47phox, and p40phox reside in a complex in resting neutrophils, we studied the role of SH3 domains in their interaction by use of an overlay technique. Wild-type and mutated 35S-labeled p67phox and p47phox were used to detect immobilized cytosolic proteins on a protein blot. A specific association of native p67phox to blotted p47phox and blotted p40phox was found. These interactions were not disturbed by deleting the only proline-rich region (amino acids 227-231) in p67phox. We also found a specific association of native p47phox with blotted p67phox. Deletions in a putative SH3-binding region of p47phox completely abrogated the interaction with p67phox. Other results suggest that the C terminus of p47phox exposes this SH3-binding domain for interaction with p67phox. Similar results were obtained when the binding of cytosolic p67phox to wild-type or mutated p47phox were studied in solution. Interestingly, mutants of p47phox unable to bind to p67phox were fully capable of supporting superoxide production under cell-free activation conditions. We conclude that an interaction between the C-terminal proline-rich region of p47phox and the second SH3 domain of p67phox is not required for oxidase activity in the cell-free assay.

NF-kappa B and related proteins: Rel/dorsal homologies meet ankyrin-like repeats.

Molecular cloning of the subunits of the transcription factor NF-kappa B and its inhibitor l kappa B revealed regions of sequence homology with two different classes of proteins: the Rel/dorsal family and a heterogeneous group of proteins containing ankyrin-like repeats. Both the Rel/dorsal homology domain and the ankyrin-like repeats appear to play important roles in protein-protein interactions that regulate localization and activity of the NF-kappa B subunits.

Phosphorylation of tyrosine prevents dityrosine formation in vitro

Treatment of L-tyrosine in a peroxidase/H 2O 2, system results in the formation of dityrosine. However, the phosphoester derivative of tyrosine, O-phospho-L-tyrosine, was unable to form dityrosine even in mixtures with free L-tyrosine. Dephosphorylation of O-phospho-L-tyrosine by alkaline phosphatase followed by horseradish peroxidase/H 2O 2, treatment resulted in the formation of dityrosine. Our in vitro results indicate that phosphorylation/dephosphorylation of L-tyrosine may regulate dityrosine formation, and is supposed to play an important role in protein-protein interactions, i.e. cross-linking.