Characterization of Protein–Protein Interfaces

We analyze the characteristics of protein–protein interfaces using the largest datasets available from the Protein Data Bank (PDB). We start with a comparison of interfaces with protein cores and non-interface surfaces. The results show that interfaces differ from protein cores and non-interface surfaces in residue composition, sequence entropy, and secondary structure. Since interfaces, protein cores, and non-interface surfaces have different solvent accessibilities, it is important to investigate whether the observed differences are due to the differences in solvent accessibility or differences in functionality. We separate out the effect of solvent accessibility by comparing interfaces with a set of residues having the same solvent accessibility as the interfaces. This strategy reveals residue distribution propensities that are not observable by comparing interfaces with protein cores and non-interface surfaces. Our conclusions are that there are larger numbers of hydrophobic residues, particularly aromatic residues, in interfaces, and the interactions apparently favored in interfaces include the opposite charge pairs and hydrophobic pairs. Surprisingly, Pro-Trp pairs are over represented in interfaces, presumably because of favorable geometries. The analysis is repeated using three datasets having different constraints on sequence similarity and structure quality. Consistent results are obtained across these datasets. We have also investigated separately the characteristics of heteromeric interfaces and homomeric interfaces.     

Computational Prediction of Protein–Protein Interactions

Recently a number of computational approaches have been developed for the prediction of protein–protein interactions. Complete genome sequencing projects have provided the vast amount of information needed for these analyses. These methods utilize the structural, genomic, and biological context of proteins and genes in complete genomes to predict protein interaction networks and functional linkages between proteins. Given that experimental techniques remain expensive, time-consuming, and labor-intensive, these methods represent an important advance in proteomics. Some of these approaches utilize sequence data alone to predict interactions, while others combine multiple computational and experimental datasets to accurately build protein interaction maps for complete genomes. These methods represent a complementary approach to current high-throughput projects whose aim is to delineate protein interaction maps in complete genomes. We will describe a number of computational protocols for protein interaction prediction based on the structural, genomic, and biological context of proteins in complete genomes, and detail methods for protein interaction network visualization and analysis.     

Antigenic Analysis of Monoclonal Antibodies against Different Epitopes of σB Protein of Avian Reovirus

Background

Avian reovirus (ARV) causes arthritis, tenosynovitis, runting-stunting syndrome (RSS), malabsorption syndrome (MAS) and immunosuppression in chickens. σB is one of the major structural proteins of ARV, which is able to induce group-specific antibodies against the virus.

Methods and Results

The present study described the identification of two linear B-cell epitopes in ARV σB through expressing a set of partially overlapping and consecutive truncated peptides spanning σB screened with two monoclonal antibodies (mAbs) 1F4 and 1H3-1.The data indicated that ²¹KTPACW²⁶ (epitope A) and ³²WDTVTFH³⁸ (epitope B) were minimal determinants of the linear B cell epitopes. Antibodies present in the serum of ARV-positive chickens recognized the minimal linear epitopes in Western blot analyses. By sequence alignment analysis, we determined that the epitopes A and B were not conserved among ARV, duck reovirus (DRV) and turkey reovirus (TRV) strains. Western blot assays, confirmed that epitopes A and B were ARV-specific epitopes, and they could not react with the corresponding peptides of DRV and TRV.

Conclusions and Significance

We identified ²¹KTPACW²⁶ and ³²WDTVTFH³⁸ as σB -specific epitopes recognized by mAbs 1F4 and 1H3-1, respectively. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against ARV groups.     

Protein kinase C-dependent dephosphorylation of tyrosine hydroxylase requires the B56δ heterotrimeric form of protein phosphatase 2A

Tyrosine hydroxylase, which plays a critical role in regulation of dopamine synthesis, is known to be controlled by phosphorylation at several critical sites. One of these sites, Ser40, is phosphorylated by a number of protein kinases, including protein kinase A. The major protein phosphatase that dephosphorylates Ser40 is protein phosphatase-2A (PP2A). A recent study has also linked protein kinase C to the dephosphorylation of Ser40 [1], but the mechanism is unclear. PP2A isoforms are comprised of catalytic, scaffold, and regulatory subunits, the regulatory B subunits being able to influence cellular localization and substrate selection. In the current study, we find that protein kinase C is able to phosphorylate a key regulatory site in the B56δ subunit leading to activation of PP2A. In turn, activation of the B56δ-containing heterotrimeric form of PP2A is responsible for enhanced dephosphorylation of Ser40 of tyrosine hydroylase in response to stimulation of PKC. In support of this mechanism, down-regulation of B56δ expression in N27 cells using RNAi was found to increase dopamine synthesis. Together these studies reveal molecular details of how protein kinase C is linked to reduced tyrosine hydroxylase activity via control of PP2A, and also add to the complexity of protein kinase/protein phosphatase interactions.     

Protein Kinase C�� Mediates Regulation of Proliferation by the Serotonin 5-Hydroxytryptamine Receptor 2B

Activation of the 5-hydroxytryptamine receptor 2B (5-HT_2B), a G_q/11 protein-coupled receptor, results in proliferation of various cell types. The 5-HT_2B receptor is also expressed on the pacemaker cells of the gastrointestinal tract, the interstitial cells of Cajal (ICC), where activation triggers ICC proliferation. The goal of this study was to characterize the mitogenic signal transduction cascade activated by the 5-HT_2B receptor. All of the experiments were performed on mouse small intestine primary cell cultures. Activation of the 5-HT_2B receptor by its agonist BW723C86 induced proliferation of ICC. Inhibition of phosphatidylinositol 3-kinase by {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 decreased base-line proliferation but had no effect on 5-HT_2B receptor-mediated proliferation. Proliferation of ICC through the 5-HT_2B receptor was inhibited by the phospholipase C inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 and by the inositol 1,4,5-trisphosphate receptor inhibitor Xestospongin C. Calphostin C, the α, β, γ, and μ protein kinase C (PKC) inhibitor Gö6976, and the α, β, γ, δ, and ζ PKC inhibitor Gö6983 inhibited 5-HT_2B receptor-mediated proliferation, indicating the involvement of PKC α, β, or γ. Of all the PKC isoforms blocked by Gö6976, PKCγ and μ mRNAs were found by single-cell PCR to be expressed in ICC. 5-HT_2B receptor activation in primary cell cultures obtained from PKCγ^−/− mice did not result in a proliferative response, further indicating the requirement for PKCγ in the proliferative response to 5-HT_2B receptor activation. The data demonstrate that the 5-HT_2B receptor-induced proliferative response of ICC is through phospholipase C, [Ca²⁺]_i, and PKCγ, implicating this PKC isoform in the regulation of cellular proliferation.Tight control of cell proliferation is essential to maintain organ size and function. Proliferation needs to be tightly regulated to maintain a critical mass of a particular cell type while preventing dysplasia or malignancy. Cell proliferation is regulated by a complex interaction between extrinsic and intrinsic factors. Extrinsic factors usually signal through cell surface receptors such as various growth factor receptors. 5-Hydroxytryptamine (5-HT,² serotonin) is well established as a neurotransmitter and a paracrine factor with over 90% of 5-HT produced by the gastrointestinal tract (1, 2). There is now substantial evidence that, together with these established functions, 5-HT is involved in the control of cell proliferation through various 5-HT receptors, in particular the 5-hydroxytryptamine receptor 2B (5-HT_2B (3–9)). The 5-HT_2B receptor is G_q/11 protein-coupled. Activation of the 5-HT_2B receptor regulates cardiac function, smooth muscle contractility, vascular physiology, and mood control. Recently it was demonstrated that activation of the 5-HT_2B receptor also induces proliferation of neurons, retinal cells (3, 4), hepatocytes (5), osteoblasts (8), and interstitial cells of Cajal (ICC) (9). ICC express the 5-HT_2B receptor, and activation by 5-HT induces proliferation of ICC (9). ICC are specialized, mesoderm-derived mesenchymal cells in the gastrointestinal tract. Their best known function is the generation of slow waves (10), but they also conduct and amplify neuronal signals (11, 12), release carbon monoxide to set the intestinal smooth muscle membrane potential gradient (13), and act as mechanosensors (14, 15). Loss of ICC has been associated with pathological conditions such as gastroparesis (16–18), infantile pyloric stenosis (19, 20), pseudo-obstruction (21, 22), and slow transit constipation (23), whereas increased proliferation of ICC or their precursors is associated with gastrointestinal stromal tumors (24).The mechanisms by which activation of the 5-HT_2B receptor results in increased proliferation are not well understood. In cultured cardiomyocytes, stimulation of the 5-HT_2B receptor activated both phosphatidylinositol 3-kinase (PI3′-K)/Akt and ERK1/2/mitogen-activated protein kinase (MAPK) signaling pathways to protect cardiomyocytes from apoptosis (25). On the other hand, the 5-HT2 subfamily of receptors are also known to couple to phospholipase C (PLC) (26–28).The objective of this study was to utilize the known expression of the 5-HT_2B receptor on ICC to determine whether proliferation through the 5-HT_2B receptor required PI3′-K or PLC. This study demonstrates that proliferation mediated by the 5-HT_2B receptor requires PLC, intracellular calcium release, and the ERK/MAPK signaling pathway and identifies the PKC isoform activated by the 5-HT_2B receptor in ICC as PKCγ.     

Rapid Production of Platelet-activating Factor Is Induced by Protein Kinase Cα-mediated Phosphorylation of Lysophosphatidylcholine Acyltransferase 2 Protein

Platelet-activating factor (PAF), a potent proinflammatory lipid mediator, is synthesized rapidly in response to extracellular stimuli by the activation of acetyl-CoA:lyso-PAF acetyltransferase (lyso-PAFAT). We have reported previously that lyso-PAFAT activity is enhanced in three distinct ways in mouse macrophages: rapid activation (30 s) after PAF stimulation and minutes to hours after LPS stimulation. Lysophosphatidylcholine acyltransferase 2 (LPCAT2) was later identified as a Ca²⁺-dependent lyso-PAFAT. However, the mechanism of rapid lyso-PAFAT activation within 30 s has not been elucidated. Here we show a new signaling pathway for rapid biosynthesis of PAF that is mediated by phosphorylation of LPCAT2 at Ser-34. Stimulation by either PAF or ATP resulted in PKCα-mediated phosphorylation of LPCAT2 to enhance lyso-PAFAT activity and rapid PAF production. Biochemical analyses showed that the phosphorylation of Ser-34 resulted in augmentation of V_max with minimal K_m change. Our results offer an answer for the previously unknown mechanism of rapid PAF production.     

Metabolic Control of Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII)-mediated Caspase-2 Suppression by the B55β/Protein Phosphatase 2A (PP2A)

High levels of metabolic activity confer resistance to apoptosis. Caspase-2, an apoptotic initiator, can be suppressed by high levels of nutrient flux through the pentose phosphate pathway. This metabolic control is exerted via inhibitory phosphorylation of the caspase-2 prodomain by activated Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). We show here that this activation of CaMKII depends, in part, on dephosphorylation of CaMKII at novel sites (Thr³⁹³/Ser³⁹⁵) and that this is mediated by metabolic activation of protein phosphatase 2A in complex with the B55β targeting subunit. This represents a novel locus of CaMKII control and also provides a mechanism contributing to metabolic control of apoptosis. These findings may have implications for metabolic control of the many CaMKII-controlled and protein phosphatase 2A-regulated physiological processes, because both enzymes appear to be responsive to alterations in glucose metabolized via the pentose phosphate pathway.     

Protein 4.1B in mouse islets of Langerhans and β-cell tumorigenesis

Protein 4.1 family proteins are thought to interact with membrane proteins and membrane skeletons. Immunohistochemical studies by light and electron microscopy were performed on mouse pancreas with a specific antibody against protein 4.1B. Specific protein 4.1B immunolabeling was observed on endocrine cells in the islets of Langerhans. Protein 4.1B localized along the plasma membranes facing adjacent cells. By immunoelectron microscopy, the immunolabeling of the cells was restricted to the cytoplasmic side just beneath their plasma membrane, including the membranes adjacent to neighboring cells, while the plasma membranes facing endothelial cells were not immunolabeled for protein 4.1B. The immunolocalization of E-cadherin was similar, if not identical, to that of protein 4.1B supporting the idea that protein 4.1B may be functionally interconnected with adhesion molecules. In a transgenic mouse model of pancreatic -cell carcinogenesis (Rip1Tag2), the loss of protein 4.1B expression coincided with the phenotypic transition from adenoma to carcinoma. Therefore, we propose a role of protein 4.1B as a connecting and/or signaling molecule between membrane architecture, cell adhesion, and tumor cell invasion in mouse pancreatic endocrine cells.     

Protein–ligand interaction-guided discovery of novel VEGFR-2 inhibitors

Abstract

As an effective target in abnormal angiogenesis-related tumor treatment, VEGFR-2 has small-molecule inhibitors of various scaffolds being approved for treating diseases such as renal carcinoma, non-small cell lung cancer, etc. However, endogenous and acquired drug resistance are still considered to be the main contributors for the failure of VEGFR-2 clinical candidates. Therefore, development of novel VEGFR-2 inhibitors is still urgently needed in the market but also challenging. In this work, residues including Asp1046, Ile1025, HIS1026, Cys919 and Lys868 were identified as the most important residues for Hbonded interaction, while His1026, Asp1046, Glu885, Ile1025 and Leu840 exhibited critical role for the nonbonded interactions through a comprehensive analysis of protein–ligand interactions, which plays critical roles in the binding of compounds and targets. Guided by the analysis of binding interactions, a total of 10 novel VEGFR-2 inhibitors based on N-methyl-4-oxo-N-propyl-1,4-dihydroquinoline-2-carboxamide scaffold were discovered through fragment-based drug design and structure-based virtual screening, which expands the chemical space of current VEGFR-2 inhibitors. Biological activity evaluation showed that even though the enzymatic activity of these compounds against VEGFR-2 were inferior to that of the positive controls sorafenib and motesanib, compound I-10 showed moderate HepG2 cell inhibitory activity with an IC₅₀ value of 33.65?μM and eight compounds exhibited moderate or higher HUVEC inhibitory activity in the range of 19.54–57.98?μM compared to the controls. Particularly, the HUVEC inhibitory activity of compound I-6 (IC₅₀ = 19.54?μM) outperformed motesanib and can be used as starting points for further optimization and development for cancer treatment.

Communicated by Ramaswamy H. Sarma     

Residues 240–250 in the C-Terminus of the Pirh2 Protein Complement the Function of the RING Domain in Self-Ubiquitination of the Pirh2 Protein

Pirh2 is a p53 inducible gene that encodes a RING-H2 domain and is proposed to be a main regulator of p53 protein, thus fine tuning the DNA damage response. Pirh2 interacts physically with p53 and promotes its MDM2-independent ubiquitination and subsequent degradation as well as participates in an auto-regulatory feedback loop that controls p53 function. Pirh2 also self-ubiquitinates. Interestingly, Pirh2 is overexpressed in a wide range of human tumors. In this study, we investigated the domains and residues essential for Pirh2 self-ubiquitination. Deletions were made in each of the three major domains of Pirh2: the N-terminal domain (NTD), Ring domain (RING), and C-terminal domain (CTD). The effects of these deletions on Pirh2 self-ubiquitination were then assessed using in vitro ubiquitination assays. Our results demonstrate that the RING domain is essential, but not sufficient, for Pirh2 self-ubiquitination and that residues 240–250 of the C-terminal domain are also essential. Our results demonstrate that Pirh2 mediated p53 polyubiquitination occurs mainly through the K48 residue of ubiquitin in vitro. Our data further our understanding of the mechanism of Pirh2 self-ubiquitination and may help identify valuable therapeutic targets that play roles in reducing the effects of the overexpression of Pirh2, thus maximizing p53''s response to DNA damage.     

Protein Phosphatase 2A Isoforms Utilizing Aβ Scaffolds Regulate Differentiation through Control of Akt Protein

Protein phosphatase 2A (PP2A) regulates almost all cell signaling pathways. It consists of a scaffolding A subunit to which a catalytic C subunit and one of many regulatory B subunits bind. Of the more than 80 PP2A isoforms, 10% use Aβ as a scaffold. This study demonstrates the isoform-specific function of the A scaffold subunits. Polyomaviruses have shown the importance of phosphotyrosine, PI3K, and p53 in transformation. Comparisons of polyoma and SV40 small T antigens implicate Aβ in the control of differentiation. Knockdown of Aβ enhanced differentiation. Akt signaling regulated differentiation; its activation or inhibition promoted or blocked it, respectively. Aβ bound Akt. Enhancement of PP2A Aβ/Akt interaction by polyoma small T antigen increased turnover of Akt Ser-473 phosphorylation. Conversely, knockdown of Aβ promoted Akt activity and reduced turnover of phosphate at Ser-473 of Akt. These data provide new insight into the regulation of Akt, a protein of extreme importance in cancer. Furthermore, our results suggest that the role for Aβ in differentiation and perhaps tumor suppression may lie partly in its ability to negatively regulate Akt.     

DC2 and Keratinocyte-associated Protein 2 (KCP2), Subunits of the Oligosaccharyltransferase Complex,Are Regulators of the γ-Secretase-directed Processing of Amyloid Precursor Protein (APP)

The oligosaccharyltransferase complex catalyzes the transfer of oligosaccharide from a dolichol pyrophosphate donor en bloc onto a free asparagine residue of a newly synthesized nascent chain during the translocation in the endoplasmic reticulum lumen. The role of the less known oligosaccharyltransferase (OST) subunits, DC2 and KCP2, recently identified still remains to be determined. Here, we have studied DC2 and KCP2, and we have established that DC2 and KCP2 are substrate-specific, affecting amyloid precursor protein (APP), indicating that they are not core components required for N-glycosylation and OST activity per se. We show for the first time that DC2 and KCP2 depletion affects APP processing, leading to an accumulation of C-terminal fragments, both C99 and C83, and a reduction in full-length mature APP. This reduction in mature APP levels was not due to a block in secretion because the levels of sAPPα secreted into the media were unaffected. We discover that DC2 and KCP2 depletion affects only the γ-secretase complex, resulting in a reduction of the PS1 active fragment blocking Aβ production. Conversely, we show that the overexpression of DC2 and KCP2 causes an increase in the active γ-secretase complex, particularly the N-terminal fragment of PS1 that is generated by endoproteolysis, leading to a stimulation of Aβ production upon overexpression of DC2 and KCP2. Our findings reveal that components of the OST complex for the first time can interact with the γ-secretase and affect the APP processing pathway.     

A Critical Assessment of Information-guided Protein–Protein Docking Predictions

The structures of protein complexes are increasingly predicted via protein–protein docking (PPD) using ambiguous interaction data to help guide the docking. These data often are incomplete and contain errors and therefore could lead to incorrect docking predictions. In this study, we performed a series of PPD simulations to examine the effects of incompletely and incorrectly assigned interface residues on the success rate of PPD predictions. The results for a widely used PPD benchmark dataset obtained using a new interface information-driven PPD (IPPD) method developed in this work showed that the success rate for an acceptable top-ranked model varied, depending on the information content used, from as high as 95% when contact relationships (though not contact distances) were known for all residues to 78% when only the interface/non-interface state of the residues was known. However, the success rates decreased rapidly to ∼40% when the interface/non-interface state of 20% of the residues was assigned incorrectly, and to less than 5% for a 40% incorrect assignment. Comparisons with results obtained by re-ranking a global search and with those reported for other data-guided PPD methods showed that, in general, IPPD performed better than re-ranking when the information used was more complete and more accurate, but worse when it was not, and that when using bioinformatics-predicted information on interface residues, IPPD and other data-guided PPD methods performed poorly, at a level similar to simulations with a 40% incorrect assignment. These results provide guidelines for using information about interface residues to improve PPD predictions and reveal a bottleneck for such improvement imposed by the low accuracy of current bioinformatic interface residue predictions.Proteins work in close association with other proteins to mediate the intricate functions of a cell. The atomic resolution of the structure of a protein complex can therefore help one understand a protein''s function in detail. Protein–protein docking (PPD),¹ a computational approach that complements experimental structure determinations, has attracted increasing research interest (1, 2), in part because it remains challenging to determine most structures of protein complexes via experimental techniques (3).To improve the performance of PPD predictions, experimentally derived data (e.g. distances) and information (e.g. the identity of interface residues) have been used either as a filter allowing less plausible docking solutions to be disregarded (4–9) or as a constraint to guide the docking process (10, 11). Various types of data and information have been used to aid PPD (12); these range from distances between, or the relative orientation of, the two interacting proteins to simple identification of the amino acid residues directly involved in the binding of the two proteins (13). Despite considerable success, the caveat for all these data-guided PPD predictions is that the data or information used must be correct in order to avoid spurious results caused by misguiding (12). It is therefore pertinent and important to evaluate the effects of errors in the incorporated data or information on the quality of PPD solutions.We have recently shown that the use of just a few distance constraints can improve the success rates of PPD such that they rival, or are even better than, those of a global search ranked using a sophisticated energy function, and that errors in the distance data significantly decrease the success rates of prediction (11). However, because distance data for interacting proteins are usually hard to obtain, other types of data or information, even if “ambiguous” (10), are increasingly used in PPD predictions (12, 14). In this study, we investigated the effects of incompletely and incorrectly assigned interface/non-interface residues, a major source of the so-called ambiguous data, on information-guided PPD predictions.As illustrated in Fig. 1, the information content of interface/non-interface residues can be rich enough to reveal the identity of every pair of residues in contact, but not their contact distances, or so poor as to reveal the interface/non-interface state of these residues but not their pairing relationship, for one or both of the two interacting proteins. To determine how these different levels of residue information content can help PPD predictions and the extent to which the use of incorrectly assigned residues degrades prediction success rates, we have developed a new interface information-driven PPD method (IPPD) and carried out a series of PPD simulations on a well-tested benchmark dataset. The results showed that when the information content was rich, excellent predictions (success rates for producing an acceptable top-ranked model > 70%) could be made via IPPD or by re-ranking a global search''s solutions using the same interface information, and that, encouragingly, the success of predictions remained respectable (top-ranked success rates > 15%) when the content was poor. However, when enough of the interface residues were incorrectly assigned, as would be the case when using interface residues predicted by a state-of-the-art bioinformatics method such as CPORT (15), few models ranked first by IPPD or other PPD methods, including HADDOCK (10), a popular ambiguous data-driven PPD method, came close to being acceptable. These results suggest that we can greatly increase the power of PPD predictions for practical applications only if the accuracy of current bioinformatics methods for predicting the interface residues of protein complexes can be significantly improved.Open in a separate windowFig. 1.Contact matrix of two interacting proteins, A and B, and the contact vectors of their residues. In the contact matrix, M_ij = 1 or 0, respectively, denotes contact or a lack of contact between residue i in protein A and residue j in protein B. In the contact vectors, V_Ai = 1 or 0, respectively, when residue Ai has, or does not have, at least one contact with any residue of protein B.     

The Role of the Phylogenetically Conserved Cochaperone Protein Droj2/DNAJA3 in NF-κB Signaling

The NF-κB pathway is a phylogenetically conserved signaling pathway with a central role in inflammatory and immune responses. Here we demonstrate that a cochaperone protein, Droj2/DNAJA3, is involved in the activation of canonical NF-κB signaling in flies and in human cultured cells. Overexpression of Droj2 induced the expression of an antimicrobial peptide in Drosophila. Conversely, Droj2 knockdown resulted in reduced expression of antimicrobial peptides and higher susceptibility to Gram-negative bacterial infection in flies. Similarly, Toll-like receptor-stimulated IκB phosphorylation and NF-κB activation were suppressed by DNAJA3 knockdown in HEK293 cells. IκB kinase overexpression-induced NF-κB phosphorylation was also compromised in DNAJA3 knockdown cells. Our study reveals a novel conserved regulator of the NF-κB pathway acting at the level of IκB phosphorylation.