Rad51–Rad52 Mediated Maintenance of Centromeric Chromatin in Candida albicans

Specification of the centromere location in most eukaryotes is not solely dependent on the DNA sequence. However, the non-genetic determinants of centromere identity are not clearly defined. While multiple mechanisms, individually or in concert, may specify centromeres epigenetically, most studies in this area are focused on a universal factor, a centromere-specific histone H3 variant CENP-A, often considered as the epigenetic determinant of centromere identity. In spite of variable timing of its loading at centromeres across species, a replication coupled early S phase deposition of CENP-A is found in most yeast centromeres. Centromeres are the earliest replicating chromosomal regions in a pathogenic budding yeast Candida albicans. Using a 2-dimensional agarose gel electrophoresis assay, we identify replication origins (ORI7-LI and ORI7-RI) proximal to an early replicating centromere (CEN7) in C. albicans. We show that the replication forks stall at CEN7 in a kinetochore dependent manner and fork stalling is reduced in the absence of the homologous recombination (HR) proteins Rad51 and Rad52. Deletion of ORI7-RI causes a significant reduction in the stalled fork signal and an increased loss rate of the altered chromosome 7. The HR proteins, Rad51 and Rad52, have been shown to play a role in fork restart. Confocal microscopy shows declustered kinetochores in rad51 and rad52 mutants, which are evidence of kinetochore disintegrity. CENP-A^CaCse4 levels at centromeres, as determined by chromatin immunoprecipitation (ChIP) experiments, are reduced in absence of Rad51/Rad52 resulting in disruption of the kinetochore structure. Moreover, western blot analysis reveals that delocalized CENP-A molecules in HR mutants degrade in a similar fashion as in other kinetochore mutants described before. Finally, co-immunoprecipitation assays indicate that Rad51 and Rad52 physically interact with CENP-A^CaCse4 in vivo. Thus, the HR proteins Rad51 and Rad52 epigenetically maintain centromere functioning by regulating CENP-A^CaCse4 levels at the programmed stall sites of early replicating centromeres.     

Absorption of Naphthalene by Leaves and Its Interaction with Chlorophyll–Protein Complexes of Pea Plants

Russian Journal of Plant Physiology - Mechanisms of photosynthesis inhibition by vaporous naphthalene, its permeation into thylakoids, and interactions with chlorophyll–protein complexes were...     

What Forces Can Determine the Formation of Highly Specific Protein–Protein Complexes?

A software package was designed and used in a detailed study of the contact regions (interfaces) of a large number of protein–protein complexes using the PDB data. It appeared that for about 75% of the complexes the amino acid composition of the subunit surface in the contact region is not essential. Thus one may suggest that, along with the amino acid residues at the interface, the residues in the interior of the globules substantially contribute to protein–protein recognition. Such interactions between quite remote residues are most probably of electrical nature, and are involved in recognition by contributing to the overall electric field created by the protein molecule; the configuration of this field is perhaps the definitive factor of recognition. The overall field of the protein molecule is additively built of the fields created by each constituent residue, and it can be calculated as a sum of the fields created by the protein multipole (aggregate of partial electric charges assigned to every atom of the protein molecule). Preliminary assessment of the remote electrostatic interaction has been performed for ribonuclease subunits in vacuum. The results are indicative of a real possibility that the electric field created by the protein multipole can strongly influence the mutual orientation of molecules before Brownian collision.     

Antiparallel Conformation of Knob and Hole Aglycosylated Half-Antibody Homodimers Is Mediated by a CH2–CH3 Hydrophobic Interaction

Bispecific antibody and antibody-like molecules are of wide interest as potential therapeutics that can recognize two distinct targets. Among the variety of ways such molecules have been engineered is by creating “knob” and “hole” heterodimerization sites in the CH3 domains of two antibody heavy chains. The molecules produced in this manner maintain their biological activities while differing very little from the native human IgG sequence. To better understand the knob-into-hole interface, the molecular mechanism of heterodimerization, and to engineer Fc domains that could improve the assembly and purity of heterodimeric reaction products, we sought crystal structures of aglycosylated heterodimeric and homodimeric “knob” and “hole” Fc fragments derived from bacterial expression. The structure of the knob-into-hole Fc was determined at 2.64 Å. Except for the sites of mutation, the structure is very similar to that of the native human IgG1 Fc, consistent with a heterodimer interaction kinetic K_D of < 1 nM. Homodimers of the “knob” and “hole” mutants were also obtained, and their X-ray structures were determined at resolutions 2.5 Å and 2.1 Å, respectively. Both kinds of homodimers adopt a head-to-tail quaternary structure and thus do not contain direct knob/knob or hole/hole CH3 interactions. The head-to-tail arrangement was disfavored by adding site-directed mutations at F241 and F243 in the CH2 domains, leading to increases in both rate and efficiency of bispecific (heterodimer) assembly.     

Vascular Smooth Muscle Cell Motility Is Mediated by a Physical and Functional Interaction of Ca2+/Calmodulin-dependent Protein Kinase IIδ2 and Fyn

In vascular smooth muscle (VSM) cells, Ca²⁺/calmodulin-dependent protein kinase IIδ₂ (CaMKIIδ₂) activates non-receptor tyrosine kinases and EGF receptor, with a Src family kinase as a required intermediate. siRNA-mediated suppression of Fyn, a Src family kinase, inhibited VSM cell motility. Simultaneous suppression of both Fyn and CaMKIIδ₂ was non-additive, suggesting coordinated regulation of cell motility. Confocal immunofluorescence microscopy indicated that CaMKIIδ₂ and Fyn selectively (compared with Src) co-localized with the Golgi in quiescent cultured VSM cells. Stimulation with PDGF resulted in a rapid (<5 min) partial redistribution and co-localization of both kinases in peripheral membrane regions. Furthermore, CaMKIIδ₂ and Fyn selectively (compared with Src) co-immunoprecipitated, suggesting a physical interaction in a signaling complex. Stimulation of VSM cells with ionomycin, a calcium ionophore, resulted in activation of CaMKIIδ₂ and Fyn and disruption of the complex. Pretreatment with KN-93, a pharmacological inhibitor of CaMKII, prevented activation-dependent disruption of CaMKIIδ₂ and Fyn, implicating CaMKIIδ₂ as an upstream mediator of Fyn. Overexpression of constitutively active CaMKII resulted in the dephosphorylation of Fyn at Tyr-527, which is required for Fyn activation. Taken together, these data demonstrate a dynamic interaction between CaMKIIδ₂ and Fyn in VSM cells and indicate a mechanism by which CaMKIIδ₂ and Fyn may coordinately regulate VSM cell motility.     

Keep Your Fingers Off My DNA: Protein–Protein Interactions Mediated by C2H2 Zinc Finger Domains

Cys2-His2 (C2H2) zinc finger domains (ZFs) were originally identified as DNA-binding domains, and uncharacterized domains are typically assumed to function in DNA binding. However, a growing body of evidence suggests an important and widespread role for these domains in protein binding. There are even examples of zinc fingers that support both DNA and protein interactions, which can be found in well-known DNA-binding proteins such as Sp1, Zif268, and Ying Yang 1 (YY1). C2H2 protein–protein interactions (PPIs) are proving to be more abundant than previously appreciated, more plastic than their DNA-binding counterparts, and more variable and complex in their interactions surfaces. Here we review the current knowledge of over 100 C2H2 zinc finger-mediated PPIs, focusing on what is known about the binding surface, contributions of individual fingers to the interaction, and function. An accurate understanding of zinc finger biology will likely require greater insights into the potential protein interaction capabilities of C2H2 ZFs.     

LRP-1 Mediated Endocytosis of EFE Across the Blood–Brain Barrier; Protein–Protein Interaction and Molecular Dynamics Analysis

International Journal of Peptide Research and Therapeutics - Additional biological protection; blood brain barrier (BBB) to neuronal tissue is essential against invading infections and unwanted...     

Computational Assessment of Protein–protein Binding Affinity by Reversely Engineering the Energetics in Protein Complexes

The cellular functions of proteins are maintained by forming diverse complexes. The stability of these complexes is quantified by the measurement of binding affinity, and mutations that alter the binding affinity can cause various diseases such as cancer and diabetes. As a result, accurate estimation of the binding stability and the effects of mutations on changes of binding affinity is a crucial step to understanding the biological functions of proteins and their dysfunctional consequences. It has been hypothesized that the stability of a protein complex is dependent not only on the residues at its binding interface by pairwise interactions but also on all other remaining residues that do not appear at the binding interface. Here, we computationally reconstruct the binding affinity by decomposing it into the contributions of interfacial residues and other non-interfacial residues in a protein complex. We further assume that the contributions of both interfacial and non-interfacial residues to the binding affinity depend on their local structural environments such as solvent-accessible surfaces and secondary structural types. The weights of all corresponding parameters are optimized by Monte-Carlo simulations. After cross-validation against a large-scale dataset, we show that the model not only shows a strong correlation between the absolute values of the experimental and calculated binding affinities, but can also be an effective approach to predict the relative changes of binding affinity from mutations. Moreover, we have found that the optimized weights of many parameters can capture the first-principle chemical and physical features of molecular recognition, therefore reversely engineering the energetics of protein complexes. These results suggest that our method can serve as a useful addition to current computational approaches for predicting binding affinity and understanding the molecular mechanism of protein–protein interactions.     

Prediction of Protein–Protein Interaction Sites in Sequences and 3D Structures by Random Forests

Identifying interaction sites in proteins provides important clues to the function of a protein and is becoming increasingly relevant in topics such as systems biology and drug discovery. Although there are numerous papers on the prediction of interaction sites using information derived from structure, there are only a few case reports on the prediction of interaction residues based solely on protein sequence. Here, a sliding window approach is combined with the Random Forests method to predict protein interaction sites using (i) a combination of sequence- and structure-derived parameters and (ii) sequence information alone. For sequence-based prediction we achieved a precision of 84% with a 26% recall and an F-measure of 40%. When combined with structural information, the prediction performance increases to a precision of 76% and a recall of 38% with an F-measure of 51%. We also present an attempt to rationalize the sliding window size and demonstrate that a nine-residue window is the most suitable for predictor construction. Finally, we demonstrate the applicability of our prediction methods by modeling the Ras–Raf complex using predicted interaction sites as target binding interfaces. Our results suggest that it is possible to predict protein interaction sites with quite a high accuracy using only sequence information.     

DNA Replication Checkpoint Signaling Depends on a Rad53–Dbf4 N-Terminal Interaction in Saccharomyces cerevisiae

Dbf4-dependent kinase (DDK) and cyclin-dependent kinase (CDK) are essential to initiate DNA replication at individual origins. During replication stress, the S-phase checkpoint inhibits the DDK- and CDK-dependent activation of late replication origins. Rad53 kinase is a central effector of the replication checkpoint and both binds to and phosphorylates Dbf4 to prevent late-origin firing. The molecular basis for the Rad53–Dbf4 physical interaction is not clear but occurs through the Dbf4 N terminus. Here we found that both Rad53 FHA1 and FHA2 domains, which specifically recognize phospho-threonine (pT), interacted with Dbf4 through an N-terminal sequence and an adjacent BRCT domain. Purified Rad53 FHA1 domain (but not FHA2) bound to a pT Dbf4 peptide in vitro, suggesting a possible phospho-threonine-dependent interaction between FHA1 and Dbf4. The Dbf4–Rad53 interaction is governed by multiple contacts that are separable from the Cdc5- and Msa1-binding sites in the Dbf4 N terminus. Importantly, abrogation of the Rad53–Dbf4 physical interaction blocked Dbf4 phosphorylation and allowed late-origin firing during replication checkpoint activation. This indicated that Rad53 must stably bind to Dbf4 to regulate its activity.     

Association of the Rad9–Rad1–Hus1 checkpoint clamp with MYH DNA glycosylase and DNA

Cell cycle checkpoints provide surveillance mechanisms to activate the DNA damage response, thus preserving genomic integrity. The heterotrimeric Rad9–Rad1–Hus1 (9–1–1) clamp is a DNA damage response sensor and can be loaded onto DNA. 9–1–1 is involved in base excision repair (BER) by interacting with nearly every enzyme in BER. Here, we show that individual 9–1–1 components play distinct roles in BER directed by MYH DNA glycosylase. Analyses of Hus1 deletion mutants revealed that the interdomain connecting loop (residues 134–155) is a key determinant of MYH binding. Both the N-(residues 1–146) and C-terminal (residues 147–280) halves of Hus1, which share structural similarity, can interact with and stimulate MYH. The Hus1^K136A mutant retains physical interaction with MYH but cannot stimulate MYH glycosylase activity. The N-terminal domain, but not the C-terminal half of Hus1 can also bind DNA with moderate affinity. Intact Rad9 expressed in bacteria binds to and stimulates MYH weakly. However, Rad9¹⁻²⁶⁶ (C-terminal truncated Rad9) can stimulate MYH activity and bind DNA with high affinity, close to that displayed by heterotrimeric 9¹⁻²⁶⁶–1–1 complexes. Conversely, Rad1 has minimal roles in stimulating MYH activity or binding to DNA. Finally, we show that preferential recruitment of 9¹⁻²⁶⁶–1–1 to 5′-recessed DNA substrates is an intrinsic property of this complex and is dependent on complex formation. Together, our findings provide a mechanistic rationale for unique contributions by individual 9–1–1 subunits to MYH-directed BER based on subunit asymmetry in protein–protein interactions and DNA binding events.     

Peptide, Peptidomimetic, and Small-molecule Antagonists of the p53–HDM2 Protein–Protein Interaction

Modulation of intracellular protein–protein interactions has been – and remains – a challenging goal for the discovery and development of small-molecule therapeutic agents. Progress in the pharmacological targeting and understanding at the molecular level of one such interaction that is relevant to cancer drug research, viz. that between the tumour suppressor protein p53 and its negative regulator HDM2, is reviewed here. The first X-ray crystal structure of a complex between a small peptide from the trans-activation domain of p53 and the N-terminal domain of HDM2 was reported almost 10 years ago. The nature of this interaction, which involves just three residue side chains in the p53 peptide ligand and a compact hydrophobic binding pocket in the HDM2 receptor, together with the attractive concept of reactivating the anti-proliferative functions of p53 in tumour cells, has spurned a great deal of effort aimed at finding drug-like antagonists of this interaction. A variety of approaches, including both structure-guided peptidomimetic and de novo design, as well as high through-put screening campaigns, have provided a wealth of leads that might be turned into actual drugs. There is still some way to go as far as optimisation and preclinical development of such leads is concerned, but it is clear already now that antagonists of the p53–HDM2 protein–protein interaction have a good chance of ultimately being successful in providing a new anti-cancer therapy modality, both in monotherapy and to potentiate the effectiveness of existing chemotherapies.     

Computational Prediction of Protein–Protein Interaction Networks: Algo-rithms and Resources

Protein interactions play an important role in the discovery of protein functions and pathways in biological processes. This is especially true in case of the diseases caused by the loss of specific protein-protein interactions in the organism. The accuracy of experimental results in finding protein-protein interactions, however, is rather dubious and high throughput experimental results have shown both high false positive beside false negative information for protein interaction. Computational methods have attracted tremendous attention among biologists because of the ability to predict protein-protein interactions and validate the obtained experimental results. In this study, we have reviewed several computational methods for protein-protein interaction prediction as well as describing major databases, which store both predicted and detected protein-protein interactions, and the tools used for analyzing protein interaction networks and improving protein-protein interaction reliability.     

Dimerization of Hepatitis E Virus Capsid Protein E2s Domain Is Essential for Virus–Host Interaction

Hepatitis E virus (HEV), a non-enveloped, positive-stranded RNA virus, is transmitted in a faecal-oral manner, and causes acute liver diseases in humans. The HEV capsid is made up of capsomeres consisting of homodimers of a single structural capsid protein forming the virus shell. These dimers are believed to protrude from the viral surface and to interact with host cells to initiate infection. To date, no structural information is available for any of the HEV proteins. Here, we report for the first time the crystal structure of the HEV capsid protein domain E2s, a protruding domain, together with functional studies to illustrate that this domain forms a tight homodimer and that this dimerization is essential for HEV–host interactions. In addition, we also show that the neutralizing antibody recognition site of HEV is located on the E2s domain. Our study will aid in the development of vaccines and, subsequently, specific inhibitors for HEV.     

Enhancement of Human DNA Polymerase η Activity and Fidelity Is Dependent Upon a Bipartite Interaction with the Werner Syndrome Protein

We have investigated the interaction between human DNA polymerase η (hpol η) and the Werner syndrome protein (WRN). Functional assays revealed that the WRN exonuclease and RecQ C-terminal (RQC) domains are necessary for full stimulation of hpol η-catalyzed formation of correct base pairs. We find that WRN does not stimulate hpol η-catalyzed formation of mispairs. Moreover, the exonuclease activity of WRN prevents stable mispair formation by hpol η. These results are consistent with a proofreading activity for WRN during single-nucleotide additions. ATP hydrolysis by WRN appears to attenuate stimulation of hpol η. Pre-steady-state kinetic results show that k_pol is increased 4-fold by WRN. Finally, pulldown assays reveal a bipartite physical interaction between hpol η and WRN that is mediated by the exonuclease and RQC domains. Taken together, these results are consistent with alteration of the rate-limiting step in polymerase catalysis by direct protein-protein interactions between WRN and hpol η. In summary, WRN improves the efficiency and fidelity of hpol η to promote more effective replication of DNA.     

Phosphorylation of Rab5a Protein by Protein Kinase C? Is Crucial for T-cell Migration

Rab GTPases control membrane traffic and receptor-mediated endocytosis. Within this context, Rab5a plays an important role in the spatial regulation of intracellular transport and signal transduction processes. Here, we report a previously uncharacterized role for Rab5a in the regulation of T-cell motility. We show that Rab5a physically associates with protein kinase Cϵ (PKCϵ) in migrating T-cells. After stimulation of T-cells through the integrin LFA-1 or the chemokine receptor CXCR4, Rab5a is phosphorylated on an N-terminal Thr-7 site by PKCϵ. Both Rab5a and PKCϵ dynamically interact at the centrosomal region of migrating cells, and PKCϵ-mediated phosphorylation on Thr-7 regulates Rab5a trafficking to the cell leading edge. Furthermore, we demonstrate that Rab5a Thr-7 phosphorylation is functionally necessary for Rac1 activation, actin rearrangement, and T-cell motility. We present a novel mechanism by which a PKCϵ-Rab5a-Rac1 axis regulates cytoskeleton remodeling and T-cell migration, both of which are central for the adaptive immune response.