Physicochemical Properties and Biological Activity of the Peptide–Protein Complex from Bovine Scleral Tissue

The peptide–protein complex from bovine sclera was studied. It is shown that it contained a protein with a molecular weight of 66387 Da with the partial N-terminal amino acid sequence DTHKSEIAHRFKDLG-, which is homologous to the mature molecule of bovine serum albumin, and polypeptides with molecular weights of 1300–5080 Da. With a model of the organotypic cultivation of posterior eye tissues of the newt Pleurodeles waltl in vitro, it was shown that the effect of this peptide–protein complex in low doses increased the viability of scleral fibroblasts.     

Chromatin Protein HP1α Interacts with the Mitotic Regulator Borealin Protein and Specifies the Centromere Localization of the Chromosomal Passenger Complex

Accurate mitosis requires the chromosomal passenger protein complex (CPC) containing Aurora B kinase, borealin, INCENP, and survivin, which orchestrates chromosome dynamics. However, the chromatin factors that specify the CPC to the centromere remain elusive. Here we show that borealin interacts directly with heterochromatin protein 1α (HP1α) and that this interaction is mediated by an evolutionarily conserved PXVXL motif in the C-terminal borealin with the chromo shadow domain of HP1α. This borealin-HP1α interaction recruits the CPC to the centromere and governs an activation of Aurora B kinase judged by phosphorylation of Ser-7 in CENP-A, a substrate of Aurora B. Consistently, modulation of the motif PXVXL leads to defects in CPC centromere targeting and aberrant Aurora B activity. On the other hand, the localization of the CPC in the midzone is independent of the borealin-HP1α interaction, demonstrating the spatial requirement of HP1α in CPC localization to the centromere. These findings reveal a previously unrecognized but direct link between HP1α and CPC localization in the centromere and illustrate the critical role of borealin-HP1α interaction in orchestrating an accurate cell division.     

Protein–Protein and Protein–Membrane Associations in the Lignin Pathway

Supramolecular organization of enzymes is proposed to orchestrate metabolic complexity and help channel intermediates in different pathways. Phenylpropanoid metabolism has to direct up to 30% of the carbon fixed by plants to the biosynthesis of lignin precursors. Effective coupling of the enzymes in the pathway thus seems to be required. Subcellular localization, mobility, protein–protein, and protein–membrane interactions of four consecutive enzymes around the main branch point leading to lignin precursors was investigated in leaf tissues of Nicotiana benthamiana and cells of Arabidopsis thaliana. CYP73A5 and CYP98A3, the two Arabidopsis cytochrome P450s (P450s) catalyzing para- and meta-hydroxylations of the phenolic ring of monolignols were found to colocalize in the endoplasmic reticulum (ER) and to form homo- and heteromers. They moved along with the fast remodeling plant ER, but their lateral diffusion on the ER surface was restricted, likely due to association with other ER proteins. The connecting soluble enzyme hydroxycinnamoyltransferase (HCT), was found partially associated with the ER. Both HCT and the 4-coumaroyl-CoA ligase relocalized closer to the membrane upon P450 expression. Fluorescence lifetime imaging microscopy supports P450 colocalization and interaction with the soluble proteins, enhanced by the expression of the partner proteins. Protein relocalization was further enhanced in tissues undergoing wound repair. CYP98A3 was the most effective in driving protein association.     

Composition of Esterase and Peroxidase Isoenzyme Complex, Zein-2 Protein Fraction, and SDS-Protein Complex of Zea Mays L. × Tripsacum Dactyloides L. Hybrids and Parents

The patterns of esterase and peroxidase isoenzymes, subunits of zein-2 fraction and protomers of SDS-protein complex of Zea mays L. × Tripsacum dactyloides L. hybrids and their parents were compared. The study has been made to detect specific to Tripsacum isoesterases and isoperoxidases, zein subunits and SDS-protein protomers which could be used as markers for introgression of gene loci encoding these proteins from Tripsacum into hybrids of Tripsacum with Zea mays. Isoesterases and isoperoxidases as well protomers of SDS-protein complex specific to Tripsacum were detected in all hybrids analyzed. Zein subunits, specific to Tripsacum were detected in some of the analyzed hybrids which i that introgression frequency of the loci encoding proteins studied was different. Chromosome counts taken on the examined hybrids showed the addition of 9 – 13 Tripsacum chromosomes to maize chromosome complement.     

Structural and Functional Analysis of the Regulator of G Protein Signaling 2-Gαq Complex

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Highlights? Two structures of the RGS2-Gα_q complex were determined by X-ray crystallography ? RGS2 binds Gα_q in a manner distinct from how other RGS proteins bind Gα_i/o ? In its distinct pose, RGS2 forms extensive contacts with the α-helical domain of Gα_q ? Helical domain contacts contribute to binding affinity and GAP potency of RGS2     

βIII Spectrin Regulates the Structural Integrity and the Secretory Protein Transport of the Golgi Complex

A spectrin-based cytoskeleton is associated with endomembranes, including the Golgi complex and cytoplasmic vesicles, but its role remains poorly understood. Using new generated antibodies to specific peptide sequences of the human βIII spectrin, we here show its distribution in the Golgi complex, where it is enriched in the trans-Golgi and trans-Golgi network. The use of a drug-inducible enzymatic assay that depletes the Golgi-associated pool of PI4P as well as the expression of PH domains of Golgi proteins that specifically recognize this phosphoinositide both displaced βIII spectrin from the Golgi. However, the interference with actin dynamics using actin toxins did not affect the localization of βIII spectrin to Golgi membranes. Depletion of βIII spectrin using siRNA technology and the microinjection of anti-βIII spectrin antibodies into the cytoplasm lead to the fragmentation of the Golgi. At ultrastructural level, Golgi fragments showed swollen distal Golgi cisternae and vesicular structures. Using a variety of protein transport assays, we show that the endoplasmic reticulum-to-Golgi and post-Golgi protein transports were impaired in βIII spectrin-depleted cells. However, the internalization of the Shiga toxin subunit B to the endoplasmic reticulum was unaffected. We state that βIII spectrin constitutes a major skeletal component of distal Golgi compartments, where it is necessary to maintain its structural integrity and secretory activity, and unlike actin, PI4P appears to be highly relevant for the association of βIII spectrin the Golgi complex.     

The Weak Complex between RhoGAP Protein ARHGAP22 and Signal Regulatory Protein 14-3-3 Has 1∶2 Stoichiometry and a Single Peptide Binding Mode

ARHGAP22 is a RhoGAP protein comprising an N-terminal PH domain, a RhoGAP domain and a C-terminal coiled-coil domain. It has recently been identified as an Akt substrate that binds 14-3-3 proteins in response to treatment with growth factors involved in cell migration. We used a range of biophysical techniques to investigate the weak interaction between 14-3-3 and a truncated form of ARHGAP22 lacking the coiled-coil domain. This weak interaction could be stabilized by chemical cross-linking which we used to show that: a monomer of ARHGAP22 binds a dimer of 14-3-3; the ARHGAP22 PH domain is required for the 14-3-3 interaction; the RhoGAP domain is unlikely to participate in the interaction; Ser16 is the more important of two predicted 14-3-3 binding sites; and, phosphorylation of Ser16 may not be necessary for 14-3-3 interaction under the conditions we used. Small angle X-ray scattering and cross-link information were used to generate solution structures of the isolated proteins and of the cross-linked ARHGAP22:14-3-3 complex, showing that no major rearrangement occurs in either protein upon binding, and supporting a role for the PH domain and N-terminal peptide of ARHGAP22 in the 14-3-3 interaction. Small-angle X-ray scattering measurements of mixtures of ARHGAP22 and 14-3-3 were used to establish that the affinity of the interaction is ～30 μM.     

Amphipol-Trapped ExbB–ExbD Membrane Protein Complex from Escherichia coli: A Biochemical and Structural Case Study

Nutrient import across Gram-negative bacteria’s outer membrane is powered by the proton-motive force, delivered by the cytoplasmic membrane protein complex ExbB–ExbD–TonB. Having purified the ExbB₄–ExbD₂ complex in the detergent dodecyl maltoside, we substituted amphipol A8-35 for detergent, forming a water-soluble membrane protein/amphipol complex. Properties of the ExbB₄–ExbD₂ complex in detergent or in amphipols were compared by gel electrophoresis, size exclusion chromatography, asymmetric flow field-flow fractionation, thermal stability assays, and electron microscopy. Bound detergent and fluorescently labeled amphipol were assayed quantitatively by 1D NMR and analytical ultracentrifugation, respectively. The structural arrangement of ExbB₄–ExbD₂ was examined by EM, small-angle X-ray scattering, and small-angle neutron scattering using a deuterated amphipol. The amphipol-trapped ExbB₄–ExbD₂ complex is slightly larger than its detergent-solubilized counterpart. We also investigated a different oligomeric form of the two proteins, ExbB₆–ExbD₄, and propose a structural arrangement of its transmembrane α-helical domains.     

The β-Barrel Outer Membrane Protein Assembly Complex of Neisseria meningitidis

The evolutionarily conserved protein Omp85 is required for outer membrane protein (OMP) assembly in gram-negative bacteria and in mitochondria. Its Escherichia coli homolog, designated BamA, functions with four accessory lipoproteins, BamB, BamC, BamD, and BamE, together forming the β-barrel assembly machinery (Bam). Here, we addressed the composition of this machinery and the function of its components in Neisseria meningitidis, a model organism for outer membrane biogenesis studies. Analysis of genome sequences revealed homologs of BamC, BamD (previously described as ComL), and BamE and a second BamE homolog, Mlp. No homolog of BamB was found. As in E. coli, ComL/BamD appeared essential for viability and for OMP assembly, and it could not be replaced by its E. coli homolog. BamE was not essential but was found to contribute to the efficiency of OMP assembly and to the maintenance of OM integrity. A bamC mutant showed only marginal OMP assembly defects, but the impossibility of creating a bamC bamE double mutant further indicated the function of BamC in OMP assembly. An mlp mutant was unaffected in OMP assembly. The results of copurification assays demonstrated the association of BamC, ComL, and BamE with Omp85. Semi-native gel electrophoresis identified the RmpM protein as an additional component of the Omp85 complex, which was confirmed in copurification assays. RmpM was not required for OMP folding but stabilized OMP complexes. Thus, the Bam complex in N. meningitidis consists of Omp85/BamA plus RmpM, BamC, ComL/BamD, and BamE, of which ComL/BamD and BamE appear to be the most important accessory components for OMP assembly.Membrane-embedded β-barrel proteins are found in the outer membranes (OMs) of gram-negative bacteria, mitochondria, and chloroplasts. Only in recent years have cellular components required for the assembly and insertion of these OM proteins (OMPs) into the OM been identified. Omp85, which was first characterized in Neisseria meningitidis, is the key protein of the OMP assembly machinery (41). The function of Omp85 has been preserved during evolution, not only in gram-negative bacteria (8, 37, 44, 46) but also in mitochondria, where an Omp85 homolog, also known as Tob55 or Sam50, was shown to mediate the assembly of β-barrel proteins into the OM (15, 23, 27). Accordingly, bacterial OMPs are still recognized by the eukaryotic assembly machinery: when expressed in yeast, bacterial OMPs were found to be assembled into the mitochondrial OM in a Tob55-dependent manner (43). Omp85 in Escherichia coli, which was recently renamed BamA, for β-barrel assembly machinery (Bam) component A, is associated with at least four lipoproteins: BamB (formerly known as YfgL), BamC (NlpB), BamD (YfiO), and BamE (SmpA) (32, 46). In E. coli, BamB, BamC, and BamE are not essential, but the phenotypes of deletion mutants suggest that these proteins contribute to the efficiency of OMP assembly. Like BamA, BamD is an essential protein in E. coli (24, 26), involved in OMP assembly (24). These lipoproteins are evolutionarily less well conserved; the mitochondrial Tob55 protein is associated with two accessory proteins, but they do not show any sequence similarity with the lipoproteins of the E. coli Bam complex (14).Besides E. coli, N. meningitidis is one of the major bacterial model organisms for studies of OM assembly. As mentioned above, it was the first organism in which the function of Omp85 was identified (41), and also, the role of an integral OMP, designated LptD (formerly Imp or OstA), in the transport of lipopolysaccharide (LPS) to the cell surface was first established in N. meningitidis (3). With regard to OM biogenesis, N. meningitidis has several features that distinguish it from E. coli. For example, in contrast to E. coli (13), N. meningitidis mutants defective in LPS synthesis or transport are viable (3, 34), and OMPs are assembled perfectly well in such mutants (33). Furthermore, in OMP assembly mutants of E. coli, the periplasmic accumulation of unassembled OMPs is limited due to the induction of the σ^E extracytoplasmic stress response, which results in the degradation of unfolded OMPs (30) and the inhibition of their synthesis by small regulatory RNAs (20). In contrast, in N. meningitidis, most of the components involved in this response are absent (4), and unassembled OMPs continue to accumulate as periplasmic aggregates when OMP assembly is halted (41). However, the composition of the Bam complex and the role of accessory components in OMP assembly have not so far been studied in this organism. Therefore, to further understand the OMP assembly process in N. meningitidis, we have now analyzed the composition of the Bam complex and addressed the roles of the different components.     

Sorting and Processing of the Alzheimer’s Disease Amyloid Precursor Protein Mediated by the AP-4 Complex

Proteolytic processing of the transmembrane amyloid precursor protein (APP) to aggregation-prone amyloid-β (Aβ) peptide underlies the development of Alzheimer’s disease.     

Protein–Protein Docking: Past,Present, and Future

The Protein Journal - The biological significance of proteins attracted the scientific community in exploring their characteristics. The studies shed light on the interaction patterns and functions...     

Cystatin B and SOD1: Protein–Protein Interaction and Possible Relation to Neurodegeneration

Cystatin B (CSTB), an inhibitor of the cysteine proteases, belongs to the cathepsin family and it is known to interact with a number of proteins involved in cytoskeletal organization. CSTB has an intrinsic tendency to form aggregates depending on the redox environment. The gene encoding for CSTB is frequently mutated in association with the rare neurodegenerative condition progressive myoclonus epilepsy. Increased levels of CSTB have been observed in the spinal cord of transgenic mice modeling SOD1-linked familial amyotrophic lateral sclerosis, a fatal neurodegenerative disease affecting motoneurons. In the present study, we have investigated the relationship occurring between the expression of SOD1 and CSTB either wild-type or double-cysteine substitution mutant (Cys 3 and Cys 64). Whether or not there is a physical interaction between the two proteins was also investigated in overexpression experiments using a human neuroblastoma cell line and mouse-immortalized motoneurons. Here we report evidences for a reciprocal influence of CSTB and SOD1 at the gene expression level and for a direct interaction of the two proteins.     

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.     

Protein kinase Cζ and glucose uptake

Protein kinase Cζ (PKCζ) is a member of the PKC family, serving downstream of insulin receptor and phosphatidylinositol (PI) 3-kinase. Many evidences suggest that PKCζ plays a very important role in activating glucose transport response. Not only insulin but also glucose and exercise can activate PKCζ through diverse pathways. PKCζ activation and activity are impaired with insulin resistance in muscle and adipose tissues of type II diabetes individuals, but heightened in liver tissue, wherein it also increases lipid synthesis mediated by SREBP-1c (sterol-regulatory element-binding protein). Many studies have focused on linkage between PKCζ and GLUT4 translocation and activation. Exploring the molecular mechanisms and pathways by which PKCζ mediates glucose transport will highlight the insulin-signaling pathway. Published in Russian in Biokhimiya, 2006, Vol. 71, No. 7, pp. 869–875. Co-first authors.