SIGLOCPRED: an algorithm to predict bacterial signal peptides and OMPS

There is a growing interest in Biological investigation to determine the location of proteins, to identify new potentially accessible drug targets. Signal peptide directs the transport of the protein to its location. Bacterial OMPs are essential for their survival in the host organism. SIGLOCPRED a signal peptide predictor for the bacterial proteins as well as OMP prediction has been developed. The signal peptide prediction is done based on the influence of the flanking residues on the signal peptide cleavage. A dataset of proteins with confirmed outer membrane location has being created, and the probable OMP polypeptide sequence is predicted. Since the algorithm uses confirmed datasets the prediction is more reliable and efficient. SIGLOCPRED is as efficient as many of the existing signal peptide predictors and can also predict OMPs in addition.     

Novel inhibitor for prolyl tripeptidyl aminopeptidase from Porphyromonas gingivalis and details of substrate-recognition mechanism

A new inhibitor, H-Ala-Ile-pyrrolidin-2-yl boronic acid, was developed as an inhibitor against prolyl tripeptidyl aminopeptidase with a K_i value of 88.1 nM. The structure of the prolyl tripeptidyl aminopeptidase complexed with the inhibitor (enzyme-inhibitor complex) was determined at 2.2 Å resolution. The inhibitor was bound to the active site through a covalent bond between Ser603 and the boron atom of the inhibitor. This structure should closely mimic the structure of the reaction intermediate between the enzyme and substrate. We previously proposed that two glutamate residues, Glu205 and Glu636, are involved in the recognition of substrates. In order to clarify the function of these glutamate residues in substrate recognition, three mutant enzymes, E205A, E205Q, and E636A were generated by site-directed mutagenesis. The E205A mutant was expressed as an inclusion body. The E205Q mutant was expressed in soluble form, but no activity was detected. Here, the structures of the E636A mutant and its complex with the inhibitor were determined. The inhibitor was located at almost the same position as in the wild-type enzyme-inhibitor complex. The amino group of the inhibitor interacted with Glu205 and the main-chain carbonyl group of Gln203. In addition, a water molecule in the place of Glu636 of the wild-type enzyme interacted with the amino group of the inhibitor. This water molecule was located near the position of Glu636 in the wild-type and formed a hydrogen bond with Gln203. The k_cat/K_M values of the E636A mutant toward the two substrates used were smaller than those of the wild-type by two orders of magnitude. The K_i value of our inhibitor for the E636A mutant was 48.8 μM, which was 554-fold higher than that against the wild-type enzyme. Consequently, it was concluded that Glu205 and Glu636 are significant residues for the N-terminal recognition of a substrate.     

Solution structure of the c-terminal dimerization domain of SARS coronavirus nucleocapsid protein solved by the SAIL-NMR method

The C-terminal domain (CTD) of the severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP) contains a potential RNA-binding region in its N-terminal portion and also serves as a dimerization domain by forming a homodimer with a molecular mass of 28 kDa. So far, the structure determination of the SARS-CoV NP CTD in solution has been impeded by the poor quality of NMR spectra, especially for aromatic resonances. We have recently developed the stereo-array isotope labeling (SAIL) method to overcome the size problem of NMR structure determination by utilizing a protein exclusively composed of stereo- and regio-specifically isotope-labeled amino acids. Here, we employed the SAIL method to determine the high-quality solution structure of the SARS-CoV NP CTD by NMR. The SAIL protein yielded less crowded and better resolved spectra than uniform ¹³C and ¹⁵N labeling, and enabled the homodimeric solution structure of this protein to be determined. The NMR structure is almost identical with the previously solved crystal structure, except for a disordered putative RNA-binding domain at the N-terminus. Studies of the chemical shift perturbations caused by the binding of single-stranded DNA and mutational analyses have identified the disordered region at the N-termini as the prime site for nucleic acid binding. In addition, residues in the β-sheet region also showed significant perturbations. Mapping of the locations of these residues onto the helical model observed in the crystal revealed that these two regions are parts of the interior lining of the positively charged helical groove, supporting the hypothesis that the helical oligomer may form in solution.     

Id-1 induces proteasome-dependent degradation of the HBX protein

Id-1 is a member of the HLH protein family that regulates a wide range of cellular processes such as cell proliferation, apoptosis, senescence and overexpression of Id-1 was recently suggested to play roles in the development and progression of different cancers. Previously, Id-1 was shown to physically interact with the viral protein E1A. Meanwhile, Id-1 expression was found to be regulated by several of the virus-encoded proteins, suggesting that Id-1 may be a common cellular target of the viral proteins. Here, we report that Id-1 interacts with the Hepatitis-B virus (HBV)-encoded protein HBX and regulates its stability in hepatocellular carcinoma (HCC) cells. We found that in HCC cells, ectopic Id-1 expression significantly decreased the half-life of the HBX protein, indicating that HBX is destabilized by Id-1. Meanwhile, the Id-1-induced HBX degradation was found to be inhibited by treatment with proteasome inhibitor, suggesting that this process is mediated through the proteasome pathway. Interestingly, while Id-1 did not induce HBX-ubiquitination, we found that removal of all the lysine residues of the HBX protein protects it from the effect of Id-1, indicating that ubiquitination is still required for the Id-1-mediated HBX degradation. Meanwhile, we found that Id-1 binds to the proteasome subunit C8 and facilitates its interaction with the HBX protein and disruption of this interaction completely abolishes the negative effect of Id-1 on HBX protein stability. Taken together, our results demonstrated a novel function of Id-1 in regulating HBX protein stability through interaction with the proteasome.     

Mutational analysis of the stability of the H2A and H2B histone monomers

The eukaryotic histone heterodimer H2A-H2B folds through an obligatory dimeric intermediate that forms in a nearly diffusion-limited association reaction in the stopped-flow dead time. It is unclear whether there is partial folding of the isolated monomers before association. To address the possible contributions of structure in the monomers to the rapid association, we characterized H2A and H2B monomers in the absence of their heterodimeric partner. By far-UV circular dichroism, the H2A and H2B monomers are 15% and 31% helical, respectively—significantly less than observed in X-ray crystal structures. Acrylamide quenching of the intrinsic Tyr fluorescence was indicative of tertiary structure. The H2A and H2B monomers exhibit free energies of unfolding of 2.5 and 2.9 kcal mol^− 1, respectively; at 10 μM, the sum of the stability of the monomers is ∼ 60% of the stability of the native dimer. The helical content, stability, and m values indicate that H2B has a more stable, compact structure than H2A. The monomer m values are larger than expected for the extended histone fold motif, suggesting that the monomers adopt an overly collapsed structure. Stopped-flow refolding—initiated from urea-denatured monomers or the partially folded monomers populated at low denaturant concentrations—yielded essentially identical rates, indicating that monomer folding is productive in the rapid association and folding of the heterodimer. A series of Ala and Gly mutations were introduced into H2A and H2B to probe the importance of helix propensity on the structure and stability of the monomers. The mutational studies show that the central α-helix of the histone fold, which makes extensive intermonomer contacts, is structured in H2B but only partially folded in H2A.     

Crystal structure of a core domain of stomatin from Pyrococcus horikoshii Illustrates a novel trimeric and coiled-coil fold

Stomatin is a major integral membrane protein of human erythrocytes, the absence of which is associated with a form of hemolytic anemia known as hereditary stomatocytosis. However, the function of stomatin is not fully understood. An open reading frame, PH1511, from the hyperthermophilic archaeon Pyrococcus horikoshii encodes p-stomatin, a prokaryotic stomatin. Here, we report the first crystal structure of a stomatin ortholog, the core domain of the p-stomatin PH1511p (residues 56-234 of PH1511p, designated as PhSto^CD). PhSto^CD forms a novel homotrimeric structure. Three α/β domains form a triangle of about 50 Å on each side, and three α-helical segments of about 60 Å in length extend from the apexes of the triangle. The α/β domain of PhSto^CD is partly similar in structure to the band-7 domain of mouse flotillin-2. While the α/β domain is relatively rigid, the α-helical segment shows conformational flexibility, adapting to the neighboring environment. One α-helical segment forms an anti-parallel coiled coil with another α-helical segment from a symmetry-related molecule. The α-helical segment shows a heptad repeat pattern, and mainly hydrophobic residues form a coiled-coil interface. According to chemical cross-linking experiments, PhSto^CD would be able to assemble into an oligomeric form. The coiled-coil fold observed in the crystal probably contributes to self-association.     

Directed evolution to low nanomolar affinity of a tumor-targeting epidermal growth factor receptor-binding affibody molecule

The epidermal growth factor receptor 1 (EGFR) is overexpressed in various malignancies and is associated with a poor patient prognosis. A small, receptor-specific, high-affinity imaging agent would be a useful tool in diagnosing malignant tumors and in deciding upon treatment and assessing the response to treatment. We describe here the affinity maturation procedure for the generation of Affibody molecules binding with high affinity and specificity to EGFR. A library for affinity maturation was constructed by rerandomization of selected positions after the alignment of first-generation binding variants. New binders were selected with phage display technology, using a single oligonucleotide in a single-library effort, and the best second-generation binders had an approximately 30-fold improvement in affinity (K_d = 5-10 nM) for the soluble extracellular domain of EGFR in biospecific interaction analysis using Biacore. The dissociation equilibrium constant, K_d, was also determined for the Affibody with highest affinity using EGFR-expressing A431 cells in flow cytometric analysis (K_d = 2.8 nM). A retained high specificity for EGFR was verified by a dot blot assay showing staining only of EGFR proteins among a panel of serum proteins and other EGFR family member proteins (HER2, HER3, and HER4). The EGFR-binding Affibody molecules were radiolabeled with indium-111, showing specific binding to EGFR-expressing A431 cells and successful targeting of the A431 tumor xenografts with 4-6% injected activity per gram accumulated in the tumor 4 h postinjection.     

Structural behavior of RNA-binding proteins in the free state and in complex with RNA: <Emphasis Type="Italic">Escherichia coli</Emphasis> ribosomal protein L25 and 5S rRNA

A novel approach was proposed to evaluate the steadiness of polar clusters containing the RNA-binding sites on the protein surface. The degree of clustering of RNA-binding polar residues was used as a measure of the steadiness of the corresponding polar clusters. Escherichia coli ribosomal protein L25 utilizes two binding sites, S1 and S2, to complexate with a 5S rRNA fragment. The cluster distribution of RNA-contacting polar residues on the protein surface was studied using the structural data on the complex (in crystal and in solution) and the free state (in solution). The degree of polar residue clustering in S1 and S2 in crystal was estimated at 71.4 and 100%, respectively. For the free state in solution, the degree of clustering of the two sites was 22.8 and 68.6%, respectively. Thus, the steadiness was quantitatively estimated for the RNA-binding sites of two different types, one preexisting in the protein and the other induced by the RNA structure upon complexation. The difference between the protein structures in crystal and in solution was found to be functionally significant. The results can be extrapolated to numerous complexes of proteins with double-stranded RNA and DNA.     

Glial precursor cell transplantation therapy for neurotrauma and multiple sclerosis

Traumatic injury to the brain or spinal cord and multiple sclerosis (MS) share a common pathophysiology with regard to axonal demyelination. Despite advances in central nervous system (CNS) repair in experimental animal models, adequate functional recovery has yet to be achieved in patients in response to any of the current strategies. Functional recovery is dependent, in large part, upon remyelination of spared or regenerating axons. The mammalian CNS maintains an endogenous reservoir of glial precursor cells (GPCs), capable of generating new oligodendrocytes and astrocytes. These GPCs are upregulated following traumatic or demyelinating lesions, followed by their differentiation into oligodendrocytes. However, this innate response does not adequately promote remyelination. As a result, researchers have been focusing their efforts on harvesting, culturing, characterizing, and transplanting GPCs into injured regions of the adult mammalian CNS in a variety of animal models of CNS trauma or demyelinating disease. The technical and logistic considerations for transplanting GPCs are extensive and crucial for optimizing and maintaining cell survival before and after transplantation, promoting myelination, and tracking the fate of transplanted cells. This is especially true in trials of GPC transplantation in combination with other strategies such as neutralization of inhibitors to axonal regeneration or remyelination. Overall, such studies improve our understanding and approach to developing clinically relevant therapies for axonal remyelination following traumatic brain injury (TBI) or spinal cord injury (SCI) and demyelinating diseases such as MS.     

Effects of litter incorporation and nitrogen fertilization on the contents of extractable aluminium in the rhizosphere soil of tea plant (Camallia sinensis (L.) O. Kuntze)

The effects of litter incorporation and nitrogen application on the properties of rhizosphere and bulk soils of tea plants (Camellia sinensis (L.) O. Kuntze) were examined in a pot experiment. Total of 8 treatments included four levels of tea litter additions at 0, 4.9, 9.8, and 24.5 g kg^–1 in combination with two N levels (154.6 mg kg^–1 and without). After 18 months of growth the rhizosphere soil was collected by removing the soil adhering to plant roots and other soil was referred to as bulk soil. The dry matter productions of tea plants were significantly increased by N fertilization and litter incorporation. The effect of litter was time-depending and significantly decreased the content of exchangeable Al (Al_ex, by 1 mol L^–1 KCl) and Al saturation at 9 months after litter incorporation whereas soil pH was not affected, although the litter contained high Al content. After 18 months, the contents of extractable Al by dilute CaCl₂, CuCl₂ + KCl, NH₄OAC, ammonium oxalate and sodium citrate (Al_CaCl2, Al_Cu/KCl, Al_NH4OAC, Al_Oxal, and Al_Cit respectively) and Al_ex, were not affected by litter application, except that of Al_CaCl2 in the rhizosphere soil which was decreased following litter additions. Nitrogen fertilization with NH₄ ⁺ (urea and (NH₄)₂SO₄) significantly reduced soil pH, the contents of exchangeable Ca, K, Mg and base saturation while raised extractable Al levels (Al_CaCl2, Al_Cu/KCl, Al_NH4OAC, and Al_ex). In the rhizosphere soils exchangeable K accumulated in all treatments while exchangeable Ca and Mg depleted in treatments without litter application. The depletions of Ca and Mg were no longer observed following litter incorporation. This change of distribution gradients in rhizosphere was possibly due to the increase of nutrient supplies from litter decomposition and/or preferable root growth in soil microsites rich in organic matter. Lower pH and higher extractable Al (Al_CaCl2, Al_ex, and Al_NH4OAC) in the rhizosphere soils, regardless of N and litter treatments, were distinct and consistent in all treatments. Such enrichments of extractable Al in the rhizosphere soil might be of importance for tea plants capable of taking up large amounts of Al.