Multistate allostery in response regulators: phosphorylation and mutagenesis activate RegA via alternate modes

Response regulators (RRs) belong to two-component signaling pathways, widely prevalent in bacteria and lower eukaryotes, for sensing and mediating responses to diverse environmental stress stimuli. RRs are modular proteins, and in most instances, a receiver domain is found connected to diverse effector domain(s). All receiver domains contain a conserved aspartate, which is the site of phosphorylation by an associated histidine kinase. RRs function as phosphorylatable signaling switches whereby histidine-kinase-mediated phosphorylation of RRs alters its output function. It is largely unknown how phosphorylation of the receiver domain triggers activation of distally positioned effector domain(s). Although crystal structures have highlighted differences in conformations from comparisons of snapshots of the unphosphorylated and phosphorylated receiver domains, how this is translated into altered activity of a distal effector domain has remained a mystery. While allosteric relays have been identified within receiver domains by NMR and X-ray crystallography, phosphorylated states of larger multidomain RRs have not yet been characterized. In this study, we have used amide hydrogen/deuterium exchange mass spectrometry to probe the conformational dynamics of a multidomain RR, RegA from Dictyostelium discoideum, by comparisons of the unphosphorylated and phosphorylated states and an activating mutant. Our results reveal allosteric coupling between the site of phosphorylation and the activating mutation. Interestingly, however, the conformations of the effector domains in both instances are distinct. Hydrogen/deuterium exchange mass spectrometry indicates that the 'inactive' and 'active' conformations exist as ensembles of multiple conformations. This is consistent with the 'conformational selection' model for describing phosphorylation-dependent regulation of multidomain RRs.     

Protein kinase D regulates RhoA activity via rhotekin phosphorylation

The members of the protein kinase D (PKD) family of serine/threonine kinases are major targets for tumor-promoting phorbol esters, G protein-coupled receptors, and activated protein kinase C isoforms (PKCs). The expanding list of cellular processes in which PKDs exert their function via phosphorylation of various substrates include proliferation, apoptosis, migration, angiogenesis, and vesicle trafficking. Therefore, identification of novel PKD substrates is necessary to understand the profound role of this kinase family in signal transduction. Here, we show that rhotekin, an effector of RhoA GTPase, is a novel substrate of PKD. We identified Ser-435 in rhotekin as the potential site targeted by PKD in vivo. Expression of a phosphomimetic S435E rhotekin mutant resulted in an increase of endogenous active RhoA GTPase levels. Phosphorylation of rhotekin by PKD2 modulates the anchoring of the RhoA in the plasma membrane. Consequently, the S435E rhotekin mutant displayed enhanced stress fiber formation when expressed in serum-starved fibroblasts. Our data thus identify a novel role of PKD as a regulator of RhoA activity and actin stress fiber formation through phosphorylation of rhotekin.     

Conformational analysis of amyloid precursor protein fragment containing amino acids 667-676, and the effect of D-Asp and iso-Asp substitution at Asp₆₇₂ residue

Amyloid precursor protein (APP) fragment containing amino acids 667–676, (APP_667–676), is a substrate for β-secretase which is responsible for generating amyloid β peptides. Conformational analysis of APP_667–676 peptide [Ac-Ser-Glu-Val-Lys-Met-Asp-Ala-Glu-Phe-Arg-NH₂] and the effect of substitution of Asp₆₇₂ with d-Asp and iso-l-Asp, studied for the first time, demonstrate that the peptide backbone of APP_667–676 is flexible and adopts different conformations in different solvent environments (water, trifluoroethanol and dimethylsulfoxide). A major conformational difference was observed in trifluoroethanol solvent when Asp₆₇₂ is substituted with d-Asp and iso-Asp. These conformational changes involved in APP_667–676 may assist in understanding the interactions between β-secretase and APP_667–676, with relevance to Alzheimer’s disease.     

Metabolically engineered Escherichia coli as a tool for the production of bioenergy and biochemicals from glycerol

Currently, a variety of feedstock is utilized by metabolically engineered bacteria for the production of bioenergy and biochemicals. Recent studies have shown that glycerol can be used as an alternative feedstock for glucose, considering its higher availability, lower price, and high degree of reduction. Hence, this review focuses on recent developments in the bioconversion of glycerol to bioenergy (ethanol and hydrogen) and biochemicals (1,3-propanediol, 1,2-propanediol, 3-hydroxypropionic acid, succinic acid, lactic acid, polyhydroxyalkanoates and Lphenyl alanine) using metabolically engineered Escherichia coli.     

Pharmacological targeting of mitochondrial complex I deficiency: the cellular level and beyond

Complex I (CI) represents a major entry point of electrons in the mitochondrial electron transport chain (ETC). It consists of 45 different subunits, encoded by the mitochondrial (mtDNA) and nuclear DNA (nDNA). In humans, mutations in nDNA-encoded subunits cause severe neurodegenerative disorders like Leigh Syndrome with onset in early childhood. The pathophysiological mechanism of these disorders is still poorly understood. Here we summarize the current knowledge concerning the consequences of nDNA-encoded CI mutations in patient-derived cells, present mouse models for human CI deficiency, and discuss potential treatment strategies for CI deficiency.     

Secretome analysis of Magnaporthe oryzae using in vitro systems

Magnaporthe oryzae is a devastating blast fungal pathogen of rice (Oryza sativa L.) that causes dramatic decreases in seed yield and quality. During the early stages of infection by this pathogen, the fungal spore senses the rice leaf surface, germinates, and penetrates the cell via an infectious structure known as an appressorium. During this process, M. oryzae secretes several proteins; however, these proteins are largely unknown mainly due to the lack of a suitable method for isolating secreted proteins during germination and appressoria formation. We examined the secretome of M. oryzae by mimicking the early stages of infection in vitro using a glass plate (GP), PVDF membrane, and liquid culture medium (LCM). Microscopic observation of M. oryzae growth revealed appressorium formation on the GP and PVDF membrane resembling natural M. oryzae-rice interactions; however, appresorium formation was not observed in the LCM. Secreted proteins were collected from the GP (3, 8, and 24 h), PVDF membrane (24 h), and LCM (48 h) and identified by two-dimensional gel electrophoresis (2DE) followed by tandem mass spectrometry. The GP, PVDF membrane, and LCM-derived 2D gels showed distinct protein patterns, indicating that they are complementary approaches. Collectively, 53 nonredundant proteins including previously known and novel secreted proteins were identified. Six biological functions were assigned to the proteins, with the predominant functional classes being cell wall modification, reactive oxygen species detoxification, lipid modification, metabolism, and protein modification. The in vitro system using GPs and PVDF membranes applied in this study to survey the M. oryzae secretome, can be used to further our understanding of the early interactions between M. oryzae and rice leaves.     

A meta-analysis and genome-wide association study of platelet count and mean platelet volume in african americans

Several genetic variants associated with platelet count and mean platelet volume (MPV) were recently reported in people of European ancestry. In this meta-analysis of 7 genome-wide association studies (GWAS) enrolling African Americans, our aim was to identify novel genetic variants associated with platelet count and MPV. For all cohorts, GWAS analysis was performed using additive models after adjusting for age, sex, and population stratification. For both platelet phenotypes, meta-analyses were conducted using inverse-variance weighted fixed-effect models. Platelet aggregation assays in whole blood were performed in the participants of the GeneSTAR cohort. Genetic variants in ten independent regions were associated with platelet count (N?=?16,388) with p<5×10(-8) of which 5 have not been associated with platelet count in previous GWAS. The novel genetic variants associated with platelet count were in the following regions (the most significant SNP, closest gene, and p-value): 6p22 (rs12526480, LRRC16A, p?=?9.1×10(-9)), 7q11 (rs13236689, CD36, p?=?2.8×10(-9)), 10q21 (rs7896518, JMJD1C, p?=?2.3×10(-12)), 11q13 (rs477895, BAD, p?=?4.9×10(-8)), and 20q13 (rs151361, SLMO2, p?=?9.4×10(-9)). Three of these loci (10q21, 11q13, and 20q13) were replicated in European Americans (N?=?14,909) and one (11q13) in Hispanic Americans (N?=?3,462). For MPV (N?=?4,531), genetic variants in 3 regions were significant at p<5×10(-8), two of which were also associated with platelet count. Previously reported regions that were also significant in this study were 6p21, 6q23, 7q22, 12q24, and 19p13 for platelet count and 7q22, 17q11, and 19p13 for MPV. The most significant SNP in 1 region was also associated with ADP-induced maximal platelet aggregation in whole blood (12q24). Thus through a meta-analysis of GWAS enrolling African Americans, we have identified 5 novel regions associated with platelet count of which 3 were replicated in other ethnic groups. In addition, we also found one region associated with platelet aggregation that may play a potential role in atherothrombosis.     

Synonymous codon usage in chloroplast genome of Coffea arabica

Synonymous codon usage of 53 protein coding genes in chloroplast genome of Coffea arabica was analyzed for the first time to find out the possible factors contributing codon bias. All preferred synonymous codons were found to use A/T ending codons as chloroplast genomes are rich in AT. No difference in preference for preferred codons was observed in any of the two strands, viz., leading and lagging strands. Complex correlations between total base compositions (A, T, G, C, GC) and silent base contents (A₃, T₃, G₃, C₃, GC₃) revealed that compositional constraints played crucial role in shaping the codon usage pattern of C. arabica chloroplast genome. ENC Vs GC₃ plot grouped majority of the analyzed genes on or just below the left side of the expected GC₃ curve indicating the influence of base compositional constraints in regulating codon usage. But some of the genes lie distantly below the continuous curve confirmed the influence of some other factors on the codon usage across those genes. Influence of compositional constraints was further confirmed by correspondence analysis as axis 1 and 3 had significant correlations with silent base contents. Correlation of ENC with axis 1, 4 and CAI with 1, 2 prognosticated the minor influence of selection in nature but exact separation of highly and lowly expressed genes could not be seen. From the present study, we concluded that mutational pressure combined with weak selection influenced the pattern of synonymous codon usage across the genes in the chloroplast genomes of C. arabica.