Negative Regulation of Phosphate Starvation-Induced Genes

Phosphate (Pi) deficiency is a major nutritional problem faced by plants in many agro-ecosystems. This deficiency results in altered gene expression leading to physiological and morphological changes in plants. Altered gene expression is presumed to be due to interaction of regulatory sequences (cis-elements) present in the promoters with DNA binding factors (trans-factors). In this study, we analyzed the expression and DNA-protein interaction of promoter regions of Pi starvation-induced genes AtPT2 and TPSI1. AtPT2 encodes the high-affinity Pi transporter in Arabidopsis, whereas TPSI1 codes for a novel gene induced in the Pi-starved tomato (Lycopersicon esculentum). Expression of AtPT2 was induced rapidly under Pi deficiency and increased with decreasing concentrations of Pi. Abiotic stresses except Pi starvation had no affect on the expression of TPSI1. DNA mobility-shift assays indicated that specific sequences of AtPT2 and TPSI1 promoter interact with nuclear protein factors. Two regions of AtPT2 and TPSI1 promoters specifically bound nuclear protein factors from Pi-sufficient plants. Interestingly, the DNA binding activity disappeared during Pi starvation, leading to the hypothesis that Pi starvation-induced genes may be under negative regulation.     

Synthetic protein design: construction of a four-stranded beta-sheet structure and evaluation of its integrity in methanol-water systems.

The characterization of a four-stranded beta-sheet structure in a designed 26-residue peptide Beta-4 is described. The sequence of Beta-4 (Arg-Gly-Thr-Ile-Lys-(D)pro-Gly-Ile-Thr-Phe-Ala-(D)Pro-Ala-Thr-Val-Leu-P he-Ala-Val-(D)Pro-Gly-Lys-Thr-Leu-Tyr-Arg) was chosen such that three strategically positioned (D)Pro-Xxx segments nucleate type II' beta-turns, which facilitate hairpin extension. A four-stranded beta-sheet structure is determined in methanol from 500 MHz 1H NMR data using a total of 100 observed NOEs, 11 dihedral restraints obtained from vicinal JCalphaH-NH values and 10 hydrogen bonding constraints obtained from H/D exchange data. The observed NOEs provide strong evidence for a stable four-stranded sheet and a nonpolar cluster involving Ile8, Phe10, Val15 and Phe17. Circular dichroism studies in water-methanol mixtures provide evidence for melting of the beta-sheet structure at high water concentrations. NMR analysis establishes that the four-stranded sheet in Beta-4 is appreciably populated in 50% (v/v) aqueous methanol. In water, the peptide structure is disorganized, although the three beta-turn nuclei appear to be maintained.     

Global impact of oncogenic Src on a phosphotyrosine proteome

Elevated activity of Src, the first characterized protein-tyrosine kinase, is associated with progression of many human cancers, and Src has attracted interest as a therapeutic target. Src is known to act in various receptor signaling systems to impact cell behavior, yet it remains likely that the spectrum of Src protein substrates relevant to cancer is incompletely understood. To better understand the cellular impact of deregulated Src kinase activity, we extensively applied a mass spectrometry shotgun phosphotyrosine (pTyr) proteomics strategy to obtain global pTyr profiles of Src-transformed mouse fibroblasts as well as their nontransformed counterparts. A total of 867 peptides representing 563 distinct pTyr sites on 374 different proteins were identified from the Src-transformed cells, while 514 peptides representing 275 pTyr sites on 167 proteins were identified from nontransformed cells. Distinct characteristics of the two profiles were revealed by spectral counting, indicative of pTyr site relative abundance, and by complementary quantitative analysis using stable isotope labeling with amino acids in cell culture (SILAC). While both pTyr profiles are replete with sites on signaling and adhesion/cytoskeletal regulatory proteins, the Src-transformed profile is more diverse with enrichment in sites on metabolic enzymes and RNA and protein synthesis and processing machinery. Forty-three pTyr sites (32 proteins) are predicted as major biologically relevant Src targets on the basis of frequent identification in both cell populations. This select group, of particular interest as diagnostic biomarkers, includes well-established Src sites on signaling/adhesion/cytoskeletal proteins, but also uncharacterized sites of potential relevance to the transformed cell phenotype.     

Chain length effects on helix-hairpin distribution in short peptides with Aib-DAla and Aib-Aib segments

The Aib-D Ala dipeptide segment has a tendency to form both type-I'/III' and type-I/III β-turns. The occurrence of prime turns facilitates the formation of β-hairpin conformations, while type-I/III turns can nucleate helix formation. The octapeptide Boc-Leu-Phe-Val-Aib-DAla-Leu-Phe-Val-OMe (1) has been previously shown to form a β-hairpin in the crystalline state and in solution. The effects of sequence truncation have been examined using the model peptides Boc-Phe-Val-Aib-Xxx-Leu-Phe-NHMe (2, 6), Boc-Val-Aib-Xxx-Leu-NHMe (3, 7), and Boc-Aib-Xxx-NHMe (4, 8), where Xxx=DAla, Aib. For peptides with central Aib-Aib segments, Boc-Phe-Val-Aib-Aib-Leu-Phe-NHMe (6), Boc-Val-Aib-Aib-Leu-NHMe (7), and Boc-Aib-Aib-NHMe (8) helical conformations have been established by NMR studies in both hydrogen bonding (CD3OH) and non-hydrogen bonding (CDCl3) solvents. In contrast, the corresponding hexapeptide Boc-Phe-Val-Aib-DAla-Leu-Phe-Val-NHMe (2) favors helical conformations in CDCl3 and β-hairpin conformations in CD3 OH. The β-turn conformations (type-I'/III) stabilized by intramolecular 4→1 hydrogen bonds are observed for the peptide Boc-Aib-D Ala-NHMe (4) and Boc-Aib-Aib-NHMe (8) in crystals. The tetrapeptide Boc-Val-Aib-Aib-Leu-NHMe (7) adopts an incipient 3(10)-helical conformation stabilized by three 4→1 hydrogen bonds. The peptide Boc-Val-Aib-DAla-Leu-NHMe (3) adopts a novel α-turn conformation, stabilized by three intramolecular hydrogen bonds (two 4→1 and one 5→1). The Aib-DAla segment adopts a type-I' β-turn conformation. The observation of an NOE between Val (1) NH?HNCH3 (5) in CD3OH suggests, that the solid state conformation is maintained in methanol solutions.     

Characterization of the phosphate starvation-induced glycerol-3-phosphate permease gene family in Arabidopsis

Phosphate (Pi) deficiency is one of the leading causes of loss in crop productivity. Plants respond to Pi deficiency by increasing Pi acquisition and remobilization involving organic and inorganic Pi transporters. Here, we report the functional characterization of a putative organic Pi transporter, Glycerol-3-phosphate permease (G3Pp) family, comprising five members (AtG3Pp1 to -5) in Arabidopsis (Arabidopsis thaliana). AtG3Pp1 and AtG3Pp2 showed 24-and 3-fold induction, respectively, in the roots of Pi-deprived seedlings, whereas Pi deficiency-mediated induction of AtG3Pp3 and -4 was evident in both roots and shoots. Furthermore, promoter-β-glucuronidase (GUS) fusion transgenics were generated for AtG3Pp2 to -5 for elucidation of their in planta role in Pi homeostasis. During Pi starvation, there was a strong expression of the reporter gene driven by AtG3Pp4 promoter in the roots, shoots, anthers, and siliques, whereas GUS expression was specific either to the roots (AtG3Pp3) or to stamens and siliques (AtG3Pp5) in other promoter-GUS fusion transgenics. Quantification of reporter gene activities further substantiated differential responses of AtG3Pp family members to Pi deprivation. A distinct pattern of reporter gene expression exhibited by AtG3Pp3 and AtG3Pp5 during early stages of germination also substantiated their potential roles during seedling ontogeny. Furthermore, an AtG3Pp4 knockdown mutant exhibited accentuated total lateral root lengths under +phosphorus and -phosphorus conditions compared with the wild type. Several Pi starvation-induced genes involved in root development and/or Pi homeostasis were up-regulated in the mutant. A 9-fold induction of AtG3Pp3 in the mutant provided some evidence for a lack of functional redundancy in the gene family. These results thus reflect differential roles of members of the G3Pp family in the maintenance of Pi homeostasis.     

Quantitative proteomics for identifying biomarkers for Rabies

Introduction

Rabies is a fatal acute viral disease of the central nervous system, which is a serious public health problem in Asian and African countries. Based on the clinical presentation, rabies can be classified into encephalitic (furious) or paralytic (numb) rabies. Early diagnosis of this disease is particularly important as rabies is invariably fatal if adequate post exposure prophylaxis is not administered immediately following the bite.

Methods

In this study, we carried out a quantitative proteomic analysis of the human brain tissue from cases of encephalitic and paralytic rabies along with normal human brain tissues using an 8-plex isobaric tags for relative and absolute quantification (iTRAQ) strategy.

Results and conclusion

We identified 402 proteins, of which a number of proteins were differentially expressed between encephalitic and paralytic rabies, including several novel proteins. The differentially expressed molecules included karyopherin alpha 4 (KPNA4), which was overexpressed only in paralytic rabies, calcium calmodulin dependent kinase 2 alpha (CAMK2A), which was upregulated in paralytic rabies group and glutamate ammonia ligase (GLUL), which was overexpressed in paralytic as well as encephalitic rabies. We validated two of the upregulated molecules, GLUL and CAMK2A, by dot blot assays and further validated CAMK2A by immunohistochemistry. These molecules need to be further investigated in body fluids such as cerebrospinal fluid in a larger cohort of rabies cases to determine their potential use as antemortem diagnostic biomarkers in rabies. This is the first study to systematically profile clinical subtypes of human rabies using an iTRAQ quantitative proteomics approach.