The combination of stable isotope labeling (SIL) with mass spectrometry (MS) allows comparison of the abundance of thousands of proteins in complex mixtures. However, interpretation of the large data sets generated by these techniques remains a challenge because appropriate statistical standards are lacking. Here, we present a generally applicable model that accurately explains the behavior of data obtained using current SIL approaches, including 18O, iTRAQ, and SILAC labeling, and different MS instruments. The model decomposes the total technical variance into the spectral, peptide, and protein variance components, and its general validity was demonstrated by confronting 48 experimental distributions against 18 different null hypotheses. In addition to its general applicability, the performance of the algorithm was at least similar than that of other existing methods. The model also provides a general framework to integrate quantitative and error information fully, allowing a comparative analysis of the results obtained from different SIL experiments. The model was applied to the global analysis of protein alterations induced by low H2O2 concentrations in yeast, demonstrating the increased statistical power that may be achieved by rigorous data integration. Our results highlight the importance of establishing an adequate and validated statistical framework for the analysis of high-throughput data.

The Escherichia coli protein Hha is a temperature- and osmolarity-dependent modulator of the expression of the hemolysin operon. The Hha protein was purified and its DNA-binding properties analyzed. Hha binds in a non-specific manner throughout the upstream regulatory region of the hemolysin operon in the recombinant hemolytic plasmid pANN202-312. A search for interacting proteins revealed that Hha interacts with H-NS. DNA-binding studies showed that, in vitro, Hha and H-NS together form a complex with DNA that differs from those formed with either protein alone. These data, together with the effects of hha and hns mutations on the expression of the hemolysin genes, suggest that in vivo H-NS and Hha form a nucleoid-protein complex that accounts for the thermo-osmotic regulation of the hemolysin operon in E. coli.

Phytochromes are widely distributed biliprotein photoreceptors. Typically, the chromophore becomes covalently linked to the protein during an autocatalytic lyase reaction. Plant and cyanobacterial phytochromes incorporate bilins with a ring A ethylidene side chain, whereas other bacterial phytochromes utilize biliverdin as chromophore, which has a vinyl ring A side chain. For Agrobacterium phytochrome Agp1, site-directed mutagenesis provided evidence that biliverdin is bound to cysteine 20. This cysteine is highly conserved within bacterial homologues, but its role as attachment site has as yet not been proven. We therefore performed mass spectrometry studies on proteolytic holopeptide fragments. For that purpose, an Agp1 expression vector was re-engineered to produce a protein with an N-terminal affinity tag. Following proteolysis, the chromophore co-purified with a ca. 5 kDa fragment during affinity chromatography, showing that the attachment site is located close to the N-terminus. Mass spectrometry analyses performed with the purified chromopeptide confirmed the role of the cysteine 20 as biliverdin attachment site. We also analyzed the role of the highly conserved histidine 250 by site-directed mutagenesis. The homologous amino acid plays an important but yet undefined role in plant phytochromes and has been proposed as chromophore attachment site of Deinococcus phytochrome. We found that in Agp1, this amino acid is dispensable for covalent attachment, but required for tight chromophore-protein interaction.

Human ras genes play central roles in coupling extracellular signals with complex intracellular networks controlling proliferation, differentiation, and apoptosis, among others processes. c-H-ras pre-mRNA can be alternatively processed into two mRNAs due to the inclusion or exclusion of the alternative exon IDX; this renders two proteins, p21H-Ras and p19H-RasIDX, which differ only at the carboxy terminus. Here, we have characterized some of the cis-acting sequences and trans-acting factors regulating IDX splicing. A downstream intronic silencer sequence (rasISS1), acting in concert with IDX, negatively regulates upstream intron splicing. This effect is mediated, at least in part, by the binding of hnRNP A1. Depletion and add-back experiments in nuclear extracts have confirmed hnRNP A1's inhibitory role in IDX splicing. Moreover, the addition of two SR proteins, SC35 and SRp40, can counteract this inhibition by strongly promoting the splicing of the upstream intron both in vivo and in vitro. Further, the RNA-dependent helicase p68 is also associated with both IDX and rasISS1 RNA, and suppression of p68 expression in HeLa cells by RNAi experiments results in a marked increase of IDX inclusion in the endogenous mRNA, suggesting a role for this protein in alternative splicing regulation.A common mechanism for gene expression regulation in metazoa is the use of alternative splice sites (SS) to produce multiple protein-coding sequences from the same pre-mRNA. It was recently predicted that nearly 60% of all human genes undergo at least one process of alternative splicing (41). The ever increasing known examples of alternative pre-mRNA processing are often tissue type or developmental state specific, indicating that complex regulation is involved in the selection of SS pairs (for a review, see reference 47). However, little is yet known about the detailed mechanisms regulating alternative splicing in mammalian cells.A number of RNA sequences that positively or negatively regulate the inclusion of alternative exons have been identified (12,22,24,26,32,35,38,42,61). Binding of certain sets of splicing factors to these regulatory sequences, together with the intrinsic strengths of the SS, dictates the specificity and efficiency of splicing, resulting in promotion or repression of each splicing event.The SR protein family is a well-characterized class of proteins involved in both constitutive and alternative splicing (29,58). Their mechanism of action involves binding to certain RNA sequences (commonly exonic enhancers) through their RNA recognition motifs and the recruitment of other splicing factors, stimulating the splicing efficiency of weak adjacent SS (1,30,33,56). SR protein binding to an exon is known to increase the binding of U2AF 65 to an upstream 3ЈSS (60) or that of U1 snRNP to a downstream 5ЈSS (6,27,36,39). SR proteins can also act in a RNA binding-independent way, promoting the assembly of general splicing factors in the proteinprotein network interactions that make up the mature spliceosome (5). Factors o...

Summary During the last decade, much has been learnt about the mechanisms by which oxidative stress is perceived by aerobic organisms. The Schizosaccharomyces pombe Pap1 protein is a transcription factor localized at the cytoplasm, which accumulates in the nucleus in response to different inducers, such as the pro‐oxidant hydrogen peroxide (H2O2) or the glutathione‐depleting agent diethylmaleate (DEM). As described for other H2O2 sensors, our genetic data indicates that H2O2 reversibly oxidizes two cysteine residues in Pap1 (Cys278 and Cys501). Surprisingly, our studies demonstrate that DEM generates a non‐reversible modification of at least two cysteine residues located in or close to the nuclear export signal of Pap1 (Cys523 and Cys532). This modification impedes the interaction of the nuclear exporter Crm1 with the nuclear export signal located at the carboxy‐terminal domain of Pap1. Mass spectrometry data suggest that DEM binds to the thiol groups of the target cysteine residues through the formation of a thioether. Here we show that DEM triggers Pap1 nuclear accumulation by a novel molecular mechanism.

Altogether, this study has important impacts on AIP care underlying that hemin needs to be restricted to severe neurovisceral crisis and suggests that alternative treatment targeting the liver such as ALAS1 and HO1 inhibitors, and anti-inflammatory therapies should be considered in patients with recurrent AIP.

Pathogen attack on plants results in numerous host-specific biochemical responses, the activation of some of them being critical for the ability of the plant to withstand disease. We have used high-resolution two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify proteins that are differentially expressed in response to fungal infection in maize embryos. Differential spots corresponding to induced or repressed proteins were apparent in silver stained 2-DE gels of proteins extracted from sterile and fungal-infected germinating embryos. Selected spots were subjected to tryptic digestion followed by identification using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry and nanospray ion-trap tandem mass spectrometry. Among the proteins induced in response to infection are proteins involved in protein synthesis, or in protein folding and stabilization, as well as proteins involved in oxidative stress tolerance. Additionally, the accumulation of specific pathogenesis-related proteins in tissues of the fungal-infected germinating embryos was studied by 2-DE and immunoblotting.

Ferná ndez, Esperanza, Montserrat Carrascal, Ferran Rousaud, Joaquín Abiá n, Antonio Zorzano, Manuel Palacín, and Josep Chillaró n. rBAT-b 0,ϩ AT heterodimer is the main apical reabsorption system for cystine in the kidney. Am J Physiol Renal Physiol 283: F540-F548, 2002. First published March 19, 2002 10.1152/ajprenal. 00071.2002Mutations in the rBAT and b 0,ϩ AT genes cause type I and non-type I cystinuria, respectively. The disulfide-linked rBAT-b 0,ϩ AT heterodimer mediates high-affinity transport of cystine and dibasic amino acids (b 0,ϩ -like activity) in heterologous cell systems. However, the significance of this heterodimer for cystine reabsorption is unknown, as direct evidence for such a complex in vivo is lacking and the expression patterns of rBAT and b 0,ϩ AT along the proximal tubule are opposite. We addressed this issue by biochemical means. Western blot analysis of mouse and human kidney brush-border membranes showed that rBAT and b 0,ϩ AT were solely expressed as heterodimers of identical size and that both proteins coprecipitated. Moreover, quantitative immunopurification of b 0,ϩ AT followed by SDS-PAGE and mass spectrometry analysis established that b 0,ϩ AT heterodimerizes exclusively with rBAT. Together with cystine reabsorption data, our results demonstrate that a decreasing expression gradient of heterodimeric rBATb 0,ϩ AT along the proximal tubule is responsible for virtually all apical cystine reabsorption. As a corollary of the above, there should be an excess of rBAT expression over that of b 0,ϩ AT protein in the kidney. Indeed, complete immunodepletion of b 0,ϩ AT did not coprecipitate Ͼ20-30% of rBAT. Therefore, another rBAT-associated subunit may be present in latter parts of the proximal tubule. proximal tubule; heterodimeric amino acid transporter; expression gradient CLASSIC CYSTINURIA IS DUE to the impaired renal reabsorption of cystine and dibasic amino acids. Low cystine solubility causes the deposition of cystine calculi, which eventually leads to kidney failure (34). Reabsorption of cystine has been the object of a great research effort (32, 39). Dent and Rose (12) proposed a defective apical cystine transporter shared with dibasic amino acids as the main cause for cystinuria. Evidence for this hypothesis came recently with the identification of the two cystinuria genes: SLC3A1, which codes for the rBAT protein and is responsible for type I cystinuria, and SLC7A9, which codes for b 0,ϩ AT and causes non-type I cystinuria (5,14,40). Together, mutations in these two genes account for ϳ80% of cystinuria patients (16,33). b 0,ϩ AT and rBAT belong to the family of heteromeric amino acid transporters (HAT), which are made up of two structurally different subunits (10,51,54). The heavy subunit is a type II membrane glycoprotein, whereas the light subunit is an unglycosylated membrane protein bearing 12 putative transmembrane domains. Two conserved cysteines form a disulfide-linked heterodimer between the two subunits (37). A trafficking role has been proposed for the heavy sub...