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Why Are tRNAs Overproduced in the Absence of Maf1, a Negative Regulator of RNAP III, Not Fully Functional?
Get PDFThe family of LSU-like proteins
Get PDFPlant response to sulfur deficiency results in extensive metabolic changes and can be monitored at various levels (transcriptome, proteome, metabolome) even before the occurrence of the first visible symptoms of sulfur starvation. The plant-specific LSU (RESPONSE TO LOW SULFUR) gene family is represented by four members in Arabidopsis thaliana (LSU1-4) and multiple members in other plant species. Three out of four LSU genes in Arabidopsis are induced in sulfur deficiency conditions. In tobacco, the LSU-like genes were characterized as UP9 (UPREGULATED BY SULFUR DEFICIT 9). The LSU-like proteins contain no characteristic domains that could help to identify their function. Despite moderate primary sequence conservation they share several common features, including small size, coiled-coil secondary structure and short conserved motifs located in particular positions. Although the precise function of these proteins is still unknown, there is some evidence that members of the LSU-like family are involved in plant response to environmental challenges, such as sulfur deficiency and possibly in plant immune responses. Interestingly, various bioinformatic approaches have identified these proteins as important hubs integrating signals from different environmental stimuli. In this paper we review a variety of published data about LSU gene expression, properties of lsu mutants and features of LSU proteins in the hope of shedding some light on their possible role in plant metabolism
Ni(II) ions cleave and inactivate human alpha-1 antitrypsin hydrolytically, implicating nickel exposure as a contributing factor in pathologies related to antitrypsin deficiency
No full textHuman alpha-1 antitrypsin (AAT) is an abundant serum protein, present at a concentration of 1.0–1.5 g x L−1. AAT deficiency is a genetic disease, manifesting itself with emphysema and liver cirrhosis, due to accumulation of a misfolded AAT mutant in hepatocytes. Lung AAT amount is inversely correlated with chronic obstructive pulmonary disease (COPD), a serious and often deadly condition, with increasing frequency in the aging population. Exposure to cigarette smoke and products of fossil fuel combustion aggravates AAT deficiency and COPD according to mechanisms that are not fully understood. Taking into account that these fumes contain particles that can release nickel to human airways and skin, we decided to investigate interactions of AAT with Ni(II) ions within the paradigm of Ni(II)-dependent peptide bond hydrolysis. We studied AAT protein derived from human blood using HPLC, SDS-PAGE, and mass spectrometry. These studies were aided by spectroscopic experiments on model peptides. As a result, we identified three hydrolysis sites in AAT. Two of them are present in the N-terminal part of the molecule next to each other (before Thr-13 and Ser-14 residues) and effectively form one N-terminal cleavage site. The single C-terminal cleavage is located before Ser-285. The N-terminal hydrolysis was more efficient than the C-terminal one, but both abolished the ability of AAT to inhibit trypsin in an additive manner. Nickel ions bound to hydrolysis products demonstrated an ability to generate ROS. These results implicate Ni(II) exposure as a contributing factor in AAT-related pathologies
A two-component enzyme complex is required for dolichol biosynthesis in tomato
Get PDFDolichol plays an indispensable role in the N-glycosylation of eukaryotic proteins. As proteins enter the secretory pathway they are decorated by a ‘glycan’, which is preassembled onto a membrane-anchored dolichol molecule embedded within the endoplasmic reticulum (ER). Genetic and biochemical evidence in yeast and animals indicate that a cis-prenyltransferase (CPT) is required for dolichol synthesis, but also point to other factor(s) that could be involved. In this study, RNAi-mediated suppression of one member of the tomato CPT family (SlCPT3) resulted in a ~60% decrease in dolichol content. We further show that the involvement of SlCPT3 in dolichol biosynthesis requires the participation of a distantly related partner protein, designated as CPT binding protein (SlCPTBP), which is a close homolog of the human Nogo-B receptor. Yeast two-hybrid and co-immunoprecipitation assays demonstrate that SlCPT3 and its partner protein interact, in vivo, and that both SlCPT3 and SlCPTBP are required to complement the growth defects and dolichol deficiency of the yeast dolichol mutant, rer2Δ. Co-expression of SlCPT3 and SlCPTBP in yeast and in E. coli confirmed that dolichol synthase activity strictly requires both proteins. Finally, organelle isolation and in vivo localization of fluorescent protein fusions showed that both SlCPT3 and SlCPTBP localize to the endoplasmic reticulum, the site of dolichol accumulation and synthesis in eukaryotes
Links Between Ethylene and Sulfur Nutrition - A Regulatory Interplay or Just Metabolite Association?
Get PDFMultiple reports demonstrate associations between ethylene and sulfur metabolisms, however the details of these links have not yet been fully characterized; the links might be at the metabolic and the regulatory levels. First, sulfur-containing metabolite, methionine, is a precursor of ethylene and is a rate limiting metabolite for ethylene synthesis; the methionine cycle contributes to both sulfur and ethylene metabolism. On the other hand, ethylene is involved in the complex response networks to various stresses and it is known that S deficiency leads to photosynthesis and C metabolism disturbances that might be responsible for oxidative stress. In several plant species, ethylene increases during sulfur starvation and might serve signaling purposes to initiate the process of metabolism reprogramming during adjustment to sulfur deficit. An elevated level of ethylene might result from increased activity of enzymes involved in its synthesis. It has been demonstrated that the alleviation of cadmium stress in plants by application of S seems to be mediated by ethylene formation. On the other hand, the ethylene-insensitive Nicotiana attenuata plants are impaired in sulfur uptake, reduction and metabolism, and they invest their already limited S into methionine needed for synthesis of ethylene constitutively emitted in large amounts to the atmosphere. Regulatory links of EIN3 and SLIM1 (both from the same family of transcriptional factors) involved in the regulation of ethylene and sulfur pathway, respectively, is also quite probable as well as the reciprocal modulation of both pathways on the enzyme activity levels
A Functional Role for Aβ in Metal Homeostasis? N-Truncation and High-Affinity Copper Binding
No full textAccumulation of the β-amyloid (Aβ) peptide in extracellular senile plaques rich in copper and zinc is a defining pathological feature of Alzheimer's disease (AD). The Aβ1-x (x=16/28/40/42) peptides have been the primary focus of Cu(II) binding studies for more than 15 years; however, the N-truncated Aβ4-42 peptide is a major Aβ isoform detected in both healthy and diseased brains, and it contains a novel N-terminal FRH sequence. Proteins with His at the third position are known to bind Cu(II) avidly, with conditional log K values at pH 7.4 in the range of 11.0-14.6, which is much higher than that determined for Aβ1-x peptides. By using Aβ4-16 as a model, it was demonstrated that its FRH sequence stoichiometrically binds Cu(II) with a conditional Kd value of 3×10(-14) M at pH 7.4, and that both Aβ4-16 and Aβ4-42 possess negligible redox activity. Combined with the predominance of Aβ4-42 in the brain, our results suggest a physiological role for this isoform in metal homeostasis within the central nervous system
Impact of OmpR on the membrane proteome of Yersinia enterocolitica in different environments: repression of major adhesin YadA and heme receptor HemR.
Get PDFEnteropathogenic Yersinia enterocolitica is able to grow within or outside the mammalian host. Previous transcriptomic studies have indicated that the regulator OmpR plays a role in the expression of hundreds of genes in enterobacteria. Here, we have examined the impact of OmpR on the production of Y. enterocolitica membrane proteins upon changes in temperature, osmolarity and pH. Proteomic analysis indicated that the loss of OmpR affects the production of 120 proteins, a third of which are involved in uptake/transport, including several that participate in iron or heme acquisition. A set of proteins associated with virulence was also affected. The influence of OmpR on the abundance of adhesin YadA and heme receptor HemR was examined in more detail. OmpR was found to repress YadA production and bind to the yadA promoter, suggesting a direct regulatory effect. In contrast, the repression of hemR expression by OmpR appears to be indirect. These findings provide new insights into the role of OmpR in remodeling the cell surface and the adaptation of Y. enterocolitica to different environmental niches, including the host
Mimicking the phosphorylation of Rsp5 in PKA site T761 affects its function and cellular localization
Get PDFRsp5 ubiquitin ligase belongs to the Nedd4 family proteins, which affect a wide variety of processes in the cell. [ ] Here we document that Rsp5 shows several phosphorylated variants of different mobility and the migration of the phosphorylated forms of Rsp5 was faster for a tpk1tpk3 mutant devoid of two alternative catalytic subunits of protein kinase A (PKA), indicating that PKA possibly phosphorylates Rsp5 in vivo. We demonstrated by immunoprecipitation and Western blot analysis of GFP-HA Rsp5 protein using an anti-PKA-phosphopeptide antibody that Rsp5 is phosphorylated in PKA sites. [ ] Rsp5 contains the sequence 758-RRFTIE-763 in the catalytic domain and four other sites with consensus RXXS/T, which might be phosphorylated by PKA. The strain bearing the T761D substitution in Rsp5 which mimics phosphorylation grew more slowly at 28oC, and did not grow at 37oC and showed defects in pre-tRNA processing and protein sorting. The rsp5-T761D strain also demonstrated a reduced ability to form colonies, an increase in the level of reactive oxygen species (ROS) and hypersensitivity to ROS-generating agents. These results indicate that PKA may downregulate many functions of Rsp5, possibly affecting its activity. Rsp5 is found in the cytoplasm, nucleus, multivesicular body and cortical patches. The rsp5-T761D mutation led to a strongly increased cortical localization while rsp5-T761A causes mutant Rsp5 to locate in internal spots. Rsp5-T761A protein is phosphorylated or not depending on the growth conditions. Thus, Rsp5 is phosphorylated by PKA in T761 and other site(s) and mimicking phosphorylation in T761 affects its localization and function
Defects in RNA polyadenylation impair both lysogenization by and lytic development of Shiga toxin-converting bacteriophages
Get PDFIn Escherichia coli, the major poly(A) polymerase (PAP I) is encoded by the pcnB gene. In this
report, a significant impairment of lysogenization by Shiga toxin-converting (Stx) bacteriophages
(W24B, 933W, P22, P27 and P32) is demonstrated in host cells with a mutant pcnB gene.
Moreover, lytic development of these phages after both infection and prophage induction was
significantly less efficient in the pcnB mutant than in the WT host. The increase in DNA
accumulation of the Stx phages was lower under conditions of defective RNA polyadenylation.
Although shortly after prophage induction, the levels of mRNAs of most phage-borne early genes
were higher in the pcnB mutant, at subsequent phases of the lytic development, a drastically
decreased abundance of certain mRNAs, including those derived from the N, O and Q genes,
was observed in PAP I-deficient cells. All of these effects observed in the pcnB cells were
significantly more strongly pronounced in the Stx phages than in bacteriophage l. Abundance of
mRNA derived from the pcnB gene was drastically increased shortly (20 min) after prophage
induction by mitomycin C and decreased after the next 20 min, while no such changes were
observed in non-lysogenic cells treated with this antibiotic. This prophage induction-dependent
transient increase in pcnB transcript may explain the polyadenylation-driven regulation of phage
gene expression