Hereditary xerocytosis - spectrum and clinical manifestations of variants in the PIEZO1 gene, including co-occurrence with a novel ß-globin mutation.

Hereditary xerocytosis (HX) is a rare, autosomal dominant congenital hemolytic anemia (CHA) characterized by erythrocyte dehydration with presentation of various degrees of hemolytic anemia. HX is often misdiagnosed as hereditary spherocytosis or other CHA. Here we report three cases of suspected HX and one case of HX associated with ß-thalassemia. Sanger method was used for sequencing cDNA of the PIEZO1 gene. Variants were evaluated for potential pathogenicity by MutationTaster, PROVEAN, PolyPhen-2 and M-CAP software, and by molecular modeling. Four different variants in the PIEZO1 gene were found, including three substitutions (p.D669H, p.D1566G, p.T1732 M) and one deletion (p.745delQ). In addition, in the patient with the p.T1732 M variant we detected a 12-nucleotide deletion in the ß-globin gene leading to a deletion of amino acids 62AHGK65. The joint presence of mutations in two different genes connected with erythrocytes markedly aggravated the presentation of the disease. Bioinformatic analysis and molecular modeling strongly indicated likely deleterious effects of all four PIEZO1 variants, but co-segregation analysis showed that the p.D1566G substitution is in fact non-pathogenic. Identification of causative mutations should improve the diagnosis and management of HX and provide a new insight into the molecular basis of this complex red blood cell abnormality.

Two mutations in mitochondrial ATP6 gene of ATP synthase, related to human cancer, affect ROS, calcium homeostasis and mitochondrial permeability transition in yeast.

The relevance of mitochondrial DNA (mtDNA) mutations in cancer process is still unknown. Since the mutagenesis of mitochondrial genome in mammals is not possible yet, we have exploited budding yeast S. cerevisiae as a model to study the effects of tumor-associated mutations in the mitochondrial MTATP6 gene, encoding subunit 6 of ATP synthase, on the energy metabolism. We previously reported that four mutations in this gene have a limited impact on the production of cellular energy. Here we show that two mutations, Atp6-P163S and Atp6-K90E (human MTATP6-P136S and MTATP6-K64E, found in prostate and thyroid cancer samples, respectively), increase sensitivity of yeast cells both to compounds inducing oxidative stress and to high concentrations of calcium ions in the medium, when Om45p, the component of porin complex in outer mitochondrial membrane (OM), was fused to GFP. In OM45-GFP background, these mutations affect the activation of yeast permeability transition pore (yPTP, also called YMUC, yeast mitochondrial unspecific channel) upon calcium induction. Moreover, we show that calcium addition to isolated mitochondria heavily induced the formation of ATP synthase dimers and oligomers, recently proposed to form the core of PTP, which was slower in the mutants. We show the genetic evidence for involvement of mitochondrial ATP synthase in calcium homeostasis and permeability transition in yeast. This paper is a first to show, although in yeast model organism, that mitochondrial ATP synthase mutations, which accumulate during carcinogenesis process, may be significant for cancer cell escape from apoptosis.

Two novel C-terminal frameshift mutations in the ß-globin gene lead to rapid mRNA decay.

BACKGROUND: The thalassemia syndromes are classified according to the globin chain or chains whose production is affected. ß-thalassemias are caused by point mutations or, more rarely, deletions or insertions of a few nucleotides in the ß-globin gene or its immediate flanking sequences. These mutations interfere with the gene function either at the transcriptional, translational or posttranslational level. METHODS: Two cases of Polish patients with hereditary hemolytic anemia suspected of thalassemia were studied. DNA sequencing and mRNA quantification were performed. Stable human cell lines which express wild-type HBB and mutated versions were used to verify that detected mutation are responsible for mRNA degradation. RESULTS: We identified two different frameshift mutations positioned in the third exon of HBB. Both patients harboring these mutations present the clinical phenotype of thalassemia intermedia and showed dominant pattern of inheritance. In both cases the mutations do not generate premature stop codon. Instead, slightly longer protein with unnatural C-terminus could be produced. Interestingly, although detected mutations are not expected to induce NMD, the mutant version of mRNA is not detectable. Restoring of the open reading frame brought back the RNA to that of the wild-type level. CONCLUSION: Our results show that a lack of natural stop codon due to the frameshift in exon 3 of ß-globin gene causes rapid degradation of its mRNA and indicate existence of novel surveillance pathway.

Mimicking the phosphorylation of Rsp5 in PKA site T761 affects its function and cellular localization.

Rsp5 ubiquitin ligase belongs to the Nedd4 family of 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 the tpk1Δ tpk3Δ 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 the anti-phospho PKA substrate antibody that Rsp5 is phosphorylated in PKA sites. Rsp5 contains the sequence 758-RRFTIE-763 with consensus RRXS/T in the catalytic HECT 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 28°C and did not grow at 37°C, 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 caused mutant Rsp5 to locate more efficiently in internal spots. Rsp5-T761A protein was phosphorylated less efficiently in PKA sites under specific growth conditions. Our data suggests that Rsp5 may be phosphorylated by PKA at position T761 and that this regulation is important for its localization and function.

Hem12, an enzyme of heme biosynthesis pathway, is monoubiquitinated by Rsp5 ubiquitin ligase in yeast cells.

Heme biosynthesis pathway is conserved in yeast and humans and hem12 yeast mutants mimic porphyria cutanea tarda (PCT), a hereditary human disease caused by mutations in the UROD gene. Even though mutations in other genes also affect UROD activity and predispose to sporadic PCT, the regulation of UROD is unknown. Here, we used yeast as a model to study regulation of Hem12 by ubiquitination and involvement of Rsp5 ubiquitin ligase in this process. We found that Hem12 is monoubiquitinated in vivo by Rsp5. Hem12 contains three conserved lysine residues located on the protein surface that can potentially be ubiquitinated and lysine K8 is close to the 36-LPEY-39 (PY) motif which binds WW domains of the Rsp5 ligase. The hem12-K8A mutation results in a defect in cell growth on a glycerol medium at 38°C but it does not affect the level of Hem12. The hem12-L36A,P37A mutations which destroy the PY motif result in a more profound growth defect on both, glycerol and glucose-containing media. However, after several passages on the glucose medium, the hem12-L36A,P37A cells adapt to the growth medium owing to higher expression of hem12-L36A,P37A gene and higher stability of the mutant Hem12-L36A,P37A protein. The Hem12 protein is downregulated upon heat stress in a Rsp5-independent way. Thus, Rsp5-dependent Hem12 monoubiquitination is important for its functioning, but not required for its degradation. Since Rsp5 has homologs among the Nedd4 family of ubiquitin ligases in humans, a similar regulation by ubiquitination might be also important for functioning of the human UROD.

Dissection of the region of Pseudomonas aeruginosa ParA that is important for dimerization and interactions with its partner ParB.

Pseudomonas aeruginosa ParA belongs to a large subfamily of Walker-type ATPases acting as partitioning proteins in bacteria. ParA has the ability to both self-associate and interact with its partner ParB. Analysis of the deletion mutants defined the part of the protein involved in dimerization and interactions with ParB. Here, a set of ParA alanine substitution mutants in the region between E67 and L85 was created and analysed in vivo and in vitro. All mutants impaired in dimerization (substitutions at positions M74, H79, Y82 and L84) were also defective in interactions with ParB, suggesting that ParA-ParB interactions depend on the ability of ParA to dimerize. Mutants with alanine substitutions at positions E67, C68, L70, E72, F76, Q83 and L85 were not impaired in dimerization, but were defective in interactions with ParB. The dimerization interface partly overlapped the pseudo-hairpin, involved in interactions with ParB. ParA mutant derivatives tested in vitro showed no defects in ATPase activity. Two parA alleles (parA84, whose product can neither self-interact nor interact with ParB, and parA67, whose product is impaired in interactions with ParB, but not in dimerization) were introduced into the P. aeruginosa chromosome by homologous gene exchange. Both mutants showed defective separation of ParB foci, but to different extents. Only PAO1161 parA84 was visibly impaired in terms of chromosome segregation, growth rate and motility, similar to a parA-null mutant.

cis-Prenyltransferase atCPT6 produces a family of very short-chain polyisoprenoids in planta.

cis-Prenyltransferases (CPTs) comprise numerous enzymes synthesizing isoprenoid hydrocarbon skeleton with isoprenoid units in the cis (Z) configuration. The chain-length specificity of a particular plant CPT is in most cases unknown despite thecomposition of the accumulated isoprenoids in the tissue of interest being well established. In this report AtCPT6, one of the nine Arabidopsis thaliana CPTs, is shown to catalyze the synthesis of a family of very short-chain polyisoprenoid alcohols of six, seven, and eight isoprenoid units, those of seven units dominating The product specificity of AtCPT6 was established in vivo following its expression in the heterologous system of the yeast Saccharomyces cerevisiae and was confirmed by the absence of specific products in AtCPT6 T-DNA insertion mutants and their overaccumulation in AtCPT6-overexpressing plants. These observations are additionally validated in silico using an AtCPT6 model obtained by homology modeling. AtCPT6 only partially complements the function of the yeast homologue of CPT-Rer2 since it restores the growth but not protein glycosylation in rer2delta yeast.This is the first in planta characterization of specific products of a plant CPT producing polyisoprenoids. Their distribution suggests that a joint activity of several CPTs is required to produce the complex mixture of polyisoprenoid alcohols found in Arabidopsis roots.

The effects of statins on the mevalonic acid pathway in recombinant yeast strains expressing human HMG-CoA reductase.

BACKGROUND: The yeast Saccharomyces cerevisiae can be a useful model for studying cellular mechanisms related to sterol synthesis in humans due to the high similarity of the mevalonate pathway between these organisms. This metabolic pathway plays a key role in multiple cellular processes by synthesizing sterol and nonsterol isoprenoids. Statins are well-known inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the cholesterol synthesis pathway. However, the effects of statins extend beyond their cholesterol-lowering action, since inhibition of HMGR decreases the synthesis of all products downstream in the mevalonate pathway. Using transgenic yeast expressing human HMGR or either yeast HMGR isoenzyme we studied the effects of simvastatin, atorvastatin, fluvastatin and rosuvastatin on the cell metabolism. RESULTS: Statins decreased sterol pools, prominently reducing sterol precursors content while only moderately lowering ergosterol level. Expression of genes encoding enzymes involved in sterol biosynthesis was induced, while genes from nonsterol isoprenoid pathways, such as coenzyme Q and dolichol biosynthesis or protein prenylation, were diversely affected by statin treatment. Statins increased the level of human HMGR protein substantially and only slightly affected the levels of Rer2 and Coq3 proteins involved in non-sterol isoprenoid biosynthesis. CONCLUSION: Statins influence the sterol pool, gene expression and protein levels of enzymes from the sterol and nonsterol isoprenoid biosynthesis branches and this effect depends on the type of statin administered. Our model system is a cheap and convenient tool for characterizing individual statins or screening for novel ones, and could also be helpful in individualized selection of the most efficient HMGR inhibitors leading to the best response and minimizing serious side effects.

A remote palm domain residue of RB69 DNA polymerase is critical for enzyme activity and influences the conformation of the active site.

Non-conserved amino acids that are far removed from the active site can sometimes have an unexpected effect on enzyme catalysis. We have investigated the effects of alanine replacement of residues distant from the active site of the replicative RB69 DNA polymerase, and identified a substitution in a weakly conserved palm residue (D714A), that renders the enzyme incapable of sustaining phage replication in vivo. D714, located several angstroms away from the active site, does not contact the DNA or the incoming dNTP, and our apoenzyme and ternary crystal structures of the Pol(D714A) mutant demonstrate that D714A does not affect the overall structure of the protein. The structures reveal a conformational change of several amino acid side chains, which cascade out from the site of the substitution towards the catalytic center, substantially perturbing the geometry of the active site. Consistent with these structural observations, the mutant has a significantly reduced k pol for correct incorporation. We propose that the observed structural changes underlie the severe polymerization defect and thus D714 is a remote, non-catalytic residue that is nevertheless critical for maintaining an optimal active site conformation. This represents a striking example of an action-at-a-distance interaction.

Direct targeting of Arabidopsis cysteine synthase complexes with synthetic polypeptides to selectively deregulate cysteine synthesis.

Biosynthesis of cysteine is one of the fundamental processes in plants providing the reduced sulfur for cell metabolism. It is accomplished by the sequential action of two enzymes, serine acetyltransferase (SAT) and O-acetylserine (thiol) lyase (OAS-TL). Together they constitute the hetero-oligomeric cysteine synthase (CS) complex through specific protein-protein interactions influencing the rate of cysteine production. The aim of our studies was to deregulate the CS complex formation in order to investigate its function in the control of sulfur homeostasis and optimize cysteine synthesis. Computational modeling was used to build a model of the Arabidopsis thaliana mitochondrial CS complex. Several polypeptides based on OAS-TL C amino-acid sequence found at SAT-OASTL interaction sites were designed as probable competitors for SAT3 binding. After verification of the binding in a yeast two-hybrid assay, the most strongly interacting polypeptide was introduced to different cellular compartments of Arabidopsis cell via genetic transformation. Moderate increase in total SAT and OAS-TL activities, but not thiols content, was observed dependent on the transgenic line and sulfur availability in the hydroponic medium. Though our studies demonstrate the proof of principle, they also suggest more complex interaction of both enzymes underlying the mechanism of their reciprocal regulation.

Nuclear import and export signals of human cohesins SA1/STAG1 and SA2/STAG2 expressed in Saccharomyces cerevisiae.

BACKGROUND: Human SA/STAG proteins, homologues of the yeast Irr1/Scc3 cohesin, are the least studied constituents of the sister chromatid cohesion complex crucial for proper chromosome segregation. The two SA paralogues, SA1 and SA2, show some specificity towards the chromosome region they stabilize, and SA2, but not SA1, has been shown to participate in transcriptional regulation as well. The molecular basis of this functional divergence is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In silico analysis indicates numerous putative nuclear localization (NLS) and export (NES) signals in the SA proteins, suggesting the possibility of their nucleocytoplasmic shuttling. We studied the functionality of those putative signals by expressing fluorescently tagged SA1 and SA2 in the yeast Saccharomyces cerevisiae. Only the N-terminal NLS turned out to be functional in SA1. In contrast, the SA2 protein has at least two functional NLS and also two functional NES. Depending on the balance between these opposing signals, SA2 resides in the nucleus or is distributed throughout the cell. Validation of the above conclusions in HeLa cells confirmed that the same N-terminal NLS of SA1 is functional in those cells. In contrast, in SA2 the principal NLS functioning in HeLa cells is different from that identified in yeast and is localized to the C-terminus. CONCLUSIONS/SIGNIFICANCE: This is the first demonstration of the possibility of non-nuclear localization of an SA protein. The reported difference in the organization between the two SA homologues may also be relevant to their partially divergent functions. The mechanisms determining subcellular localization of cohesins are only partially conserved between yeast and human cells.

Different statins produce highly divergent changes in gene expression profiles of human hepatoma cells: a pilot study.

Statins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the sterol biosynthesis pathway. Statin therapy is commonly regarded as well tolerated. However, serious adverse effects have also been reported, especially during high-dose statin therapy. The aim of our study was to investigate the effect of statins on gene expression profiles in human hepatoma HepG2 cells using Affymetrix Human Genome U133 Plus 2.0 arrays. Expression of 102, 857 and 1091 genes was changed substantially in HepG2 cells treated with simvastatin, fluvastatin and atorvastatin, respectively. Pathway and gene ontology analysis showed that many of the genes with changed expression levels were involved in a broad range of metabolic processes. The presented data clearly indicate substantial differences between the tested statins.

Yeast ubiquitin ligase Rsp5 contains nuclear localization and export signals.

The Rsp5 ubiquitin ligase regulates numerous cellular processes. Rsp5 is mainly localized to the cytoplasm but nuclear localization was also reported. A potential nuclear export signal was tested for activity by using a GFP(2) reporter. The 687-LIGGIAEIDI-696 sequence located in the Hect domain was identified as a nuclear export signal active in a Crm1-dependent manner, and its importance for the localization of Rsp5 was documented by using fluorescence microscopy and a lacZ-based reporter system. Analysis of the cellular location of other Rsp5 fragments fused with GFP(2) indicated two independent potential nuclear localization signals, both located in the Hect domain. We also uncovered Rsp5 fragments that are important to targeting/tethering Rsp5 to various regions in the cytoplasm. The presented data indicate that Rsp5 ligase is a shuttling protein whose distribution within the cytoplasm and partitioning between cytoplasmic and nuclear locations is determined by a balance between the actions of several targeting sequences and domains.

Binding and spreading of ParB on DNA determine its biological function in Pseudomonas aeruginosa.

ParB protein of Pseudomonas aeruginosa belongs to a widely represented ParB family of chromosomally and plasmid-encoded partitioning type IA proteins. Ten putative parS sites are dispersed in the P. aeruginosa chromosome, with eight of them localizing in the oriC domain. After binding to parS, ParB spreads on the DNA, causing transcriptional silencing of nearby genes (A. A. Bartosik et al., J. Bacteriol. 186:6983-6998, 2004). We have studied ParB derivatives impaired in spreading either due to loss of DNA-binding ability or oligomerization. We defined specific determinants outside of the helix-turn-helix motif responsible for DNA binding. Analysis confirmed the localization of the main dimerization domain in the C terminus of ParB but also mapped another self-interactive domain in the N-terminal domain. Reverse genetics were used to introduce five parB alleles impaired in spreading into the P. aeruginosa chromosome. The single amino acid substitutions in ParB causing a defect in oligomerization but not in DNA binding caused a chromosome segregation defect, slowed the growth rate, and impaired motilities, similarly to the pleiotropic phenotype of parB-null mutants, indicating that the ability to spread is vital for ParB function in the cell. The toxicity of ParB overproduction in Pseudomonas spp. is not due to the spreading since several ParB derivatives defective in oligomerization were still toxic for P. aeruginosa when provided in excess.

Investigating the effects of statins on cellular lipid metabolism using a yeast expression system.

In humans, defects in lipid metabolism are associated with a number of severe diseases such as atherosclerosis, obesity and type II diabetes. Hypercholesterolemia is a primary risk factor for coronary artery disease, the major cause of premature deaths in developed countries. Statins are inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the key enzyme of the sterol synthesis pathway. Since yeast Saccharomyces cerevisiae harbours many counterparts of mammalian enzymes involved in lipid-synthesizing pathways, conclusions drawn from research with this single cell eukaryotic organism can be readily applied to higher eukaryotes. Using a yeast strain with deletions of both HMG1 and HMG2 genes (i.e. completely devoid of HMGR activity) with introduced wild-type or mutant form of human HMGR (hHMGR) gene we investigated the effects of statins on the lipid metabolism of the cell. The relative quantification of mRNA demonstrated a different effect of simvastatin on the expression of the wild-type and mutated hHMGR gene. GC/MS analyses showed a significant decrease of sterols and enhanced conversion of squalene and sterol precursors into ergosterol. This was accompanied by the mobilization of ergosterol precursors localized in lipid particles in the form of steryl esters visualized by confocal microscopy. Changes in the level of ergosterol and its precursors in cells treated with simvastatin depend on the mutation in the hHMGR gene. HPLC/MS analyses indicated a reduced level of phospholipids not connected with the mevalonic acid pathway. We detected two significant phenomena. First, cells treated with simvastatin develop an adaptive response compensating the lower activity of HMGR. This includes enhanced conversion of sterol precursors into ergosterol, mobilization of steryl esters and increased expression of the hHMGR gene. Second, statins cause a substantial drop in the level of glycerophospholipids.

[The C1155G mutation of the red blood cell glucose-6-phosphate dehydrogenase gene in a subject with severe hereditary chronic nonspherocytic anaemia]. / Mutacja C1155G genu dehydrogenazy 6-glukozo-fosforanowej krwinek czerwonych u chorego z ciezka wrodzona niesferocytowa niedokrwistoscia hemolityczna.

UNLABELLED: THE AIM of the study is a genetic analysis of hereditary chronic nonspherocytic anaemia in a case, caused by mutation in the glucose-6-phosphate dehydrogenase gene. MATERIALS AND METHODS: The activity of G6PD enzyme was established. PCR method and DNA sequencing were implemented for molecular studies. Bioinformatic methods were used to check the effect of the mutation on the enzyme structure. RESULTS: Direct sequencing of g6pd gene revealed the presence of 1155 C > G mutation which results in cysteine to tryptophan substitution at position 385. Bioinformatic analysis established that this mutation may be responsible for protein destabilization. CONCLUSIONS: 1. G6PD deficiency should be considered in patients with haemolytic anaemia of unknown etiology. 2. Molecular tests are necessary, especially in cases of suspected mutation carriers in G6PD gene.

Novel beta-spectrin mutations in hereditary spherocytosis associated with decreased levels of mRNA.

Hereditary spherocytosis (HS) is one of the most frequent and heterogeneous inherited haemolytic anaemias. It is associated with abnormalities of several erythrocyte membrane proteins. We investigated relative mRNA quantification of red blood cell membrane protein genes using real-time quantitative polymerase chain reaction (qPCR) in order to better characterize HS cases and to select genes to search for mutations in patients with spherocytosis. qPCR experiments indicated that the spectrin beta gene (SPTB) could be involved in anaemia pathogenesis. DNA analysis of SPTB in the HS subjects with decreased SPTB mRNA levels revealed the presence of five previously undescribed mutations: R1756X, 781delT and IVS22nt-4G>A, 1502insA and IVS20nt-2A>G.

Different behaviors in vivo of mutations in the beta hairpin loop of the DNA polymerases of the closely related phages T4 and RB69.

The T4 and RB69 DNA replicative polymerases are members of the B family and are highly similar. Both replicate DNA with high fidelity and employ the same mechanism that allows efficient switching of the primer terminus between the polymerase and exonuclease sites. Both polymerases have a beta hairpin loop (hereafter called the beta loop) in their exonuclease domains that plays an important role in active-site switching. The beta loop is involved in strand separation and is needed to stabilize partially strand-separated exonuclease complexes. In T4 DNA polymerase, modification of the beta-loop residue G255 to Ser confers a strong mutator phenotype in vivo due to a reduced ability to form editing complexes. Here, we describe the RB69 DNA polymerase mutant with the equivalent residue (G258) changed to Ser but showing only mild mutator activity in vivo. On the other hand, deletion of the tip of the RB69 beta loop confers a strong mutator phenotype in vivo. Based on detailed mutational spectral analyses, DNA binding activities, and coupled polymerase/exonuclease assays, we define the differences between the T4 and RB69 polymerases. We propose that their beta loops facilitate strand separation in both polymerases, while the residues that form the loop have low structural constraints.