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Results for “Loss of Function Mutation”

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PubMed1999

Functional characterization of the MODY1 gene mutations HNF4(R127W), HNF4(V255M), and HNF4(E276Q).

Genetic studies have shown that mutations in the gene encoding hepatocyte nuclear factor (HNF)-4alpha, a member of the steroid/thyroid hormone receptor superfamily, give rise to early-onset type 2 diabetes (MODY1). The functional properties of mutant HNF-4alpha proteins and the molecular mechanisms by which they impair insulin secretion are largely unknown. In the present study, we have investigated transcriptional activation, DNA binding properties, and protein dimerization activity of three HNF-4alpha missense mutations--HNF4(R127W), HNF4(V255M), and HNF4(E276Q)--that have been associated with type 2 diabetes. We demonstrate that HNF4(E276Q) has lost its ability to bind to HNF-4 consensus binding sites and activate transcription. HNF4(E276Q) had no effect on the functional activity of wild-type HNF-4alpha in the pancreatic beta-cell line HIT-T15, but it exhibited weak dominant-negative activity in other cell types. Analysis of HNF4(E276Q) protein showed that it exists in two forms: a full length 54-kDa protein and a 40-kDa COOH-terminal protein lacking the NH2-terminal transactivation domain and the DNA binding domain. Immunoprecipitation experiments indicate that this truncated protein can bind to wild-type HNF-4alpha and may be responsible for the weak dominant-negative effects seen in these cells. In addition, we show that the transcriptional transactivation of HNF4(R127W) and HNF4(V255M) is indistinguishable from that of wild-type HNF-4alpha, suggesting that they are sequence polymorphisms. Our results demonstrate that HNF4(E276Q) is a loss-of-function mutation and that it identifies glutamic acid 276 in alpha-helix 8 of the ligand-binding domain of HNF-4alpha protein as a critical residue for DNA binding, transcriptional activation, and protein stability in vivo.

Animals↗
PubMedDate not supplied

Reconstitution of TGF-beta sensitivity in the VACO-411 human colon carcinoma line by somatic cell fusion with MCF-7.

We characterized the mechanism of transforming growth factor beta (TGF-beta) resistance in the VACO-411 human colon carcinoma line. VACO-411 is unique for several reasons, including having a novel mutator phenotype and wild-type p53. Like many colon tumors, VACO-411 is not growth inhibited by TGF-beta. However, VACO-411 represents a subset of colon tumors that are resistant to TGF-beta-mediated growth inhibition, despite the expression of functional TGF-beta receptors. VACO-411 expresses cell surface TGF-beta receptor types I and II, and the coding regions of these receptors are wild type. To further characterize the nature of the VACO-411 defect, we fused VACO-411 with the human breast carcinoma line MCF-7. MCF-7 is also resistant to TGF-beta-mediated growth inhibition. However, unlike VACO-411, MCF-7 lacks cell surface expression of TGF-beta receptor type II, but does contain an intact postreceptor signaling pathway, as shown by regeneration of TGF-beta sensitivity following wild-type TGF-beta receptor type II transfection. In contrast to parental VACO-411 and MCF-7, the morphologically distinct cell hybrids were growth inhibited by TGF-beta. Therefore, the TGF-beta defect in VACO-411 is a postreceptor, loss-of-function mutation which can be genetically complemented. The data suggest that the VACO-411 defect in TGF-beta signaling will be able to be further complemented by microcell-mediated chromosome transfer.

Breast Neoplasms↗
PubMed2004

Genetic and biochemical analyses of Pfh1 DNA helicase function in fission yeast.

The Schizosaccharomyces pombe pfh1+ gene (PIF1 homolog) encodes an essential enzyme that has both DNA helicase and ATPase activities and is implicated in lagging strand DNA processing. Mutations in the pfh1+ gene suppress a temperature-sensitive allele of cdc24+, which encodes a protein that functions with Schizosaccharomyces pombe Dna2 in Okazaki fragment processing. In this study, we describe the enzymatic properties of the Pfh1 helicase and the genetic interactions between pfh1 and cdc24, dna2, cdc27 or pol 3, all of which are involved in the Okazaki fragment metabolism. We show that a full-length Pfh1 fusion protein is active as a monomer. The helicase activity of Pfh1 displaced only short (<30 bp) duplex DNA regions efficiently in a highly distributive manner and was markedly stimulated by the presence of a replication-fork-like structure in the substrate. The temperature-sensitive phenotype of a dna2-C2 or a cdc24-M38 mutant was suppressed by pfh1-R20 (a cold-sensitive mutant allele of pfh1) and overexpression of wild-type pfh1+ abolished the ability of the pfh1 mutant alleles to suppress dna2-C2 and cdc24-M38. Purified Pfh1-R20 mutant protein displayed significantly reduced ATPase and helicase activities. These results indicate that the simultaneous loss-of-function mutations of pfh1+ and dna2+ (or cdc24+) are essential to restore the growth defect. Our genetic data indicate that the Pfh1 DNA helicase acts in concert with Cdc24 and Dna2 to process single-stranded DNA flaps generated in vivo by pol -mediated lagging strand displacement DNA synthesis.

Adenosine Triphosphatases↗
PubMed2004

Basal cell carcinoma and its development: insights from radiation-induced tumors in Ptch1-deficient mice.

Loss-of-function mutations in Patched (Ptch1) are implicated in constitutive activation of the Sonic hedgehog pathway in human basal cell carcinomas (BCCs), and inherited Ptch1 mutations underlie basal cell nevus syndrome in which a typical feature is multiple BCC occurring with greater incidence in portals of radiotherapy. Mice in which one copy of Ptch1 is inactivated show increased susceptibility to spontaneous tumor development and hypersensitivity to radiation-induced tumorigenesis, providing an ideal in vivo model to study the typical pathologies associated with basal cell nevus syndrome. We therefore examined BCC development in control and irradiated Ptch1(neo67/+) mice. We show that unirradiated mice develop putative BCC precursor lesions, i.e., basaloid hyperproliferation areas arising from both follicular and interfollicular epithelium, and that these lesions progress to nodular and infiltrative BCCs only in irradiated mice. Data of BCC incidence, multiplicity, and latency support the notion of epidermal hyperproliferations, nodular and infiltrative BCC-like tumors representing different stages of tumor development. This is additionally supported by the pattern of p53 protein expression observed in BCC subtypes and by the finding of retention of the normal remaining Ptch1 allele in all nodular, circumscribed BCCs analyzed compared with its constant loss in infiltrative BCCs. Our data suggest chronological tumor progression from basaloid hyperproliferations to nodular and then infiltrative BCC occurring in a stepwise fashion through the accumulation of sequential genetic alterations.

Alleles↗
PubMed2004

A novel 1p36.2 located gene, APITD1, with tumour-suppressive properties and a putative p53-binding domain, shows low expression in neuroblastoma tumours.

Neuroblastoma is characterised by a lack of TP53 mutations and no other tumour suppressor gene consistently inactivated has yet been identified in this childhood cancer form. Characterisation of a new gene, denoted APITD1, in the neuroblastoma tumour suppressor candidate region in chromosome 1p36.22 reveals that APITD1 contains a predicted TFIID-31 domain, representing the TATA box-binding protein-associated factor, TAF(II)31, which is required for p53-mediated transcription activation. Two different transcripts of this gene were shown to be ubiquitously expressed, one of them with an elevated expression in foetal tissues. Primary neuroblastoma tumours of all different stages showed either very weak or no measurable APITD1 expression, contrary to the level of expression observed in neuroblastoma cell lines. A reduced pattern of expression was also observed in a set of various tumour types. APITD1 was functionally tested by adding APITD1 mRNA to neuroblastoma cells, leading to the cell growth to be reduced up to 90% compared to control cells, suggesting APITD1 to have a role in a cell death pathway. Furthermore, we determined the genomic organisation of APITD1. Automated genomic DNA sequencing of the coding region of the gene as well as the promoter sequence in 44 neuroblastoma tumours did not reveal any loss-of-function mutations, indicating that mutations in APITD1 is not a common abnormality of neuroblastoma tumours. We suggest that low expression of this gene might interfere with the ability for apoptosis through the p53 pathway.

Amino Acid Sequence↗
PubMed2001

ATP-binding cassette transporter A1 contains an NH2-terminal signal anchor sequence that translocates the protein's first hydrophilic domain to the exoplasmic space.

Mutations in the ATP-binding cassette transporter A1 (ABCA1) transporter are associated with Tangier disease and a defect in cellular cholesterol efflux. The amino terminus of the ABCA1 transporter has two putative in-frame translation initiation sites, 60 amino acids apart. A cluster of hydrophobic amino acids form a potentially cleavable signal sequence in this 60-residue extension. We investigated the functional role of this extension and found that it was required for stable protein expression of transporter constructs containing any downstream transmembrane domains. The extension directed transporter translocation across the ER membrane with an orientation that resulted in glycosylation of amino acids immediately distal to the signal sequence. Neither the native signal sequence nor a green fluorescent protein tag, fused at the amino terminus, was cleaved from ABCA1. The green fluorescent protein fusion protein had efflux activity comparable with wild type ABCA1 and demonstrated a predominantly plasma membrane distribution in transfected cells. These data establish a requirement for the upstream 60 amino acids of ABCA1. This region contains an uncleaved signal anchor sequence that positions the amino terminus in a type II orientation leading to the extracellular presentation of an approximately 600-amino acid loop in which loss-of-function mutations cluster in Tangier disease patients.

ATP-Binding Cassette Transporters↗
PubMed2006

DJ-1 transcriptionally up-regulates the human tyrosine hydroxylase by inhibiting the sumoylation of pyrimidine tract-binding protein-associated splicing factor.

Loss-of-function mutations in DJ-1 cause a subset of familial Parkinson disease (PD). However, the mechanism underlying the selective vulnerability in dopaminergic pathway due to the inactivation of DJ-1 is unclear. Previously, we have reported that DJ-1 is a neuroprotective transcriptional co-activator interacting with the transcriptional co-repressor pyrimidine tract-binding protein-associated splicing factor (PSF). Here we show that DJ-1 and PSF bind and regulate the human tyrosine hydroxylase (TH) promoter. Inactivation of DJ-1 by small interference RNA (siRNA) results in decreased TH expression and l-DOPA production in human dopaminergic cell lines. Consistent with its role as a transcriptional regulator, DJ-1 specifically suppresses the global SUMO-1 modification. High molecular weight sumoylated protein species, including PSF, accumulate in the lymphoblast cells from the patients carrying pathogenic DJ-1 mutations. DJ-1 elevates the TH expression by inhibiting the sumoylation of PSF and preventing its sumoylation-dependent recruitment of histone deacetylase 1. Furthermore, siRNA silencing of DJ-1 decreases the acetylation of TH promoter-bound histones, and histone deacetylase inhibitors restore the DJ-1 siRNA-induced repression of TH. Therefore, our results suggest DJ-1 as a regulator of protein sumoylation and directly link the loss of DJ-1 expression and transcriptional dysfunction to impaired dopamine synthesis.

Humans↗
PubMed2003

A mutation causing a reduced level of expression of six beta4-galactosyltransferase genes is the basis of the Lec19 CHO glycosylation mutant.

To identify factors required for the synthesis of complex glycans, we have isolated Chinese hamster ovary (CHO) cell mutants resistant to plant lectins. We previously identified Lec19 CHO cells as resistant to the Gal-binding lectins ricin, abrin, and modeccin and hypersensitive to the toxicity of other lectins that bind Gal, including L-PHA and E-PHA. Here we show that Lec19 cell extracts have a decreased ability to transfer Gal to simple sugar, oligosaccharide, and glycopeptide acceptors, particularly to biantennary, GlcNAc-terminated acceptors. Ricin(II)-agarose lectin affinity chromatography, oligomapping, and monosaccharide analyses provided evidence that Lec19 N-glycans have fewer Gal residues than CHO N-glycans. MALDI-TOF mass spectra of N-glycans released from Lec19 cell glycoproteins by peptide N-glycanase F revealed species with the predicted masses of neutral N-glycans with few Gal residues. Such truncated species are essentially absent from CHO cell glycoproteins. However, the complement of fully galactosylated or sialylated bi-, tri-, and tetra-antennary N-glycans was largely equivalent in Lec19 and CHO cells. In addition, the coding region sequences of the beta4GalT-1, -T-2, -T-3, -T-4, -T-5, and -T-6 genes were identical in CHO and Lec19 cells. However, Northern analyses revealed an approximately 2-4-fold reduction in the level of transcripts of all six beta4GalT genes in Lec19 cells. Since the recessive Lec19 phenotype is the result of a loss-of-function mutation, the combined data predict the existence of a trans-acting regulator of the steady-state level of transcripts that derive from these six mammalian beta4GalT genes.

Animals↗
PubMed2005

Genetic models show that parathyroid hormone and 1,25-dihydroxyvitamin D3 play distinct and synergistic roles in postnatal mineral ion homeostasis and skeletal development.

In humans, loss-of-function mutations in parathyroid hormone (PTH) and 25-hydroxyvitamin D3-1alpha-hydroxylase [1alpha(OH)ase] genes lead to isolated hypoparathyroidism and vitamin D-dependent rickets type I, respectively. To better understand the relative contributions of PTH and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] to skeletal and calcium homeostasis, we compared mice with targeted disruption of the PTH or 1alpha(OH)ase genes to the double null mutants. Although PTH-/- and 1alpha(OH)ase-/- mice displayed only moderate hypocalcemia, PTH-/-1alpha(OH)ase-/- mice died of tetany with severe hypocalcemia by 3 weeks of age. At 2 weeks, PTH-/- mice exhibited only minimal dysmorphic changes, whereas 1alpha(OH)ase-/- mice displayed epiphyseal dysgenesis which was most severe in the double mutants. Although reduced osteoblastic bone formation was seen in both mutants, PTH deficiency caused only a slight reduction in long bone length but a marked reduction in trabecular bone volume, whereas 1alpha(OH)ase ablation caused a smaller reduction in trabecular bone volume but a significant decrease in bone length. The results therefore show that PTH plays a predominant role in appositional bone growth, whereas 1,25(OH)2D3 acts predominantly on endochondral bone formation. Although PTH and 1,25(OH)2D3 independently, but not additively, regulate osteoclastic bone resorption, they do affect the renal calcium transport pathway cooperatively. Consequently, PTH and 1,25(OH)2D3 exhibit discrete and collaborative roles in modulating skeletal and calcium homeostasis and loss of the renal component of calcium conservation might be the major factor contributing to the lethal hypocalcemia in double mutants.

Animals↗
PubMed2003

ROMK is required for expression of the 70-pS K channel in the thick ascending limb.

Apical potassium recycling is crucial for salt transport by the thick ascending limb (TAL). Loss-of-function mutations in the K channel, ROMK (Kir1.1; KCNJ1), cause Bartter syndrome, a genetically heterogeneous disorder characterized by severe reduction in salt absorption by the TAL, Na wasting, polyuria, and hypokalemic alkalosis. ROMK(-/-) null mice exhibit a Bartter phenotype and lack the small-conductance (30-pS) apical K channel (SK) in the TAL. However, a distinct 70-pS K channel can also significantly contribute to the apical conductance of TAL. We now examine the effect of ROMK deletion on the functional expression of the 70-pS K channel in the TAL. Functional expression of the 70-pS K channel was low [average channel activity (NP(o)) = 0.02] in ROMK(+/+) mice on a control K diet but increased to 0.27 by high-K intake for 2 wk. In contrast, the high-K diet decreased NP(o) of SK by approximately 30%, from 2.04 to 1.44. In ROMK heterozygous (+/-) mice on a control K diet, SK activity was about one-half of that observed in ROMK(+/+) mice (0.95 vs. 2.04). The high-K diet also reduced SK activity in ROMK(+/-) mice by approximately 40% (from 0.95 to 0.55) but increased NP(o) of the 70-pS K channel from 0 to 0.09 in ROMK(+/-) mice. This corresponds to approximately 30% of channel activity (NP(o) = 0.27) observed in ROMK(+/+) mice. Neither the 70-pS nor the 30-pS K channels were observed in TAL cells from ROMK(-/-) mice on either the normal or high-K diets. Thus functional expression of the 70-pS K channel is enhanced by increasing dietary K and requires expression of ROMK. It is likely that ROMK forms a critical subunit of the 70-pS K channel, accounting for the loss of apical K secretory channel activity in ROMK Bartter syndrome.

Administration, Oral↗
PubMed1999

A PDR5-independent pathway of multi-drug resistance regulated by the SIN4 gene product.

The SIN4 locus encodes a global transcriptional regulator of various yeast genes. In this report, we demonstrate that loss of function mutations in SIN4 create a multi-drug hypersensitive phenotype that is independent of PDR5 mediated resistance. Thus, double sin4, pdr5 mutants are more sensitive than single mutants. Furthermore, SIN4 does not regulate the PDR5 locus. These observations establish that yeast cells have two genetically distinct pathways conferring resistance towards similar substrates.

ATP-Binding Cassette Transporters↗
PubMed1998

Midline Fasciclin: a Drosophila Fasciclin-I-related membrane protein localized to the CNS midline cells and trachea.

Drosophila Fasciclin I is the prototype of a family of vertebrate and invertebrate proteins that mediate cell adhesion and signaling. The midline fasciclin gene encodes a second Drosophila member of the Fasciclin I family. Midline Fasciclin largely consists of four 150 amino acid repeats characteristic of the Fasciclin I family of proteins. Immunostaining and biochemical analysis using Midline Fasciclin antibodies indicates that it is a membrane-associated protein, although the sequence does not reveal a transmembrane domain. The gene is expressed in a dynamic fashion during embryogenesis in the blastoderm, central nervous system midline cells, and trachea, suggesting it plays multiple developmental roles. Protein localization studies indicate that Midline Fasciclin is found within cell bodies of midline neurons and glia, and on midline axons. Initial cellular analysis of a midline fasciclin loss-of-function mutation reveals only weak defects in axonogenesis. However, embryos mutant for both midline fasciclin and the abelson nonreceptor tyrosine kinase, show more severe defects in axonogenesis that resemble fasciclin I abelson double mutant phenotypes.

Amino Acid Sequence↗
PubMed1998

V2 vasopressin receptor dysfunction in nephrogenic diabetes insipidus caused by different molecular mechanisms.

Loss-of-function mutations in the V2 vasopressin receptor (AVPR2) gene have been identified as a molecular basis for X-linked nephrogenic diabetes insipidus (NDI). Herein, we describe a novel deletion mutation at nucleotide position 102 (delG102) found in a Russian family resulting in a frameshift and a truncated receptor protein. Furthermore, we analyzed the AVPR2 gene of two other unrelated boys with NDI from our patient clientele. These patients showed previously described mutations (R137H, R181C). In-depth characterization of the three mutant AVPR2s by a combination of functional and immunological techniques permitted further insight into molecular mechanisms leading to receptor dysfunction. Premature truncation of the AVPR2 (delG102) led to a drastically reduced receptor protein expression in transfected COS-7 cells and, as expected, precluded specific AVPR2 functions. As indicated by different ELISA and binding studies, the R137H mutant was almost completely retained in the cell interior. In contrast to previous studies, the few mutant receptors in the plasma membrane displayed a low (2.3-fold above basal) but significant ability to stimulate the Gs/adenylyl cyclase system. In contrast to the latter mutation, the R181C mutant is properly delivered to the cell surface but the mutation interferes with high affinity vasopressin binding. Impaired ligand binding is reflected in an about 100-fold shift of the concentration-response curve toward higher vasopressin concentrations with only slightly reduced agonist potency.

Amino Acid Sequence↗
PubMed1998

Targeting of MOF, a putative histone acetyl transferase, to the X chromosome of Drosophila melanogaster.

Dosage compensation ensures that males with a single X chromosome have the same amount of most X-linked gene products as females with two X chromosomes. In Drosophila, this equalization is achieved by a twofold enhancement of the level of transcription of the X in males relative to each X chromosome in females. The products of at least five genes, maleless (mle), male-specific lethal 1, 2, and 3 (msl-1, msl-2, msl-3) and males absent on the first (mof), are necessary for dosage compensation. The proteins produced by these genes form a complex that is preferentially associated with numerous sites on the X chromosome in somatic cells of males but not of females. Binding of the dosage compensation complex to the X chromosome is correlated with a significant increase in the presence of a specific histone isoform, histone 4 acetylated at lysine 16, on this chromosome. Experimental results and sequence analysis suggest that the mof gene encodes an acetyl transferase that plays a direct role in the specific histone acetylation associated with dosage compensation. Recently, RNA transcripts encoded by at least two different genes have also been found associated with the X chromosome in males. We have studied the role played by the various components of the complex in the targeting of MOF to the X chromosome. To this end, we have used indirect cytoimmunofluorescence to monitor the binding of these components in males carrying complete or partial loss-of-function mutations as well as in XX individuals in which formation of the dosage compensation complex has been induced by genetic means.

Acetyltransferases↗
PubMed1999

Segment polarity genes in neuroblast formation and identity specification during Drosophila neurogenesis.

The relatively simple central nervous system (CNS) of the Drosophila embryo provides a useful model system for investigating the mechanisms that generate and pattern complex nervous systems. Central to the generation of different types of neurons by precursor neuroblasts is the initial specification of neuroblast identity and the Drosophila segment polarity genes, genes that specify regions within a segment or repeating unit of the Drosophila embryo, have emerged recently as significant players in this process. During neurogenesis the segment polarity genes are expressed in the neuroectodermal cells from which neuroblasts delaminate and they continue to be expressed in neuroblasts and their progeny. Loss-of-function mutations in these genes lead to a failure in the formation of neuroblasts and/or specification of neuroblast identity. Results from several recent studies suggest that regulatory interactions between segment polarity genes during neurogenesis lead to an increase in the number of neuroblasts and specification of different identities to neuroblasts within a population of cells.

Animals↗
PubMed2000

Fas (CD95/APO-1) plays a role in the pathophysiology of focal cerebral ischemia.

The purpose of this study was to investigate the role of fas antigen, a member of the TNF receptor family, in cell death after focal cerebral ischemia. Focal ischemia was induced in the Sprague-Dawley rat. Evidence for apoptosis was determined by morphology as well as the presence of DNA fragmentation by the end labeling technique (TUNEL). Immunohistochemistry was performed to detect expression of both fas and fas ligand (fasL). In a separate set of experiments, two groups of mice were studied: lpr (that have a loss of function mutation for fas) and wild type. Infarct volume was measured at 24 hr as well as evidence for apoptosis. Twenty-four hours after ischemia, there was evidence for apoptosis based on morphological criteria as well as the TUNEL technique in the rat. Immunohistochemistry demonstrated increased expression of both fas and fasL in the ischemic region, with maximal staining occurring between 24-48 hr for both. Twenty-four hours after ischemia in the mice, there was evidence of apoptosis in both groups, however, the mutant mice (lpr) had significantly smaller infarcts as compared to the wild type. There was no difference in the cerebrovasculature of the two groups of mice. These data support the hypothesis that apoptosis plays a role in the pathophysiology of focal cerebral ischemia. Furthermore, these data suggest that fas-mediated apoptosis contributes to this process.

Animals↗
PubMed2002

Requirement of Fas expression in B cells for tolerance induction.

Fas is a death receptor that belongs to the tumor necrosis factor receptor family and is expressed in various cell types, in particular, in lymphoid cells. A loss-of-function mutation in the Fas gene (lpr mutation) causes lymphadenopathy and splenomegaly, and accelerates autoimmune diseases in some strains of mice such as MRL. In this report, Fas cDNA driven by murine lck distal promoter was used to establish transgenic MRL-lpr mouse lines. The transgenic mice expressed functional Fas in mature T cells and B cells. The lymphadenopathy and splenomegaly caused by accumulation of abnormal T cells in the lpr mice were rescued in the transgenic mice. The number of B cells in the periphery as well as the serum IgG level were significantly reduced, and the autoimmune symptoms and mortality were ameliorated. These results indicate that both mature B cells and T cells must undergo Fas-mediated apoptosis to prevent the development of autoimmune diseases.

Animals↗
PubMed2001

Identification of differentially regulated proteins in metronidozole resistant Helicobacter pylori by proteome techniques.

Resistance to metronidazole (MTZ) is common among Helicobacter pylori strains in many societies, and results from loss of function mutations in genes for one or more cellular nitroreductases. When functional, these enzymes convert MTZ from a harmless prodrug to mutagenic and bacteriocidal products (probably hydroxylamine-type compounds), and in the process may generate active reactive oxygen metabolites. Here we examine the protein profiles of a derivative of strain 26695 that is resistant to moderate levels of MTZ because of mutation in rdxA (HP0954), the gene for the most important of these nitroreductases. The strain was grown with and without 18 micrograms/mL of MTZ to assess whether sublethal exposure triggers an adaptive response. Bacterial lysates were subjected to two-dimensional (2-D) electrophoresis and protein bands were identified by mass spectrometry and sequence analysis. Several proteins were decreased at least two-fold during growth with MTZ, yet the levels of various isoforms of alkylhydroperoxide reductase (AHP) (encoded by ahpC HP1563) were increased. AHP is an essential enzyme, and had been linked to resistance to oxygen toxicity in various prokaryotic and eukaryotic systems; we propose that the ability of an rdxA mutant strain to increase AHP abundance during exposure to MTZ is critically important in the realization of the resistance phenotype. More generally, these results highlight the potential of proteome analysis to tracing out how pathogenic bacteria cope with the challenges imposed on them by therapy or host responses to infection.

Bacterial Proteins↗