MANF brakes TLR4 signaling by competitively binding S100A8 with S100A9 to regulate macrophage phenotypes in hepatic fibrosis.

The mesencephalic astrocyte-derived neurotrophic factor (MANF) has been recently identified as a neurotrophic factor, but its role in hepatic fibrosis is unknown. Here, we found that MANF was upregulated in the fibrotic liver tissues of the patients with chronic liver diseases and of mice treated with CCl4. MANF deficiency in either hepatocytes or hepatic mono-macrophages, particularly in hepatic mono-macrophages, clearly exacerbated hepatic fibrosis. Myeloid-specific MANF knockout increased the population of hepatic Ly6Chigh macrophages and promoted HSCs activation. Furthermore, MANF-sufficient macrophages (from WT mice) transfusion ameliorated CCl4-induced hepatic fibrosis in myeloid cells-specific MANF knockout (MKO) mice. Mechanistically, MANF interacted with S100A8 to competitively block S100A8/A9 heterodimer formation and inhibited S100A8/A9-mediated TLR4-NF-κB signal activation. Pharmacologically, systemic administration of recombinant human MANF significantly alleviated CCl4-induced hepatic fibrosis in both WT and hepatocytes-specific MANF knockout (HKO) mice. This study reveals a mechanism by which MANF targets S100A8/A9-TLR4 as a "brake" on the upstream of NF-κB pathway, which exerts an impact on macrophage differentiation and shed light on hepatic fibrosis treatment.

Androgen Mediated Regulation of Endoplasmic Reticulum-Associated Degradation and its Effects on Prostate Cancer.

The endoplasmic reticulum (ER) comprises thirty percent of the newly translated proteins in eukaryotic cells. The quality control mechanism within the ER distinguishes between properly and improperly folded proteins and ensures that unwanted proteins are retained in the ER and subsequently degraded through ER-associated degradation (ERAD). Besides cleaning of misfolded proteins ERAD is also important for physiological processes by regulating the abundance of normal proteins of the ER. Thus it is important to unreveal the regulation patterns of ERAD. Here, we describe that ERAD pathway is regulated by androgen, where its inhibitor SVIP was downregulated, all other ERAD genes were upregulated. Consistently, androgen treatment increased the degradation rate of ERAD substrates. Using several independent techniques, we showed that this regulation is through androgen receptor transactivation. ERAD genes found to be upregulated in prostate cancer tissues and silencing expression of Hrd1, SVIP, and gp78 reduced the in vitro migration and malignant transformation of LNCaP cells. Our data suggests that expression levels of ERAD components are regulated by androgens, that promotes ERAD proteolytic activity, which is positively related with prostate tumorigenesis.

The E3 ubiquitin ligase gp78 protects against ER stress in zebrafish liver.

Enhanced endoplasmic reticulum (ER)-associated protein degradation (ERAD) activity by the unfolded protein response (UPR) represents one of the mechanisms for restoring ER homeostasis. In vitro evidence indicates that the mammalian gp78 protein is an E3 ubiquitin ligase that facilitates ERAD by polyubiquitinating and targeting proteins for proteasomal degradation under both physiologic and stress conditions. However, the in vivo function of gp78 in maintaining ER protein homeostasis remains untested. Here we show that like its mammalian counterpart, the zebrafish gp78 is also an E3 ubiquitin ligase as revealed by in vitro ubiquitination assays. Expression analysis uncovered that gp78 is highly expressed in several organs, including liver and brain, of both larval and adult fish. Treatment of larvae or adult fish with tunicamycin induces ER stress and upregulates the expression of several key components of the gp78 ERAD complex in the liver. Moreover, liver-specific overexpression of the dominant-negative form of gp78 (gp78-R2M) renders liver more sensitive to tunicamycin-induced ER stress and enhances the expression of sterol response element binding protein (Srebp)-target genes, which was largely suppressed in fish overexpressing wild-type gp78. Together, these data indicate that gp78 plays a critical role in protecting against ER stress in liver.

Bioactivity screening of five Centaurea species and in vivo anti-inflammatory activity of C. athoa.

CONTEXT: Centaurea L. (Asteraceae) species used as herbal remedies in Turkish traditional medicine have shown several biological properties. OBJECTIVE: Extracts obtained from the aerial parts of Centaurea aphrodisea Boiss., Centaurea athoa DC., Centaurea hyalolepis Boiss., Centaurea iberica Trev. and Centaurea polyclada DC. were evaluated for their antioxidant, cytotoxic and anti-inflammatory activities. MATERIALS AND METHODS: Extracts of Centaurea species were tested for their antioxidant activity in the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) screening assays and for in vitro anti-inflammatory activity by Nf-κB and iNOS inhibition assays. The extracts were tested for their in vitro cytotoxicities against a panel of human solid tumor cell lines (SK-MEL: malignant melanoma, KB: oral epidermal carcinoma, BT-549: breast ductal carcinoma and SK-OV-3: ovary carcinoma) as well as non-cancerous kidney fibroblast (Vero) and kidney epithelial cells (LLC-PK1) by Neutral Red assay. In vivo anti-inflammatory activity of C. athoa was evaluated by the carrageenan-induced paw edema test in rats. RESULTS: Antioxidant activities were observed for methanol extracts of plants. C. polyclada had the strongest effect on BT-549, KB and SK-OV-3 cell lines (30, 33 and 47 µg/ml, respectively). Nf-κB inhibition of chloroform extract of C. athoa was determined equivalent to positive control parthenolide (IC50: 6 µg/ml). This extract also showed anti-inflammatory activity by the carrageenan-induced paw edema test in rats, in all hours at a dose of 50 mg/kg compared to the control group. DISCUSSION AND CONCLUSION: C. athoa is suggested to be a potential source of lead compounds for inflammatory diseases due to the significant in vitro and in vivo anti-inflammatory results.

A unique IBMPFD-related P97/VCP mutation with differential binding pattern and subcellular localization.

p97/VCP is a hexameric AAA type ATPase that functions in a variety of cellular processes such as endoplasmic reticulum associated degradation (ERAD), organelle biogenesis, autophagy and cell-cycle regulation. Inclusion body myopathy associated with Paget disease of the bone and frontotemporal dementia (IBMPFD) is an autosomal dominant disorder which has been attributed to mutations in p97/VCP. Several missense mutations affecting twelve different amino acids have been identified in IBMPFD patients and some of them were suggested to be involved in the observed pathology. Here, we analyzed the effect of all twelve p97/VCP variants on ERAD substrates and their cofactor binding abilities. While all mutants cause ERAD substrate accumulation, P137L mutant p97/VCP differs from other IBMPFD mutants by having a unique solubility profile and subcellular localization. Intriguingly, although almost all mutants exhibit enhanced p47 and Ufd1-Npl4 binding, the P137L mutation completely abolishes p97/VCP interactions with Ufd1, Npl4 and p47, while retaining its gp78 binding. While recombinant R155C mutant protein consistently interacts with both Ufd1 and VIM of gp78, P137L mutant protein lost binding ability to Ufd1 but not to VIM in vitro. The differential impairments in p97/VCP interactions with its functional partners and function should help our understanding of the molecular pathogenesis of IBMPFD.

Electrochemical based detection of microRNA, mir21 in breast cancer cells.

In this work, a novel electrochemical microRNA (miRNA) detection method based on enzyme amplified biosensing of mir21 from cell lysate of total RNA was demonstrated. The proposed enzymatic detection method was detailed and compared with the conventional guanine oxidation based assay in terms of detection limit and specificity. For the detection of mir21, capture probes and/or cell lysates were covalently attached onto the pencil graphite electrode (PGE) by coupling agents of N-(dimethylamino)propyl-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (NHS). Having immobilized the capture probe onto the surface of PGE, hybridization was achieved with a biotinylated (from its 3' end) complementary target. Extravidin labeled alkaline phosphatase (Ex-Ap) binds to the biotinylated target due to the interaction between biotin-avidin and the enzyme converts electro-inactive alpha naphtyl phosphate (the substrate) to electro-active alpha naphtol (α-NAP, the product). α-NAP was oxidized at +0.23 V vs Ag/AgCl and this signal was measured by Differential Pulse Voltammetry (DPV). The signals obtained from α-NAP oxidation were compared for the probe and hybrid DNA. The specificity of the designed biosensor was proved by using non-complementary sequences instead of complementary sequences and the detection limit of the assay was calculated to be 6 pmol for cell lysates.

SVIP induces localization of p97/VCP to the plasma and lysosomal membranes and regulates autophagy.

The small p97/VCP-interacting protein (SVIP) functions as an inhibitor of the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway. Here we show that overexpression of SVIP in HeLa cells leads to localization of p97/VCP at the plasma membrane, intracellular foci and juxtanuclear vacuoles. The p97/VCP-positive vacuolar structures colocalized or associated with LC3 and lamp1, suggesting that SVIP may regulate autophagy. In support of this possibility, knockdown of SVIP diminished, whereas overexpression of SVIP enhanced LC3 lipidation. Surprisingly, knockdown of SVIP reduced the levels of p62 protein at least partially through downregulation of its mRNA, which was accompanied by a decrease in starvation-induced formation of p62 bodies. Overexpression of SVIP, on the other hand, increased the levels of p62 protein and enhanced starvation-activated autophagy as well as promoted sequestration of polyubiquitinated proteins and p62 in autophagosomes. These results suggest that SVIP plays a regulatory role in p97 subcellular localization and is a novel regulator of autophagy.

Importin beta interacts with the endoplasmic reticulum-associated degradation machinery and promotes ubiquitination and degradation of mutant alpha1-antitrypsin.

The mechanism by which misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytosol for proteasomal degradation is still poorly understood. Here, we show that importin ß, a well established nucleocytoplasmic transport protein, interacts with components of the retrotranslocation complex and promotes ER-associated degradation (ERAD). Knockdown of importin ß specifically inhibited the degradation of misfolded ERAD substrates but did not affect turnover of non-ERAD proteasome substrates. Genetic studies and in vitro reconstitution assays demonstrate that importin ß is critically required for ubiquitination of mutant α1-antitrypsin, a luminal ERAD substrate. Furthermore, we show that importin ß cooperates with Ran GTPase to promote ubiquitination and proteasomal degradation of mutant α1-antitrypsin. These results establish an unanticipated role for importin ß in ER protein quality control.

Synthesis and screening of cyclooxygenase inhibitory activity of some 1,3-dioxoisoindoline derivatives.

In this study, 15 compounds bearing N,N-phthaloylacetamide structure designed by the molecular simplification approach based on thalidomide structure were synthesized and evaluated for inhibitory potencies against cyclooxgenase (COX) isoenzymes, namely COX-1 and COX-2. The results suggested that the N,N-phthaloylacetamide structure, as a primary amide, has inhibitory activity against cyclooxygenase isoenzymes with a higher COX-1 selectivity. The conversion of the primary amide to secondary or tertiary derivatives lowered the potency but favored the COX-2 selectivity thus yielding the compounds with stronger COX-2 inhibiting activity.

Site-directed mutagenesis.

The technique of site-directed mutagenesis has been used to characterize gene and protein structure-function relationships, protein-protein interactions, binding domains of proteins, or active sites of enzymes for the last three decades. In this technique, a nucleotide sequence of interest is experimentally altered using synthetic oligonucleotides. The most commonly used approach is to use an oligonucleotide that is complementary to part of a single-stranded DNA template, but containing an internal mismatch to direct the mutation. In addition to single point mutations, this approach may also be used to construct multiple mutations, insertions, or deletions. As a result of its broad applicability in disease gene characterization studies, numerous commercial kits are now available, making this technique quick, straightforward, and reliable. In this chapter, we detail the steps involved in site-directed mutagenesis and highlight the essentials of this versatile technique based upon our experience.

Different p97/VCP complexes function in retrotranslocation step of mammalian ER-associated degradation (ERAD).

Studies in yeast indicate that three specialized endoplasmic reticulum-associated degradation (ERAD) pathways, namely ERAD-L, -M, or -C, dispose substrates with structural lesions in the lumenal, transmembrane, or cytosolic domains, respectively. The ubiquitin ligase (E3) Hrd1p and its cooperating partners are required for ERAD-L and -M pathways, whereas Doa10p complex is required for the ERAD-C pathway. We investigated these pathways in mammalian cells by assessing the requirements of the mammalian ERAD E3s, gp78 and Hrd1, in degradation of four substrates each with different type of structural lesions: CD3δ, Z-variant α1-antitrypsin, tyrosinase (C89R) and mutant cystic fibrosis transmembrane conductance regulator (CFTRΔF508). We demonstrated that tyrosinase (C89R) is a substrate for Hrd1 while all others are gp78 substrates. Knockdown of Hrd1 diminished gp78 substrate levels, but silencing of gp78 had no effect on Hrd1's substrate, suggesting that the functional interaction between Hrd1 and gp78 is unidirectional. Furthermore, while Ufd1 is dispensable for gp78-mediated ERAD, it is essential for Hrd1-mediated ERAD. Interestingly, Npl4 was found to be a key component for both pathways. These results suggest that the Hrd1-mediated ERAD requires a well-established retrotranslocation machinery, the p97/VCP-Ufd1-Npl4 complex, whereas the gp78 pathway needs only p97/VCP and Npl4. In addition, the three distinct ERAD pathways described in yeast may not be strictly conserved in mammalian cells as gp78 can function on three substrates with different structural lesions.

Phenolic Glycosides with antiproteasomal activity from Centaurea urvillei DC. subsp. urvillei.

A new flavanone glycoside, naringenin-7-O-ß-D-glucuronopyranoside, and a new flavonol glycoside, 6-hydroxykaempferol-7-O-ß-D-glucuronopyranoside were isolated together with 12 known compounds, 5 flavone glycoside; hispidulin-7-O-ß-D-glucuronopyranoside, apigenin-7-O-ß-D-methylglucuronopyranoside, hispidulin-7-O-ß-D-methylglucuronopyranoside, hispidulin-7-O-ß-D-glucopyranoside, apigenin-7-O-ß-D-glucopyranoside, a flavonol; kaempferol, two flavone; apigenin, and luteolin, a flavanone glycoside; eriodictyol-7-O-ß-D-glucuronopyranoside, and three phenol glycoside; arbutin, salidroside, and 3,5-dihydroxyphenethyl alcohol-3-O-ß-D-glucopyranoside from Centaurea urvillei subsp. urvillei. The structure elucidation of the new compounds was achieved by a combination of one- ((1)H and (13)C) and two-dimensional NMR techniques (G-COSY, G-HMQC, and G-HMBC) and LC-ESI-MS. The isolated compounds were tested for their antiproteasomal activity. The results indicated that kaempferol, a well known and widely distributed flavonoid in the plant kingdom, was the most active antiproteasomal agent, followed by apigenin, eriodictyol-7-O-ß-D-glucuronopyranoside, 3,5-dihydroxyphenethyl alcohol-3-O-ß-D-glucopyranoside, and salidroside, respectively.

Differential regulation of CFTRDeltaF508 degradation by ubiquitin ligases gp78 and Hrd1.

The most common mutation associated with cystic fibrosis is the deletion of phenylalanine 508 of cystic fibrosis transmembrane conductance regulator (CFTRDeltaF508). This mutation renders otherwise functional protein susceptible to ER-associated degradation (ERAD) and prevents CFTR from exiting the ER and trafficking to the plasma membrane. In this study, we demonstrate that RNAi-mediated silencing of gp78, an established ubiquitin ligase (E3) involved in ERAD, leads to accumulation of CFTRDeltaF508 protein in cells. gp78 facilitates the degradation of CFTRDeltaF508 by enhancing both its ubiquitination and interaction with p97/VCP. SVIP, which is the inhibitor of gp78, causes accumulation of CFTRDeltaF508. We showed that endogenous gp78 co-immunoprecipitates with Hrd1. Furthermore, the results indicate that silencing the expression of another ERAD E3, Hrd1, leads to stabilization of gp78 and decline in gp78 ubiquitination; thereby enhancing CFTRDeltaF508 degradation. The results support that gp78 is an E3 targeting CFTRDeltaF508 for degradation and Hrd1 inhibits CFTRDeltaF508 degradation by acting as an E3 for gp78.

Regulation of ER-associated degradation via p97/VCP-interacting motif.

p97/VCP (valosin-containing protein) is a cytosolic AAA (ATPase associated with various cellular activities) essential for retrotranslocation of misfolded proteins during ERAD [ER (endoplasmic reticulum)-associated degradation]. gp78, an ERAD ubiquitin ligase, is one of the p97/VCP recruitment proteins localized to the ER membrane. A newly identified VIM (p97/VCP-interacting motif) in gp78 has brought about novel insights into mechanisms of ERAD, such as the presence of a p97/VCP-dependent but Ufd1-independent retrotranslocation during gp78-mediated ERAD. Additionally, SVIP (small p97/VCP-interacting protein), which contains a VIM in its N-terminal region, negatively regulates ERAD by uncoupling p97/VCP and Derlin1 from gp78. Thus SVIP may protect cells from damage by extravagant ERAD.

Identification of SVIP as an endogenous inhibitor of endoplasmic reticulum-associated degradation.

Misfolded proteins in the endoplasmic reticulum (ER) are eliminated by a process known as ER-associated degradation (ERAD), which starts with misfolded protein recognition, followed by ubiquitination, retrotranslocation to the cytosol, deglycosylation, and targeting to the proteasome for degradation. Actions of multisubunit protein machineries in the ER membrane integrate these steps. We hypothesized that regulation of the multisubunit machinery assembly is a mechanism by which ERAD activity is regulated. To test this hypothesis, we investigated the potential regulatory role of the small p97/VCP-interacting protein (SVIP) on the formation of the ERAD machinery that includes ubiquitin ligase gp78, AAA ATPase p97/VCP, and the putative channel Derlin1. We found that SVIP is anchored to microsomal membrane via myristoylation and co-fractionated with gp78, Derlin1, p97/VCP, and calnexin to the ER. Like gp78, SVIP also physically interacts with p97/VCP and Derlin1. Overexpression of SVIP blocks unassembled CD3delta from association with gp78 and p97/VCP, which is accompanied by decreases in CD3delta ubiquitination and degradation. Silencing SVIP expression markedly enhances the formation of gp78-p97/VCP-Derlin1 complex, which correlates with increased degradation of CD3delta and misfolded Z variant of alpha-1-antitrypsin, established substrates of gp78. These results suggest that SVIP is an endogenous inhibitor of ERAD that acts through regulating the assembly of the gp78-p97/VCP-Derlin1 complex.

Ubiquitin ligase Hrd1 enhances the degradation and suppresses the toxicity of polyglutamine-expanded huntingtin.

E3 ubiquitin ligases catalyze the conjugation of ubiquitin onto proteins, which acts as a signal for targeting proteins for degradation by the proteasome. Hrd1 is an endoplasmic reticulum (ER) membrane-spanning E3 with its catalytic active RING finger facing the cytosol. We speculated that this topology might allow Hrd1 to ubiquitinate misfolded proteins in the cytosol. We tested this idea by using polyglutamine (polyQ)-containing huntingtin (htt) protein as a model substrate. We found that the protein levels of Hrd1 were increased in cells overexpressing the N-terminal fragment of htt containig an expanded polyQ tract (httN). Forced expression of Hrd1 enhanced the degradation of httN in a RING finger-dependent manner, whereas silencing of endogenous Hrd1 expression by RNA interference stabilized httN. Degradation of httN was found to be p97/VCP-dependent, but independent of Ufd1 and Npl4, all of which are thought to form a complex with Hrd1 during ER-associated degradation. Consistent with its role as an E3 for httN, we demonstrate that Hrd1 interacts with and ubiquitinates httN. Subcellular fractionation and confocal microscopy revealed that Hrd1recruits HttN to the ER and co-localizes with juxtanuclear aggregates of httN in cells. Interaction of Hrd1 with httN was found to be independent of the length of the polyglutamine tract. However, httN with expanded polyglutamine tracts appeared to be a preferred substrate for Hrd1. Functionally, we found that Hrd1 protects cells against the httN-induced cell death. These results suggest that Hrd1 is a novel htt-interacting protein that can target pathogenic httN for degradation and is able to protect cells against httN-induced cell death.

ER stress differentially regulates the stabilities of ERAD ubiquitin ligases and their substrates.

Endoplasmic reticulum (ER) stress-induced accumulation of misfolded proteins in the ER stimulates the ER-associated degradation (ERAD) process. ERAD in turn eliminates those misfolded proteins. Upregulation of ubiquitination enzymes is an essential mechanism by which ER stress enhances ERAD. However, ectopic overexpression of ubiquitination enzymes often fails to increase, and sometimes, inhibits ERAD. To further understand how ER stress regulates ERAD, we studied the effects of ER stress on ubiquitin ligase (E3) gp78-mediated ERAD and on the stabilities of gp78 and another ERAD E3 Hrd1. The results showed that ER stress-inducing agent tunicamycin significantly enhanced ERAD in cells that either express endogenous or overexpress gp78. Importantly, ER stress could increase ERAD even when new protein synthesis was inhibited by cycloheximide. Surprisingly, tunicamycin treatment stabilized gp78, an established ERAD E3 and an ERAD substrate as well, for up to 8h. By contrast, ER stress had little effects on the stability of another E3 Hrd1 except that it reduced the total ubiquitination level of Hrd1. Our data suggest that ER stress differentially regulates the stabilities of ERAD E3s and their substrates, which may represent a novel mechanism by which ER stress increases ERAD.

The role of a novel p97/valosin-containing protein-interacting motif of gp78 in endoplasmic reticulum-associated degradation.

Improperly folded proteins in the endoplasmic reticulum (ER) are eliminated via ER-associated degradation, a process that dislocates misfolded proteins from the ER membrane into the cytosol, where they undergo proteasomal degradation. Dislocation requires a subclass of ubiquitin ligases that includes gp78 in addition to the AAA ATPase p97/VCP and its cofactor, the Ufd1-Npl4 dimer. We have previously reported that gp78 interacts directly with p97/VCP. Here, we identify a novel p97/VCP-interacting motif (VIM) within gp78 that mediates this interaction. We demonstrate that the VIM of gp78 recruits p97/VCP to the ER, but has no effect on Ufd1 localization. We also show that gp78 VIM interacts with the ND1 domain of p97/VCP that was shown previously to be the binding site for Ufd1. To evaluate the role of Ufd1 in gp78-p97/VCP-mediated degradation of CD3delta, a known substrate of gp78, RNA interference was used to silence the expression of Ufd1 and p97/VCP. Inhibition of p97/VCP, but not Ufd1, stabilized CD3delta in cells that overexpress gp78. However, both p97/VCP and Ufd1 appear to be required for CD3delta degradation in cells expressing physiological levels of gp78. These results raise the possibility that Ufd1 and gp78 may bind p97/VCP in a mutually exclusive manner and suggest that gp78 might act in a Ufd1-independent degradation pathway for misfolded ER proteins, which operates in parallel with the previously established p97/VCP-Ufd1-Npl4-mediated mechanism.

Ubiquitin ligase gp78 increases solubility and facilitates degradation of the Z variant of alpha-1-antitrypsin.

Deficiency of circulating alpha-1-antitrypsin (AAT) is the most widely recognized abnormality of a proteinase inhibitor that causes lung disease. AAT-deficiency is caused by mutations of the AAT gene that lead to AAT protein retention in the endoplasmic reticulum (ER). Moreover, the mutant AAT accumulated in the ER predisposes the homozygote to severe liver injuries, such as neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. Despite the fact that mutant AAT protein is subject to ER-associated degradation (ERAD), yeast genetic studies have determined that the ubiquitination machinery, Hrd1/Der3p-cue1p-Ubc7/6p, which plays a prominent role in ERAD, is not involved in degradation of mutant AAT. Here we report that gp78, a ubiquitin ligase (E3) pairing with mammalian Ubc7 for ERAD, ubiquitinates and facilitates degradation of ATZ, the classic deficiency variant of AAT having a Z mutation (Glu 342 Lys). Unexpectedly, gp78 over-expression also significantly increases ATZ solubility. p97/VCP, an AAA ATPase essential for retrotranslocation of misfolded proteins from the ER during ERAD, is involved in gp78-mediated degradation of ATZ. Surprisingly, unlike other ERAD substrates that cause ER stress leading to apoptosis when accumulated in the ER, ATZ, in fact, increases cell proliferation when over-expressed in cells. This effect can be partially inhibited by gp78 over-expression. These data indicate that gp78 assumes multiple unique quality control roles over ATZ, including the facilitation of degradation and inhibition of aggregation of ATZ.

The DNA-binding specificity of the Bacillus anthracis AbrB protein.

The Bacillus subtilis AbrB protein is a DNA-binding global regulator of a plethora of functions that are expressed during the transition from exponential growth to stationary phase and under suboptimal growth conditions. AbrB orthologues have been identified in a variety of prokaryotic organisms, notably in all species of Bacillus, Clostridium and Listeria that have been examined. Based on amino acid sequence identity in the N-terminal domains of the orthologues from B. subtilis and Bacillus anthracis, it was predicted that the proteins might display identical DNA-binding specificities. The binding of purified B. anthracis AbrB (AbrB(BA)) and purified B. subtilis AbrB (AbrB(BS)) at DNA targets of B. subtilis, B. anthracis and a synthetic origin was compared. In all cases examined, DNA-binding specificity was identical as judged by DNase I footprinting. In B. subtilis cells, the B. anthracis promoters from the atxA and abrB genes were regulated by AbrB(BS), and the B. subtilis promoter from the yxbB operon was regulated by AbrB(BA).