Mimicking cotranslational folding of prosubtilisin E in vitro.

Nascent polypeptides are synthesized on ribosomes starting at the N-terminus and simultaneously begin to fold during translation. We constructed N-terminal fragments of prosubtilisin E containing an intramolecular chaperone (IMC) at N-terminus to mimic cotranslational folding intermediates of prosubtilisin. The IMC-fragments of prosubtilisin exhibited progressive enhancement of their secondary structures and thermostabilities with increasing polypeptide length. However, even the largest IMC-fragment with 72 residues truncated from the C-terminus behaved as a molten globule, indicating the requirement of the C-terminal region to have a stable tertiary structure. Furthermore, truncation of the IMC in the IMC-fragments resulted in aggregation, suggesting that the IMC plays a crucial role to prevent misfolding and aggregation of cotranslational folding intermediates during translation of prosubtilisin polypeptide.

Regression of solid tumors by induction of MazF, a bacterial mRNA endoribonuclease.

MazF from Escherichia coli is an endoribonuclease that specifically cleaves mRNAs at ACA sequences. Its induction in mammalian cells has been shown to cause programmed cell death. Here we explored if a bacterial MazF-MazE toxin-antitoxin system can be used for gene therapy. For this, we first constructed a tetracycline-inducible MazF expression system in human embryonic kidney cells (T-Rex 293-mazF). Solid tumors were formed by injecting T-Rex 293-mazF cells into nude mice. All 8 mice injected with the cells developed solid tumors, which regressed upon induction of MazF. In 4 mice, tumors completely regressed, while in the remaining 4 mice, tumors reappeared after apparent significant regression, which was found to be due to the lack of presence of functional MazF. Notably, the MazF-mediated regression of the tumors was counteracted by the expression of its cognate antitoxin MazE. These results indicate that a bacterial MazF-MazE toxin-antitoxin system may have potential to be used as a therapeutic tool.

Characterization of LdrA (long direct repeat A) protein of Escherichia coli.

Reminiscent of apoptotic genes in higher systems, almost all bacteria contain 'suicide' genes, which encode toxins. Toxins inhibit cell growth and may cause cell death. These are coexpressed with their cognate antitoxins from a toxin-antitoxin (TA) operon in normally growing cells. The cellular targets of toxins are diverse. The study of the TA system is important as in addition to their involvement in the normal bacterial physiology, TA systems may also play a role in bacterial pathogenicity. The long direct repeat (Ldr) family of proteins belongs to one such TA system. Here we report characterization of LdrA, the prototypical protein of the Ldr family, and show that it is highly toxic for cell growth. The data suggests that LdrA may exert toxicity by inhibiting ATP synthesis, possibly due to its localization in the cell membrane. This inhibition of energy production leads to the inhibition of biosynthetic reactions in the cell such as DNA replication, transcription and translation, and eventually cell growth.

ACA-specific RNA sequence recognition is acquired via the loop 2 region of MazF mRNA interferase.

MazF is an mRNA interferase that cleaves mRNAs at a specific RNA sequence. MazF from E. coli (MazF-ec) cleaves RNA at A^CA. To date, a large number of MazF homologs that cleave RNA at specific three- to seven-base sequences have been identified from bacteria to archaea. MazF-ec forms a dimer, in which the interface between the two subunits is known to be the RNA substrate-binding site. Here, we investigated the role of the two loops in MazF-ec, which are closely associated with the interface of the MazF-ec dimer. We examined whether exchanging the loop regions of MazF-ec with those from other MazF homologs, such as MazF from Myxococcus xanthus (MazF-mx) and MazF from Mycobacterium tuberculosis (MazF-mt3), affects RNA cleavage specificity. We found that exchanging loop 2 of MazF-ec with loop 2 regions from either MazF-mx or MazF-mt3 created a new cleavage sequence at (A/U)(A/U)AA^C in addition to the original cleavage site, A^CA, whereas exchanging loop 1 did not alter cleavage specificity. Intriguingly, exchange of loop 2 with 8 or 12 consecutive Gly residues also resulted in a new RNA cleavage site at (A/U)(A/U)AA^C. The present study suggests a method for expanding the RNA cleavage repertoire of mRNA interferases, which is crucial for potential use in the regulation of specific gene expression and for biotechnological applications.

Intramolecular regulation of the sequence-specific mRNA interferase activity of MazF fused to a MazE fragment with a linker cleavable by specific proteases.

The genomes of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) consist of single-stranded RNA encoding polyproteins, which are processed to individual functional proteins by virus-encoded specific proteases. These proteases have been used as targets for drug development. Here, instead of targeting these proteases to inhibit viral infection, we utilized the protease activity to activate a toxic protein to prevent viral infection. We engineered the MazE-MazF antitoxin-toxin system of Escherichia coli to fuse a C-terminal 41-residue fragment of antitoxin MazE to the N-terminal end of toxin MazF with a linker having a specific protease cleavage site for either HIV PR (HIV-1 protease), NS3 protease (HCV protease), or factor Xa. These fusion proteins formed a stable dimer (instead of the MazF(2)-MazE(2)-MazF(2) heterohexamer in nature) to inactivate the ACA (sequence)-specific mRNA interferase activity of MazF. When the fusion proteins were incubated with the corresponding proteases, the MazE fragment was cleaved from the fusion proteins, releasing active MazF, which then acted as an ACA-specific mRNA interferase cleaving single-stranded MS2 phage RNA. The intramolecular regulation of MazF toxicity by proteases as demonstrated may provide a novel approach for preventive and therapeutic treatments of infection by HIV-1, HCV, and other single-stranded RNA viruses.

Toxin-antitoxin systems in bacteria and archaea.

Almost all bacteria and many archaea contain genes whose expression inhibits cell growth and may lead to cell death when overproduced, reminiscent of apoptotic genes in higher systems. The cellular targets of these toxins are quite diverse and include DNA replication, mRNA stability, protein synthesis, cell-wall biosynthesis, and ATP synthesis. These toxins are co-expressed and neutralized with their cognate antitoxins from a TA (toxin-antitoxin) operon in normally growing cells. Antitoxins are more labile than toxins and are readily degraded under stress conditions, allowing the toxins to exert their toxic effect. Presence of at least 33 TA systems in Escherichia coli and more than 60 TA systems in Mycobacterium tuberculosis suggests that the TA systems are involved not only in normal bacterial physiology but also in pathogenicity of bacteria. The elucidation of their cellular function and regulation is thus crucial for our understanding of bacterial physiology under various stress conditions.

Solution NMR structure of the NlpC/P60 domain of lipoprotein Spr from Escherichia coli: structural evidence for a novel cysteine peptidase catalytic triad.

Escherichia coli Spr is a membrane-anchored cell wall hydrolase. The solution NMR structure of the C-terminal NlpC/P60 domain of E. coli Spr described here reveals that the protein adopts a papain-like alpha+beta fold and identifies a substrate-binding cleft featuring several highly conserved residues. The active site features a novel Cys-His-His catalytic triad that appears to be a unique structural signature of this cysteine peptidase family. Moreover, the relative orientation of these catalytic residues is similar to that observed in the analogous Ser-His-His triad, a variant of the classic Ser-His-Asp charge relay system, suggesting the convergent evolution of a catalytic mechanism in quite distinct peptidase families.

RelA functionally suppresses the growth defect caused by a mutation in the G domain of the essential Der protein.

A unique bacterial GTPase, Der, containing two tandem GTP-binding domains, is essential for cell growth and plays a crucial role in a large ribosomal subunit in Escherichia coli. The depletion of Der resulted in accumulation of both large and small ribosomal subunits and also affected the stability of large ribosomal subunits. However, its exact cellular function still remains elusive. Previously, we have shown that two G domain mutants, DerN118D and DerN321D, cannot support cell growth at low temperatures, suggesting that both GTP-binding domains are indispensable. In this study, we show that both Der variants are defective in ribosome biogenesis. Genetic screening of an E. coli genomic library was performed to identify the genes which, when expressed from a multicopy plasmid, can restore the growth defect of the DerN321D mutant at restrictive temperatures. Among seven suppressors isolated, four were located at 62.7 min on the E. coli genomic map, and the gene responsible for the suppression of DerN321D was identified as the relA gene which encodes a ribosome-associated (p)ppGpp synthetase. The synthetic activity of RelA was found to be essential for its DerN321D suppressor activity. Overexpression of RelA in a suppressor strain did not affect the expression of DerN321D but suppressed the polysome defects caused by the DerN321D mutant. This is the first demonstration of suppression of impaired function of Der by a functional enzyme. A possible mechanism of the suppression of DerN321D by RelA overproduction is discussed.

Dissection of 16S rRNA methyltransferase (KsgA) function in Escherichia coli.

A 16S rRNA methyltransferase, KsgA, identified originally in Escherichia coli is highly conserved in all living cells, from bacteria to humans. KsgA orthologs in eukaryotes possess functions in addition to their rRNA methyltransferase activity. E. coli Era is an essential GTP-binding protein. We recently observed that KsgA functions as a multicopy suppressor for the cold-sensitive cell growth of an era mutant [Era(E200K)] strain (Q. Lu and M. Inouye, J. Bacteriol. 180:5243-5246, 1998). Here we observed that although KsgA(E43A), KsgA(G47A), and KsgA(E66A) mutations located in the S-adenosylmethionine-binding motifs severely reduced its methyltransferase activity, these mutations retained the ability to suppress the growth defect of the Era(E200K) strain at a low temperature. On the other hand, a KsgA(R248A) mutation at the C-terminal domain that does not affect the methyltransferase activity failed to suppress the growth defect. Surprisingly, E. coli cells overexpressing wild-type KsgA, but not KsgA(R248A), were found to be highly sensitive to acetate even at neutral pH. Such growth inhibition also was observed in the presence of other weak organic acids, such as propionate and benzoate. These chemicals are known to be highly toxic at acidic pH by lowering the intracellular pH. We found that KsgA-induced cells had increased sensitivity to extreme acid conditions (pH 3.0) compared to that of noninduced cells. These results suggest that E. coli KsgA, in addition to its methyltransferase activity, has another unidentified function that plays a role in the suppression of the cold-sensitive phenotype of the Era(E200K) strain and that the additional function may be involved in the acid shock response. We discuss a possible mechanism of the KsgA-induced acid-sensitive phenotype.

Functional and structural characterization of EnvZ, an osmosensing histidine kinase of E. coli.

EnvZ is an osmosensing histidine kinase located in the inner membrane, and one of the most extensively studied Escherichia coli histidine kinases. Because of its structural complexity, functional and structural studies have been quite challenging. It is a multidomain transmembrane protein consisting of 450 amino acid residues. In addition, it must form a dimer to function as a histidine kinase like all the other histidine kinases. EnvZ consists of the 115-residue periplasmic domain, two transmembrane domains (TM1 and TM2), and the cytoplasmic domain consisting of the 43-residue linker (HAMP) domain and the 228-residue kinase domain. It has been shown that the kinase domain of EnvZ, responsible for its enzymatic activities, contains all of the conserved regions of histidine kinases such as H, F, N, G1, G2, and G3 boxes. Therefore, the 271-residue cytoplasmic domain of EnvZ (termed EnvZc) has been used as a model system to establish fundamental characteristics of histidine kinases. The DNA fragment encoding EnvZc was cloned in pET vector and EnvZc was expressed and purified. It is highly soluble and retains all the enzymatic activities of EnvZ. We demonstrated that it consists of two functional domains, domain A and domain B. NMR spectroscopic studies of these two domains revealed, for the first time, the structure of a histidine kinase. Domain A is responsible for dimerization of EnvZc forming a four-helical bundle containing two alpha-helical hairpin structures, while domain B is a monomer and has an ATP-binding pocket formed by regions conserved among the histidine kinases. In this chapter, we describe functional and structural studies of EnvZc, which can be applied to characterize other histidine kinases.

Complementation analysis of the cold-sensitive phenotype of the Escherichia coli csdA deletion strain.

The cold shock response of Escherichia coli is elicited by downshift of temperature from 37 degrees C to 15 degrees C and is characterized by induction of several cold shock proteins, including CsdA, during the acclimation phase. CsdA, a DEAD-box protein, has been proposed to participate in a variety of processes, such as ribosome biogenesis, mRNA decay, translation initiation, and gene regulation. It is not clear which of the functions of CsdA play a role in its essential cold shock function or whether all do, and so far no protein has been shown to complement its function in vivo. Our screening of an E. coli genomic library for an in vivo counterpart of CsdA that can compensate for its absence at low temperature revealed only one protein, RhlE, another DEAD-box RNA helicase. We also observed that although not detected in our genetic screening, two cold shock-inducible proteins, namely, CspA, an RNA chaperone, and RNase R, an exonuclease, can also complement the cold shock function of CsdA. Interestingly, the absence of CsdA and RNase R leads to increased sensitivity of the cells to even moderate temperature downshifts. The correlation between the helicase activity of CsdA and the stability of mRNAs of cold-inducible genes was shown using cspA mRNA, which was significantly stabilized in the DeltacsdA cells, an effect counteracted by overexpression of wild-type CsdA or RNase R but not by that of the helicase-deficient mutant of CsdA. These results suggest that the primary role of CsdA in cold acclimation of cells is in mRNA decay and that its helicase activity is pivotal for promoting degradation of mRNAs stabilized at low temperature.

NBK/BIK antagonizes MCL-1 and BCL-XL and activates BAK-mediated apoptosis in response to protein synthesis inhibition.

Ribonucleases, antibiotics, bacterial toxins, and viruses inhibit protein synthesis, which results in apoptosis in mammalian cells. How the BCL-2 family of proteins regulates apoptosis in response to the shutoff of protein synthesis is not known. Here we demonstrate that an Escherichia coli toxin, MazF, inhibited protein synthesis by cleavage of cellular mRNA and induced apoptosis in mammalian cells. MazF-induced apoptosis required proapoptotic BAK and its upstream regulator, the proapoptotic BH3-only protein NBK/BIK, but not BIM, PUMA, or NOXA. Interestingly, in response to MazF induction, NBK/BIK activated BAK by displacing it from anti-apoptotic proteins MCL-1 and BCL-X(L) that sequester BAK. Furthermore, NBK/BIK- or BAK-deficient cells were resistant to cell death induced by pharmacologic inhibition of translation and by virus-mediated shutoff of protein synthesis. Thus, the BH3-only protein NBK/BIK is the apical regulator of a BAK-dependent apoptotic pathway in response to shutoff of protein synthesis that functions to displace BAK from sequestration by MCL1 and BCL-X(L). Although NBK/BIK is dispensable for development, it is the BH3-only protein targeted for inactivation by viruses, suggesting that it plays a role in pathogen/toxin response through apoptosis activation.

The discovery of mRNA interferases: implication in bacterial physiology and application to biotechnology.

Escherichia coli contains a large number of suicide or toxin genes, whose expression leads to cell growth arrest and eventual cell death. This raises intriguing questions as to why E. coli contains so many toxin genes and what are their roles in bacterial physiology. Among these, MazF has been shown to be a sequence-specific endoribonuclease, which cleaves mRNAs at ACA sequences to completely inhibit protein synthesis. MazF is therefore called mRNA interferase. A number of other mRNA interferases with different cleavage specificities have been discovered not only in E. coli, but also in other bacteria including Mycobacterium tuberculosis. Induction of MazF in the cell leads to cellular dormancy termed quasi-dormancy. In spite of complete cell growth inhibition, cells in the quasi-dormant state are fully capable of energy metabolism, amino acids and nucleic acids biosynthesis and RNA and protein synthesis. The quasi-dormancy may be implicated in cell survival under stress conditions and may play a major role in pathogenicity of M. tuberculosis. The quasi-dormant cells provide an intriguing novel biotechnological system producing only a protein of interest in a high yield. MazF causing Bak-dependent programmed cell death in mammalian cells may be used as a tool for gene therapy against cancer and AIDS. The discovery of a novel way to interfere with mRNA function by mRNA interferases opens a wide variety of avenues in basic as well as applied and clinical sciences.

Superoxide dismutase expression attenuates cigarette smoke- or elastase-generated emphysema in mice.

RATIONALE: Oxidants are believed to play a major role in the development of emphysema. OBJECTIVES: This study aimed to determine if the expression of human copper-zinc superoxide dismutase (CuZnSOD) within the lungs of mice protects against the development of emphysema. METHODS: Transgenic CuZnSOD and littermate mice were exposed to cigarette smoke (6 h/d, 5 d/wk, for 1 yr) and compared with nonexposed mice. A second group was treated with intratracheal elastase to induce emphysema. MEASUREMENTS: Lung inflammation was measured by cell counts and myeloperoxidase levels. Oxidative damage was assessed by immunofluorescence for 3-nitrotyrosine and 8-hydroxydeoxyguanosine and lipid peroxidation levels. The development of emphysema was determined by measuring the mean linear intercept (Lm). MAIN RESULTS: Smoke exposure caused a fourfold increase in neutrophilic inflammation and doubled lung myeloperoxidase activity. This inflammatory response did not occur in the smoke-exposed CuZnSOD mice. Similarly, CuZnSOD expression prevented the 58% increase in lung lipid peroxidation products that occurred after smoke exposure. Most important, CuZnSOD prevented the onset of emphysema in both the smoke-induced model (Lm, 68 exposed control vs. 58 exposed transgenic; p < 0.04) and elastase-generated model (Lm, 80 exposed control vs. 63 exposed transgenic; p < 0.03). These results demonstrate for the first time that antioxidants can prevent smoke-induced inflammation and can counteract the proteolytic cascade that leads to emphysema formation in two separate animal models of the disease. CONCLUSIONS: These findings indicate that strategies aimed at enhancing or supplementing lung antioxidants could be effective for the prevention and treatment of this disease.

Human organic anion transporter hOAT1 forms homooligomers.

Human organic anion transporter hOAT1 belongs to a superfamily of organic anion transporters, which play critical roles in the body disposition of clinically important drugs, including anti-human immunodeficiency virus therapeutics, anti-tumor drugs, antibiotics, anti-hypertensives, and anti-inflammatories. To gain insight into the regulation of hOAT1, detailed information on its structural assembly is essential. In the present study, we investigate the quaternary structure of hOAT1 using combined approaches of chemical cross-linking, gel filtration chromatography, co-immunoprecipitation, cell surface biotinylation, and metabolic labeling. Chemical cross-linking of intact membrane proteins from LLC-PK1 cells stably expressing hOAT1 converted quantitatively hOAT1 monomer to putative trimer and higher order of oligomer, indicating that hOAT1 is present in the membrane as multimeric complexes. When co-expressed in LLC-PK1 cells, FLAG-tagged hOAT1 co-immunoprecipitated with myc-tagged hOAT1. The hOAT1 oligomer was also detected in gel filtration chromatography of total membranes from hOAT1-expressing LLC-PK1 cells. Cell surface biotinylation with membrane-impermeable reagents and metabolic labeling with [(35)S]methionine followed by immunoprecipitation showed that the oligomeric hOAT1 did not contain any other proteins. Taken together, this is the first study demonstrating that hOAT1 exists in the plasma membrane as a homooligomer, possibly trimer, and higher order of oligomer.

Cysteine-scanning analysis of the dimerization domain of EnvZ, an osmosensing histidine kinase.

EnvZ and OmpR are a transmembrane sensor and its cognate response regulator, respectively, regulating the transcription of porin genes in response to medium osmolarity in Escherichia coli. The cytoplasmic domain of EnvZ (EnvZc) possesses both kinase and phosphatase activities and can be dissected into two functional domains, A and B. Here, we performed a cysteine-scanning analysis of domain A, a 67-residue central dimerization and phosphatase domain containing His-243 as the phosphorylation site, and we examined the effects of the cysteine substitution mutations on the enzymatic activities of domain A. The substitution mutations were made at 31 residues, from which 24 mutant domain A proteins were biochemically characterized. From the analysis of the phosphatase activity of purified mutant proteins, it was found that there are two regions in domain A which are important for this activity. Cysteine mutations in these regions dramatically reduce or completely abolish the phosphatase activity of domain A. The mutations that have the most-severe effects on domain A phosphatase activity also significantly reduce the phosphatase activity of EnvZc containing the same mutation. Using an in vitro complementation system with EnvZc(H243V), these cysteine mutants were further characterized for their autophosphorylation activities as well as their phosphotransfer activities. The results indicate that some mutations are specific either for the phosphatase activity or for the kinase activity.

Spontaneous subunit exchange and biochemical evidence for trans-autophosphorylation in a dimer of Escherichia coli histidine kinase (EnvZ).

The EnvZ/OmpR histidyl-aspartyl phosphorelay (HAP) system in Escherichia coli regulates the expression of ompF and ompC, the major outer membrane porin genes, in response to environmental osmolarity changes. Here, we report that dimers of EnvZc, the cytoplasmic domain of EnvZ, undergo spontaneous subunit exchange in solution. By introducing a cysteine substitution (S260C) in the dimerization domain of EnvZc, we were able to crosslink the two subunits in a dimer and trap the heterodimer formed between two different mutant EnvZc. By using a complementing system with two autophosphorylation-defective EnvZc mutants, one containing the H243V mutation at the autophosphorylation site and the other containing the G405A mutation in the ATP-binding domain, we demonstrated that an EnvZc(G405A) subunit can be phosphorylated by an EnvZc(H243V) subunit only when a heterodimer is formed. The rate of subunit exchange is concentration-dependent, with higher rates at higher concentrations of protein. The disulfide-crosslinked EnvZc(G405A) homodimer could not be phosphorylated by EnvZc(H243V), since the heterodimer formation between the two mutant proteins was blocked, indicating that autophosphorylation cannot occur by dimer-dimer interaction. By using MBP-deltaL-EnvZc(S260C) fusion protein (deltaL: the linker region, spanning residues 180-222, was deleted), it was found that in the disulfide-crosslinked MBP-deltaL-EnvZc(S260C)/deltaL-EnvZc(S260C/G405A) heterodimer, only the deltaL-EnvZc(S260C/G405A) subunit was phosphorylated but not the MBP-deltaL-EnvZc(S260C) subunit. Together, the present results provide biochemical evidence that EnvZ autophosphorylation occurs in trans and only within an EnvZ dimer.

Probing catalytically essential domain orientation in histidine kinase EnvZ by targeted disulfide crosslinking.

EnvZ, a dimeric transmembrane histidine kinase, belongs to the family of His-Asp phosphorelay signal transduction systems. The cytoplasmic kinase domain of EnvZ can be dissected into two independently functioning domains, A and B, whose NMR solution structures have been individually determined. Here, we examined the topological arrangement of these two domains in the EnvZ dimer, a structure that is key to understanding the mechanism underlying the autophosphorylation activity of the kinase. A series of cysteine substitution mutants were constructed to test the feasibility of chemical crosslinking between the two domains. These crosslinking data demonstrate that helix I of domain A of one subunit in the EnvZc dimer is in close proximity to domain B of the other subunit in the same dimer, while helix II of domain A of one subunit interacts with domain B of the same subunit in the EnvZc dimer. This is the first demonstration of the topological arrangement between the central dimerization domain containing the active center His residues (domain A) and the ATP-binding catalysis assisting domain (domain B) in a class I histidine kinase.

Thermotoga maritima MazG protein has both nucleoside triphosphate pyrophosphohydrolase and pyrophosphatase activities.

MazG proteins form a widely conserved family among bacteria, but their cellular function is still unknown. Here we report that Thermotoga maritima MazG protein (Tm-MazG), the product of the TM0913 gene, has both nucleoside triphosphate pyrophosphohydrolase (NTPase) and pyrophosphatase activities. Tm-MazG catalyzes the hydrolysis of all eight canonical ribo- and deoxyribonucleoside triphosphates to their corresponding nucleoside monophosphates and PPi and subsequently hydrolyzes the resultant PPi to Pi. The NTPase activity with deoxyribonucleoside triphosphates as substrate is higher than corresponding ribonucleoside triphosphates. dGTP is the best substrate among the deoxyribonucleoside triphosphates, and GTP is the best among the ribonucleoside triphosphates. Both NTPase and pyrophosphatase activities were enhanced at higher temperatures and blocked by the alpha,beta-methyleneadenosine triphosphate, which cannot be hydrolyzed by Tm-MazG. Furthermore, PPi is an inhibitor for the Tm-MazG NTPase activity. Significant decreases in the NTPase activity and concomitant increases in the pyrophosphatase activity were observed when mutations were introduced at the highly conserved amino acid residues in Tm-MazG N-terminal region (E41Q/E42Q, E45Q, E61Q, R97A/R98A, and K118A). These results demonstrated that Tm-MazG has dual enzymatic functions, NTPase and pyrophosphatase, and that these two enzymatic activities are coordinated.