| 2019 | Drug Inducible CRISPR/Cas Systems. | Comput Struct Biotechnol J | Clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems have been employed as a powerful versatile technology for programmable gene editing, transcriptional modulation, epigenetic modulation, and genome labeling, Yet better control of their activity is important to accomplish greater precision and to reduce undesired outcomes such as off-target events. The use of small molecules to control CRISPR/Cas activity represents a promising direction. Here, we provide an updated review on multiple drug inducible CRISPR/Cas systems and discuss their distinct properties. We arbitrarily divided the emerging drug inducible CRISPR/Cas systems into two categories based on whether at transcription or protein level does chemical control occurs. The first category includes Tet-On/Off system and Cre-dependent system. The second category includes chemically induced proximity systems, intein splicing system, 4-Hydroxytamoxifen-Estrogen Receptor based nuclear localization systems, allosterically regulated Cas9 system, and destabilizing domain mediated protein degradation systems. Finally, the advantages and limitations of each system were summarized. |
| 2019 | Luminespib plus pemetrexed in patients with non-squamous non-small cell lung cancer. | Lung Cancer | Luminespib (AUY922) is a second-generation heat shock protein 90 (HSP90) inhibitor with demonstrated activity in non-small cell lung cancer (NSCLC). Since luminespib reduces levels of dihydrofolate reductase (DHFR), a key enzymatic target of pemetrexed, we assessed the safety and tolerability of luminespib in combination with pemetrexed in patients with previously treated metastatic non-squamous non-small cell lung cancer (NSCLC). We also sought to study the pharmacokinetics and correlate tumor dihydrofolate reductase (DHFR) expression with clinical response.Patients received weekly luminespib at either 40 mg/m, 55 mg/m, or 70 mg/m according to a standard 3 + 3 dose-escalation design along with pemetrexed at 500 mg/m followed by an expansion at the maximum tolerated dose (MTD).Two-dose limiting toxicities (DLTs) were experienced in the 70 mg/m2 cohort, therefore the MTD was determined to be 55 mg/m. 69% (N = 9) of patients experienced ophthalmologic toxicity related to luminespib. Maximum serum concentration (Cmax) of luminespib was associated with increased grade 2 drug related adverse events (DRAEs) (r = 0.74, P < 0.01), with volume of distribution (V) inversely associated with the number of DRAEs (r = - 0.81, P = 0.004) and ophthalmologic related DRAEs (r = - 0.65, P = 0.04). The best response was partial response in one patient for 20 months, prior to expiration of all luminespib. Amongst patients treated at the MTD, the objective response rate was 14%.In patients with previously treated metastatic NSCLC, the MTD of luminespib in combination with pemetrexed was 55 mg/m per week. The combination of luminespib and pemetrexed demonstrated clinical activity. Tolerability of luminespib with pemetrexed is limited by ocular toxicity. |
| 2018 | Iclaprim: a differentiated option for the treatment of skin and skin structure infections. | Expert Rev Anti Infect Ther | Iclaprim is a selective bacterial dihydrofolate reductase (DHFR) inhibitor. Although there are alternative options for the treatment of acute bacterial skin and skin structure infections (ABSSSI), iclaprim is differentiated from other available antibiotics. Areas covered: Iclaprim is under clinical development for ABSSSI. This review summarizes the mechanism of action, pharmacokinetics, microbiology, clinical development program, and the differentiation of iclaprim from other antibiotics. Expert commentary: Iclaprim has a different mechanism of action (DHFR inhibitor) compared to most other antibiotics, is active and rapidly bactericidal against Gram-positive pathogens including antibiotic-resistant pathogens, and suppresses bacterial exotoxins (alpha hemolysin, Panton Valentine leukocidin, and toxic shock syndrome toxin-1). Compared to trimethoprim, iclaprim has lower MIC, can be given without a sulfonamide, overcomes select trimethoprim resistance, and does not cause hyperkalemia. Iclaprim is administered as a fixed dose, does not require dose adjustment in renally-impaired or obese patients, and was not associated with nephrotoxicity in the Phase 3 pivotal REVIVE studies. Iclaprim represents a novel, alternative option for the treatment of severe skin and skin structure infections due to Gram-positive bacteria, particularly in patients at risk of acute kidney injury. |
| 2015 | PGC-1α overexpression suppresses blood pressure elevation in DOCA-salt hypertensive mice. | Biosci Rep | Increasing evidences have accumulated that endothelial dysfunction is involved in the pathogenesis of hypertension. Peroxisome proliferator-activated receptor γ (PPARγ) coactivator-1α (PGC-1α) has been identified as an essential factor that protects against endothelial dysfunction in vascular pathologies. However, the functional role of PGC-1α in hypertension is not well understood. Using an adenovirus infection model, we tested the hypothesis that PGC-1α overexpression retards the progression of hypertension in deoxycorticosterone acetate (DOCA)-salt mice model through preservation of the function of endothelium. We first demonstrated that PGC-1α expression not only in conductance and resistance arteries but also in endothelial cells was decreased after DOCA-salt treatment. In PGC-1α adenovirus-infected mice, the elevation of blood pressure in DOCA-salt mice was attenuated, as determined using tail-cuff measurement. Furthermore, PGC-1α overexpression inhibited the decrease in nitric oxide (NO) generation and the increase in superoxide anion (O2 (-)) production in DOCA-salt-treated mice, in parallel with improved endothelium-dependent relaxation. Rather than affecting endothelial NO synthase (eNOS) total expression and phosphorylation, PGC-1α significantly inhibited eNOS uncoupling, as evidenced by increased eNOS homodimerization, BH4 levels, GTP-cyclohydrolase 1 (GTPCH1) and dihydrofolate reductase (DHFR) expression and heat-shock protein (Hsp)90-eNOS interaction. Our findings demonstrate that PGC-1α overexpression preserves eNOS coupling, enhances NO generation, improves endothelium-dependent relaxation and thus lowers blood pressure, suggesting that up-regulation of PGC-1α may be a novel strategy to prevent and treat hypertension. |
| 2013 | Nitric oxide synthase inhibition and oxidative stress in cardiovascular diseases: possible therapeutic targets? | Pharmacol Ther | Nitric oxide (NO) is synthetized enzymatically from l-arginine (l-Arg) by three NO synthase isoforms, iNOS, eNOS and nNOS. The synthesis of NO is selectively inhibited by guanidino-substituted analogs of l-Arg or methylarginines such as asymmetric dimethylarginine (ADMA), which results from protein degradation in cells. Many disease states, including cardiovascular diseases and diabetes, are associated with increased plasma levels of ADMA. The N-terminal catalytic domain of these NOS isoforms binds the heme prosthetic group as well as the redox cofactor, tetrahydrobiopterin (BH(4)) associated with a regulatory protein, calmodulin (CaM). The enzymatic activity of NOS depends on substrate and cofactor availability. The importance of BH(4) as a critical regulator of eNOS function suggests that BH(4) may be a rational therapeutic target in vascular disease states. BH(4) oxidation appears to be a major contributor to vascular dysfunction associated with hypertension, ischemia/reperfusion injury, diabetes and other cardiovascular diseases as it leads to the increased formation of oxygen-derived radicals due to NOS uncoupling rather than NO. Accordingly, abnormalities in vascular NO production and transport result in endothelial dysfunction leading to various cardiovascular disorders. However, some disorders including a wide range of functions in the neuronal, immune and cardiovascular system were associated with the over-production of NO. Inhibition of the enzyme should be a useful approach to treat these pathologies. Therefore, it appears that both a lack and excess of NO production in diseases can have various important pathological implications. In this context, NOS modulators (exogenous and endogenous) and their therapeutic effects are discussed. |
| 2013 | Genetic linkage of blaNDM among nosocomial isolates of Acinetobacter baumannii from a tertiary referral hospital in northern India. | Int J Antimicrob Agents | The aim of this study was to evaluate the emergence of blaNDM-1 among clinical isolates of Acinetobacter baumannii isolated from a north Indian tertiary care hospital and to assess the gene cassettes and resistance determinants located within them. In total, 74 A. baumannii were screened for MBL production by the imipenem-EDTA method and were characterised for antibiotic sensitivity. PCR was performed to detect the presence of blaNDM and the co-existence of ESBL and AmpC genes. NDM-producing isolates were typed by ERIC-PCR, and the association of integrons with blaNDM-1 and the presence of gene cassettes were determined using specific primers. The genetic location of blaNDM in ISAba125 was also determined. Transformation was performed using a heat-shock method. Three isolates were found to harbour blaNDM, all of which co-produced blaEBC, blaDHA and blaCIT AmpC β-lactamases. All MBL-producers showed resistance to cephalosporins, carbapenems, aminoglycosides, fluoroquinolones and tigecycline but were susceptible to polymyxin B. Presence of class 1 integrons was demonstrated in all three blaNDM-harbouring isolates, whilst linkage between the integron and blaNDM could not be established. Detection of gene cassettes revealed the presence of dihydrofolate reductase (dhfr) and aminoglycoside 6'-N-acetyltransferase [aac(6')] genes. Presence of blaNDM in ISAba125 was also observed. These findings suggest that ISAba125 appears to be the main genetic component for dissemination of blaNDM in A. baumannii. The association of blaNDM-1 with ISAba125 and the mobility of other multiresistance region (gene cassette)-carrying integrons provide an easy way to cross species barriers and reach a level that places the patients at risk. |
| 2012 | Mechanism of reversal of high glucose-induced endothelial nitric oxide synthase uncoupling by tanshinone IIA in human endothelial cell line EA.hy926. | Eur J Pharmacol | Endothelial nitric oxide synthase (eNOS) uncoupling plays a causal role in endothelial dysfunction in many cardiovascular and metabolic diseases. Tanshinone IIA (Tan IIA), an active compound from Salvia miltiorrhiza, has been used to treat cardiovascular and metabolic diseases. However, the effects of Tan IIA on eNOS uncoupling have not been reported. We hypothesize that Tan IIA can regulate eNOS uncoupling in endothelium cells under oxidative stress. The results showed that eNOS-mediated NO generation was significantly decreased, accompanied by increased superoxide production and NOX4 expression. The ratio of eNOS dimer to monomer and NOS cofactor tetrahydrobiopterin (BH4) to 7,8-dihydrobiopterin (BH2) as well as expressions of heat-shock protein of 90kDa (HSP90), GTP cyclohydrolase-1 (GTPCH1) and dihydrofolate reductase (DHFR) were significantly decreased. Tan IIA significantly inhibited superoxide production and expression of NOX4, and increased NO generation and eNOS homodimerization, as well as expressions of HSP90, GTPCH1 and DHFR in a concentration-dependent manner. The ratio of BH4 to BH2 was also elevated by Tan IIA. In addition, Tan IIA significantly inhibited the increase in expression of PI3K in high glucose treated cells. Wortmannin, a PI3K inhibitor, significantly inhibited the high glucose induced NOX4 expression. The results demonstrated that Tan IIA restored eNOS uncoupling induced by high glucose by targeting NADPH oxidase, HSP90, GTPCH1 and DHFR, and PI3K pathway, which leads to reduced intracellular oxidative stress and increased NO generation. Tan IIA may be used as a prototype agent to restore eNOS coupling under certain cardiovascular and metabolic diseases. |
| 2012 | Role of the AAA protease Yme1 in folding of proteins in the intermembrane space of mitochondria. | Mol Biol Cell | The vast majority of mitochondrial proteins are synthesized in the cytosol and transported into the organelle in a largely, if not completely, unfolded state. The proper function of mitochondria thus depends on folding of several hundreds of proteins in the various subcompartments of the organelle. Whereas folding of proteins in the mitochondrial matrix is supported by members of several chaperone families, very little is known about folding of proteins in the intermembrane space (IMS). We targeted dihydrofolate reductase (DHFR) as a model substrate to the IMS of yeast mitochondria and analyzed its folding. DHFR can fold in this compartment, and its aggregation upon heat shock can be prevented in an ATP-dependent manner. Yme1, an AAA (ATPases associated with diverse cellular activities) protease of the IMS, prevented aggregation of DHFR. Analysis of protein aggregates in mitochondria lacking Yme1 revealed the presence of a number of proteins involved in the establishment of mitochondrial ultrastructure, lipid metabolism, protein import, and respiratory growth. These findings explain the pleiotropic effects of deletion of YME1 and suggest an important role for Yme1 as a folding assistant, in addition to its proteolytic function, in the protein homeostasis of mitochondria. |
| 2009 | Large-scale chromatin structure of inducible genes: transcription on a condensed, linear template. | J Cell Biol | The structure of interphase chromosomes, and in particular the changes in large-scale chromatin structure accompanying transcriptional activation, remain poorly characterized. Here we use light microscopy and in vivo immunogold labeling to directly visualize the interphase chromosome conformation of 1-2 Mbp chromatin domains formed by multi-copy BAC transgenes containing 130-220 kb of genomic DNA surrounding the DHFR, Hsp70, or MT gene loci. We demonstrate near-endogenous transcription levels in the context of large-scale chromatin fibers compacted nonuniformly well above the 30-nm chromatin fiber. An approximately 1.5-3-fold extension of these large-scale chromatin fibers accompanies transcriptional induction and active genes remain mobile. Heat shock-induced Hsp70 transgenes associate with the exterior of nuclear speckles, with Hsp70 transcripts accumulating within the speckle. Live-cell imaging reveals distinct dynamic events, with Hsp70 transgenes associating with adjacent speckles, nucleating new speckles, or moving to preexisting speckles. Our results call for reexamination of classical models of interphase chromosome organization. |
| 2005 | ARF directly binds DP1: interaction with DP1 coincides with the G1 arrest function of ARF. | Mol Cell Biol | The tumor suppressor ARF inhibits cell growth in response to oncogenic stress in a p53-dependent manner. Also, there is an increasing appreciation of ARF's ability to inhibit cell growth via multiple p53-independent mechanisms, including its ability to regulate the E2F pathway. We have investigated the interaction between the tumor suppressor ARF and DP1, the DNA binding partner of the E2F family of factors (E2Fs). We show that ARF directly binds to DP1. Interestingly, binding of ARF to DP1 results in an inhibition of the interaction between DP1 and E2F1. Moreover, ARF regulates the association of DP1 with its target gene, as evidenced by a chromatin immunoprecipitation assay with the dhfr promoter. By analyzing a series of ARF mutants, we demonstrate a strong correlation between ARF's ability to regulate DP1 and its ability to cause cell cycle arrest. S-phase inhibition by ARF is preceded by an inhibition of the E2F-activated genes. Moreover, we provide evidence that ARF inhibits the E2F-activated genes independently of p53 and Mdm2. Also, the interaction between ARF and DP1 is enhanced during oncogenic stress and "culture shock." Taken together, our results show that DP1 is a critical direct target of ARF. |
| 2003 | Cellular uptake of Clostridium botulinum C2 toxin: membrane translocation of a fusion toxin requires unfolding of its dihydrofolate reductase domain. | Biochemistry | The Clostridium botulinum C2 toxin is the prototype of the family of binary actin-ADP-ribosylating toxins. C2 toxin is composed of two separated nonlinked proteins. The enzyme component C2I ADP-ribosylates actin in the cytosol of target cells. The binding/translocation component C2II mediates cell binding of the enzyme component and its translocation from acidic endosomes into the cytosol. After proteolytic activation, C2II forms heptameric pores in endosomal membranes, and most likely, C2I translocates through these pores into the cytosol. For this step, the cellular heat shock protein Hsp90 is essential. We analyzed the effect of methotrexate on the cellular uptake of a fusion toxin in which the enzyme dihydrofolate reductase (DHFR) was fused to the C-terminus of C2I. Here, we report that unfolding of C2I-DHFR is required for cellular uptake of the toxin via the C2IIa component. The C2I-DHFR fusion toxin catalyzed ADP-ribosylation of actin in vitro and was able to intoxicate cultured cells when applied together with C2IIa. Binding of the folate analogue methotrexate favors a stable three-dimensional structure of the dihydrofolate reductase domain. Pretreatment of C2I-DHFR with methotrexate prevented cleavage of C2I-DHFR by trypsin. In the presence of methotrexate, intoxication of cells with C2I-DHFR/C2II was inhibited. The presence of methotrexate diminished the translocation of the C2I-DHFR fusion toxin from endosomal compartments into the cytosol and the direct C2IIa-mediated translocation of C2I-DHFR across cell membranes. Methotrexate had no influence on the intoxication of cells with C2I/C2IIa and did not alter the C2IIa-mediated binding of C2I-DHFR to cells. The data indicate that methotrexate prevented unfolding of the C2I-DHFR fusion toxin, and thereby the translocation of methotrexate-bound C2I-DHFR from endosomes into the cytosol of target cells is inhibited. |
| 2004 | Heat shock protein 72 binds and protects dihydrofolate reductase against oxidative injury. | Biochem Biophys Res Commun | Although heat shock protein Hsp72 confers resistance to oxidative injury, the mechanisms are unknown. These studies demonstrate that Hsp72 protects dihydrofolate reductase (DHFR) against injury caused by the thiol oxidant monochloramine (NH(2)Cl). When exposed to NH(2)Cl, DHFR catalytic activity is impaired and SDS-PAGE migration retarded. These may be blocked by prior addition of Hsp72 or the folate analog methotrexate. Methotrexate binding to DHFR is diminished by oxidant treatment, preventable by prior Hsp72 incubation. Hsp72 also protects DHFR in IEC-18 cells following oxidant exposure. Hsp72 co-immunoprecipitates with DHFR, especially after partial oxidation. The DHFR-Hsp72 interaction is modulated by cofactor/substrate binding for both Hsp72 (ATP) and DHFR (methotrexate). Thiol oxidation of DHFR increases susceptibility for tryptic proteolysis. Preincubation of DHFR with Hsp72 prevents the NH(2)Cl-induced sensitivity to proteolysis. Thus, Hsp72 binds DHFR through enhanced protein-chaperone interactions upon oxidant exposure, a process that may protect against irreversible modification of DHFR catalytic and structural integrity. |
| 2001 | Quantification of expression of Staphylococcus epidermidis housekeeping genes with Taqman quantitative PCR during in vitro growth and under different conditions. | J Bacteriol | The aims of the present study were (i) to develop and test a sensitive and reproducible method for the study of gene expression in staphylococci and (ii) to study the expression of five housekeeping genes which are involved in nucleic acid metabolism (gmk, guanylate kinase; the dihydrofolate reductase [DHFR] gene), glucose metabolism (tpi, triosephosphate isomerase), and protein metabolism (the 16S rRNA gene; hsp-60, heat-shock protein 60) during in vitro exponential and stationary growth. A modified method for instant mRNA isolation was combined with gene quantification via Taqman real-time quantitative PCR. The detection limit of our method was 10 copies of RNA. The average intersample variability was 16%. A 10-fold increase in the expression of the hsp-60 gene was induced by exposure to a 10 degrees C heat shock (37 to 47 degrees C) for 10 min. During in vitro growth, the expression of all five housekeeping genes showed rapid up-regulation after inoculation of the bacteria in brain heart infusion medum and started to decline during the mid-exponential-growth phase. Maximal gene expression was 110- to 300-fold higher than gene expression during stationary phase. This indicates that housekeeping metabolism is a very dynamic process that is extremely capable of adapting to different growth conditions. Expression of the 16S rRNA gene decreases significantly earlier than that of other housekeeping genes. This confirms earlier findings for Escherichia coli that a decline in bacterial ribosomal content (measured by 16S rRNA gene expression) precedes the decline in protein synthesis (measured by mRNA expression). |
| 2000 | Import of a cytosolic protein into lysosomes by chaperone-mediated autophagy depends on its folding state. | J Biol Chem | We have analyzed the folding state of cytosolic proteins imported in vitro into lysosomes, using an approach originally developed by Eilers and Schatz, (Eilers, M., and Schatz, G. (1986) Nature 322, 228-232) to investigate protein import into mitochondria. The susceptibility toward proteases of mouse dihydrofolate reductase (DHFR), synthesized in a coupled transcription-translation system with rabbit reticulocytes, decreased in the presence of its substrate analogue, methotrexate. This analogue complexes with high affinity with the in vitro synthesized DHFR and locks it into a protease-resistant folded conformation. DHFR was taken up by freshly isolated rat liver lysosomes and methotrexate reduced this uptake by about 80%. A chimeric DHFR protein, which carries the N-terminal presequence of subunit 9 of ATP synthase preprotein from Neurospora crassa fused to its N terminus, was taken up by lysosomes more efficiently. Again, methotrexate abolished the lysosomal uptake of the fusion protein, which was partially restored by washing of methotrexate from DHFR or by adding together methotrexate and dihydrofolate, the natural substrate of DHFR. Immunoblot analysis with anti-DHFR of liver lysosomes and of other fractions, isolated from rats starved for 88 h and treated with lysosomal inhibitors, suggests that DHFR is degraded by chaperone-mediated autophagy. Competition with ribonuclease A and stimulation by ATP/Mg(2+) and the heat shock cognate protein of 73 kDa show that the lysosomal uptake of the fusion protein also occurs by this pathway. It is concluded that the lysosomal uptake of cytosolic proteins by chaperone-mediated autophagy mainly occurs by passage of the unfolded proteins through the lysosomal membrane. Therefore, this mechanism is different from protein transport into peroxisomes, but similar to the import of proteins into the endoplasmic reticulum and mitochondria. |
| 2000 | Biogenesis of the yeast frataxin homolog Yfh1p. Tim44-dependent transfer to mtHsp70 facilitates folding of newly imported proteins in mitochondria. | Eur J Biochem | Tim44 is an essential component of the mitochondrial inner membrane protein import machinery. In this study we asked if Tim44 is of relevance in intramitochondrial protein folding. We investigated the role of Tim44 in the biogenesis of the authentic mitochondrial protein Yfh1p, the yeast homolog of mammalian frataxin, which was recently implicated in Friedreich ataxia. After inactivation of Tim44, binding of mitochondrial heat shock protein (mtHsp)70 to translocating Yfh1p and subsequent folding to the native state was nearly completely blocked. Residual amounts of imported Yfh1p showed an increased tendency to aggregate. To further characterize the functions of Tim44 in the matrix, we imported dihydrofolate reductase (DHFR) as a model protein. Depletion of Tim44 allowed import of DHFR, although folding of the newly imported DHFR was delayed. Moreover, the depletion of Tim44 caused a strongly reduced binding of mtHsp70 and Mge1 to the translocating polypeptide. Subsequent dissociation of mtHsp70 from imported DHFR was delayed, indicating that mtHsp70-substrate complexes formed independently of Tim44 differ from the complexes that form under the control of Tim44. We conclude that Tim44 not only plays a role in protein translocation but also in the pathways of mitochondrial protein folding. |
| 1993 | Presequence and mature part of preproteins strongly influence the dependence of mitochondrial protein import on heat shock protein 70 in the matrix. | J Cell Biol | To test the hypothesis that 70-kD mitochondrial heat shock protein (mt-hsp70) has a dual role in membrane translocation of preproteins we screened preproteins in an attempt to find examples which required either only the unfoldase or only the translocase function of mt-hsp70. We found that a series of fusion proteins containing amino-terminal portions of the intermembrane space protein cytochrome b2 (cyt. b2) fused to dihydrofolate reductase (DHFR) were differentially imported into mitochondria containing mutant hsp70s. A fusion protein between the amino-terminal 167 residues of the precursor of cyt. b2 and DHFR was efficiently transported into mitochondria independently of both hsp70 functions. When the length of the cyt. b2 portion was increased and included the heme binding domain, the fusion protein became dependent on the unfoldase function of mt-hsp70, presumably caused by a conformational restriction of the heme-bound preprotein. In the absence of heme the noncovalent heme binding domain in the longer fusion proteins no longer conferred a dependence on the unfoldase function. When the cyt. b2 portion of the fusion protein was less than 167 residues, its import was still independent of mt-hsp70 function; however, deletion of the intermembrane space sorting signal resulted in preproteins that ended up in the matrix of wild-type mitochondria and whose translocation was strictly dependent on the translocase function of mt-hsp70. These findings provide strong evidence for a dual role of mt-hsp70 in membrane translocation and indicate that preproteins with an intermembrane space sorting signal can be correctly imported even in mutants with severely impaired hsp70 function. |
| 1993 | Identification of an uncleavable targeting signal in the 70-kilodalton spinach chloroplast outer envelope membrane protein. | J Biol Chem | A cDNA clone encoding a cognate 70-kDa heat shock protein from the spinach chloroplast outer envelope (SCE70) was recently characterized (Ko, K., Bornemisza, O., Kourtz, L., Ko, Z. W., Plaxton, W. C., and Cashmore, A. R. (1992) J. Biol. Chem. 267, 2986-2993). Initial studies revealed that SCE70 is targeted to the chloroplast outer envelope membrane without further processing. To determine whether SCE70 possesses a "targeting domain," we tested the targeting ability of SCE70 proteins with various carboxyl- and amino-terminal deletions. Carboxyl-terminal deletions of up to 60% of the protein had no apparent effect on the targeting ability of SCE70. Amino-terminal deletions abolished targeting to the chloroplast except when the extreme NH2-terminal 48-amino acid sequence was retained. We further assessed the chloroplast-targeting ability of the NH2-terminal 48 amino acids by fusing to the foreign protein, mouse dihydrofolate reductase (DHFR). The resulting fusion protein, SCE70-DHFR, was localized to the outer envelope membrane of isolated chloroplasts. SCE70-DHFR exhibited targeting characteristics similar to native SCE70. The targeting of SCE70-DHFR was inhibited effectively by anti-SCE70 antibodies. Immunoprecipitation and chemical cross-linking experiments revealed that SCE70-DHFR is targeted to the same complex as SCE70 in the chloroplast envelope. These results suggest that the extreme NH2 terminus of SCE70 is required for directing SCE70 to a destination in the chloroplast outer envelope membrane, possibly through assembling the polypeptide into a protein complex. |
| 1992 | Prevention of protein denaturation under heat stress by the chaperonin Hsp60. | Science | The increased synthesis of heat shock proteins is a ubiquitous physiological response of cells to environmental stress. How these proteins function in protecting cellular structures is not yet understood. The mitochondrial heat shock protein 60 (Hsp60) has now been shown to form complexes with a variety of polypeptides in organelles exposed to heat stress. The Hsp60 was required to prevent the thermal inactivation in vivo of native dihydrofolate reductase (DHFR) imported into mitochondria. In vitro, Hsp60 bound to DHFR in the course of thermal denaturation, preventing its aggregation, and mediated its adenosine triphosphate-dependent refolding at increased temperatures. These results suggest a general mechanism by which heat shock proteins of the Hsp60 family stabilize preexisting proteins under stress conditions. |
| 1990 | Mitochondrial precursor protein. Effects of 70-kilodalton heat shock protein on polypeptide folding, aggregation, and import competence. | J Biol Chem | A hybrid precursor protein constructed by fusing the mitochondrial matrix-targeting signal of rat preornithine carbamyl transferase to murine cytosolic dihydrofolate reductase (designated pO-DHFR) was expressed in Escherichia coli. Following purification under denaturing conditions, pO-DHFR was capable of membrane translocation when diluted directly into import medium containing purified mitochondria but lacking cytosolic extracts. This import competence was lost with time, however, when the precursor was diluted and preincubated in medium lacking mitochondria, unless cytosolic proteins (provided by rabbit reticulocyte lysate) were present. Identical results were obtained for purified precursor made by in vitro translation. The ability of the cytosolic proteins to maintain the purified precursor in an import-competent state was sensitive to protease, N-ethylmaleimide (NEM), and was heat labile. Further, this activity appeared to be signal sequence dependent. ATP was not required for the maintenance of pO-DHFR competence, nor did purified 70-kDa heat shock protein (the constitutive form of Hsp70) substitute for this activity. Interestingly, however, purified Hsp70 prevented aggregation of the precursor in an ATP-dependent manner and, as well, retarded the apparent rate and extent of pO-DHFR folding. Partial purification of reticulocyte lysate proteins indicated that competence activity resides within a large mass protein fraction (200-250 kDa) that contains Hsp70. Sucrose density gradient analysis revealed that pO-DHFR reversibly interacts with components of this fraction. Pretreatment of the fraction with NEM, however, significantly stabilized the subsequent formation of a complex with the precursor. The results indicate that Hsp70 can retard precursor polypeptide folding and prevent precursor aggregation; however, by itself, Hsp70 cannot confer import competence to pO-DHFR. Maintenance of import competence correlates with interactions between the precursor and an NEM-sensitive cytosolic protein fraction. Efficient dissociation of the precursor from this complex appears to require a reactive thiol moiety on the cytosolic protein(s). |
| 1987 | Molecular karyotype of five species of Leishmania and analysis of gene locations and chromosomal rearrangements. | Mol Biochem Parasitol | The molecular karyotypes of five species of Leishmania were studied by pulsed field gradient gel electrophoresis (PFGGE) of chromosome-sized DNA bands. Each species exhibits a unique pattern of 22-28 bands in the size range approximately 200-2200 kb whereas strains of one species exhibit similar karyotypes. Analysis of the behaviour of kinetoplast DNA during PFGGE showed that minicircle DNA remains confined to the gel slot but a proportion of the maxicircle DNA fractionates as a low molecular weight band below band 1. The band location of genes for alpha and beta tubulin, the 5' spliced leader sequence (5'SL), heat shock proteins 70 (hsp 70) and 83 (hsp 83) and thymidylate synthase-dihydrofolate reductase (TS-DHFR) were analysed. Housekeeping genes are not clustered in Leishmania but are found on at least 7 bands in L. major. The hsp 83 gene is linked to the tandemly repeated beta tubulin allele on band 21 in L. major. Among different species, the location of the unlinked hsp 83 and hsp 70 genes is conserved whereas the TS-DHFR and 5'SL sequences are found on bands of varying size. The 5'SL gene may be rearranged in L. enriettii and two 5'SL loci were identified in L. donovani and L. tropica. The conservation of loci in strains of L. major suggests that the chromosomal genetic linkage map should be a reliable marker for identifying unknown isolates of Leishmania. Sequences on one band in L. mexicana sp. were shared among several bands and distributed on homologous and non-homologous bands in other species showing that DNA sequences are rearranged during speciation in Leishmania. |