The respiratory syncytial virus (RSV) L protein is a viral RNA-dependent RNA polymerase that contains multiple enzyme activities required for RSV replication. The RSV L inhibitors described in literature are limited by their cytotoxicity or the lack of RSV B subtype coverage. Here, we characterize a new RSV L inhibitor with strong antiviral activity against both RSV A and B subtypes and no detectable cytotoxicity. This compound, AZ-27, was equally active against RSV live viruses and subgenomic replicons and demonstrated advantages over other classes of RSV inhibitors in time-of-addition and cell line dependency studies. Resistance studies identified a dominant mutation in the putative capping enzyme domain of L protein, which conferred strong resistance to the AZ-27 series but not other classes of RSV inhibitors, supporting RSV L protein as the direct target for AZ-27. This novel and broad-spectrum RSV L polymerase inhibitor may pave the way toward an efficacious RSV therapeutic and provide a new tool for interrogation of the L protein function.
Urinary exosome-like vesicles (ELVs) are a heterogenous mixture (diameter 40–200nm) containing vesicles shed from all segments of the nephron including glomerular podocytes. Contamination with Tamm Horsfall protein (THP) oligomers has hampered their isolation and proteomic analysis. Here we improved ELV isolation protocols employing density centrifugation to remove THP and albumin, and isolated a glomerular membranous vesicle (GMV) enriched subfraction from 7 individuals identifying 1830 proteins and in 3 patients with glomerular disease identifying 5657 unique proteins. The GMV fraction was composed of podocin/podocalyxin positive irregularly shaped membranous vesicles and podocin/podocalyxin negative classical exosomes. Ingenuity pathway analysis identified integrin, actin cytoskeleton and RhoGDI signaling in the top three canonical represented signaling pathways and 19 other proteins associated with inherited glomerular diseases. The GMVs are of podocyte origin and the density gradient technique allowed isolation in a reproducible manner. We show many nephrotic syndrome proteins, proteases and complement proteins involved in glomerular disease are in GMVs and some were shed in the disease state (nephrin, TRPC6 and INF2 and PLA2R). We calculated sample sizes required to identify new glomerular disease biomarkers, expand the ELV proteome and provide a reference proteome in a database that may prove useful in the search for biomarkers of glomerular disease.
Exosome; proteomics; podocyte; glomerular disease; Integrin; actin cytoskeleton; Rho GDI. Q-Exactive mass spectrometer
Evidence indicating an association between cigarette smoke exposure and an increase in breast cancer risk highlights the need for health messages that aim to prevent smoking initiation and reduce secondhand smoke (SHS) exposure among adolescent girls.
This study aimed to evaluate the efficacy of targeted gender-sensitive, breast cancer-specific, Web-based messages about the increased risk of breast cancer associated with cigarette smoke exposure. Outcomes assessed 6 months postmessage delivery included nonsmoking adolescent girls’ knowledge of the link between cigarette smoke exposure and breast cancer, perceptions of breast cancer risk associated with cigarette smoke, smoking behavior and intentions, and stage of change related to avoidance of secondhand smoke.
A prospective randomized controlled trial was used to compare standard (control) messages with targeted gender- and Aboriginal status-sensitive, breast cancer-specific (intervention) messages. Messages were delivered online to 618 nonsmoking girls, aged 13 to 15 years, clustered in 74 Canadian secondary schools.
Compared with the control group, girls in the intervention group were significantly more likely to report that breast cancer is an illness caused by cigarette smoke (adjusted relative risk [ARR] 1.33, 95% CI 1.05-1.68) and to agree that exposure to SHS increases their risk of breast cancer (ARR 1.10, 95% CI 1.02-1.20). No significant effects were observed for a change in smoking status, intention to try smoking, or stage of change related to avoidance of SHS.
Compared with standard messages, targeted gender-sensitive, breast cancer-specific messages had a stronger influence on girls’ knowledge and perceived risk of cigarette smoke exposure as a risk factor for breast cancer. Brief information-based interventions delivered over the Internet have the potential to provide effective health promotion that could be broadly disseminated and lead to long-term effects on girls’ knowledge and risk perceptions about cigarette exposure and breast cancer.
secondhand smoke; breast cancer; Web-based health promotion; adolescents
A structural and biophysical study of the interactions between cod and human uracil-DNA N-glycosylase (UNG) and their inhibitor Ugi is presented. The stronger interaction between cod UNG and Ugi can be explained by a greater positive electrostatic surface potential.
Uracil-DNA N-glycosylase from Atlantic cod (cUNG) shows cold-adapted features such as high catalytic efficiency, a low temperature optimum for activity and reduced thermal stability compared with its mesophilic homologue human UNG (hUNG). In order to understand the role of the enzyme–substrate interaction related to the cold-adapted properties, the structure of cUNG in complex with a bacteriophage encoded natural UNG inhibitor (Ugi) has been determined. The interaction has also been analyzed by isothermal titration calorimetry (ITC). The crystal structure of cUNG–Ugi was determined to a resolution of 1.9 Å with eight complexes in the asymmetric unit related through noncrystallographic symmetry. A comparison of the cUNG–Ugi complex with previously determined structures of UNG–Ugi shows that they are very similar, and confirmed the nucleotide-mimicking properties of Ugi. Biophysically, the interaction between cUNG and Ugi is very strong and shows a binding constant (K
b) which is one order of magnitude larger than that for hUNG–Ugi. The binding of both cUNG and hUNG to Ugi was shown to be favoured by both enthalpic and entropic forces; however, the binding of cUNG to Ugi is mainly dominated by enthalpy, while the entropic term is dominant for hUNG. The observed differences in the binding properties may be explained by an overall greater positive electrostatic surface potential in the protein–Ugi interface of cUNG and the slightly more hydrophobic surface of hUNG.
cold adaptation; uracil-DNA N-glycosylase; Atlantic cod; protein–inhibitor complex; protein–protein interactions; isothermal titration calorimetry; binding affinity
Crystal structures of methyl, ethyl, propyl and butyl isocyanide bound to sperm whale myoglobin (Mb) reveal two major conformations. In the in conformer, His(E7) is in a “closed” position, forcing the ligand alkyl chain to point inward. In the out conformer, His(E7) is in an “open” position, allowing the ligand side chain to point outward. A progressive increase in the population of the out conformer is observed with increasing ligand length in P21 crystals of native Mb at pH 7.0. This switch from in to out with increasing ligand size also occurs in solution as measured by the decrease in the relative intensity of the low (~2075 cm 1) versus high frequency (~2125 cm 1) isocyano bands. In contrast, all four isocyanides in P6 crystals of wild type recombinant Mb occupy the in conformation. However, mutating either His64 to Ala, creating a “hole” to solvent, or Phe46 to Val, freeing rotation of His64, causes bound butyl isocyanide to point completely outward in P6 crystals. Thus, the unfavorable hindrance caused with crowding a large alkyl side chain into the distal pocket appears to be roughly equal to that for pushing open the His(E7) gate and is easily affected by crystal packing. This structural conclusion supports the “side path” kinetic mechanism for O2 release, in which the dissociated ligand first moves toward the protein interior and then encounters steric resistance, which is roughly equal to that for escaping to solvent through the His(E7) channel.
myoglobin; alkyl isocyanide; isonitrile; X-ray crystallography; FTIR; infrared; vibrational spectroscopy; iron coordination
Early cochlear development is marked by an exuberant outgrowth of neurites that innervate multiple targets. The establishment of mature cochlear neural circuits is, however, dependent on the pruning of inappropriate axons and synaptic connections. Such refinement also occurs in the central nervous system (CNS), and recently, genes ordinarily associated with immune and inflammatory processes have been shown to play roles in synaptic pruning in the brain. These molecules include the major histocompatibility complex class I (MHCI) genes, H2-Kb and H2-Db, and the complement cascade gene, C1qa. Since the mechanisms involved in synaptic refinement in the cochlea are not well understood, we investigated whether these immune system genes may be involved in this process and whether they are required for normal hearing function. Here we report that these genes are not necessary for normal synapse formation and refinement in the mouse cochlea. We further demonstrate that C1qa expression is not necessary for normal hearing in mice but the lack of expression of H2-Kb and H2-Db causes hearing impairment. These data underscore the importance of the highly polymorphic family of MHCI genes in hearing in mice and also suggest that factors and mechanisms regulating synaptic refinement in the cochlea may be distinct from those in the CNS.
CorA is a copper repressible protein previously identified in the methanotrophic bacterium Methylomicrobium album BG8. In this work, we demonstrate that CorA is located on the cell surface and binds one copper ion per protein molecule, which, based on X-ray Absorption Near Edge Structure analysis, is in the reduced state (Cu(I)). The structure of endogenously expressed CorA was solved using X-ray crystallography. The 1.6 Å three-dimensional structure confirmed the binding of copper and revealed that the copper atom was coordinated in a mononuclear binding site defined by two histidines, one water molecule, and the tryptophan metabolite, kynurenine. This arrangement of the copper-binding site is similar to that of its homologous protein MopE* from Metylococcus capsulatus Bath, confirming the importance of kynurenine for copper binding in these proteins. Our findings show that CorA has an overall fold similar to MopE, including the unique copper(I)-binding site and most of the secondary structure elements. We suggest that CorA plays a role in the M. album BG8 copper acquisition.
bioinformatics and chemoinformatics; chemical and biological space; drug design; physicochemical properties; neuroprotective agents
This paper evaluates comparative patterns of fertility in new Hispanic destinations and established gateways using pooled cross-sectional data from the 2005–2009 microdata files of the American Community Survey. Changing Hispanic fertility provides a useful indicator of cultural incorporation. Analyses show that high fertility among Hispanics has been driven in part by the Mexican-origin and other new immigrant populations (e.g., noncitizens, those with poor English language skills, etc.). However, high fertility rates among Hispanics – and Mexican-origin Hispanics in particular – cannot be explained entirely by socio-demographic characteristics that place them at higher risk of fertility. For 2005–2009, Hispanic fertility rates were 48 percent higher than fertility among whites; they were roughly 25 percent higher after accounting for differences in key social characteristics, such as age, nativity, county of origin, and education. Contrary to most previous findings of spatial assimilation among in-migrants, fertility rates among Hispanics in new destinations exceeded fertility in established gateways by 18 percent. In the multivariate analyses, Hispanics in new destinations were roughly 10 percent more likely to have had a child in the past year than those living in established gateways. Results are consistent with sub-cultural explanations of Hispanic fertility and raise new questions about the spatial patterning of assimilation and the formation of ethnic enclaves outside traditional settlement areas.
Recent evidence indicates a causal link between both active smoking and secondhand smoke (SHS) exposure and breast cancer (BC).
The objective of the present study was to evaluate the initial reactions of girls and boys to tailored Web-based messages that describe the relationship between SHS and BC, using a parallel, single-blinded cluster randomized controlled trial.
This trial was nested within a cycle of an ongoing longitudinal study of 1498 students from 74 secondary schools. Self-reported assessments were used to evaluate the impact of study messages on participants’ risk perception and interest in obtaining additional information after participants were randomized by schools to control or intervention groups. The intervention group received a tailored visual message (based on gender and Aboriginal status) about BC and tobacco smoke. The control group received a standard visual message about smoking and cancer.
SHS exposure was identified as a BC risk factor by 380/1488 (25.54%) participants, during the preintervention analysis. Compared to the female participants in the control group (491/839, 58.5%), girls who received the intervention (339/649, 52.2%) were 14% more likely to agree that exposure to SHS increased their BC risk (relative risk [RR] 1.14, 95% CI 1.07-1.21). Nonsmoking girls who received the intervention were 14% more likely to agree that starting smoking would increase their BC risk (RR 1.14, 95% CI 1.07-1.21). Compared to the male participants in control group (348/839, 41.5%), boys who received the intervention (310/649, 47.8%) were 10% more likely to agree that girls’ exposure to SHS increased their BC risk (RR 1.10, 95% CI 1.02-1.18). Compared to controls, girls who received the intervention were 52% more likely to request additional information about SHS and BC (RR 1.52, 95% CI 1.12-2.06).
Brief gender-sensitive messages delivered via the Internet have the potential to increase awareness and to stimulate information seeking about the risk for BC associated with SHS.
breast cancer; secondhand smoke; cancer prevention; youth; gender
Hair cells are mechanosensors for the perception of sound, acceleration and fluid motion. Mechanotransduction channels in hair cells are gated by tip links, which connect the stereocilia of a hair cell in the direction of their mechanical sensitivity. The molecular constituents of the mechanotransduction channels of hair cells are not known. Here we show that mechanotransduction is impaired in mice lacking the tetraspan TMHS. TMHS binds to the tip-link component PCDH15 and regulates tip-link assembly, a process that is disrupted by deafness-causing Tmhs mutations. TMHS also regulates transducer channel conductance and is required for fast channel adaptation. TMHS therefore resembles other ion channel regulatory subunits such as the TARPs of AMPA receptors that facilitate channel transport and regulate the properties of pore-forming channel subunits. We conclude that TMHS is an integral component of the hair cells mechanotransduction machinery that functionally couples PCDH15 to the transduction channel.
The bacterial degradation of the nematicide 1,3-dichloropropene, an isomeric mixture, requires the action of trans- and cis-3-chloracrylic acid dehalogenase (CaaD and cis-CaaD, respectively). Both enzymes are tautomerase superfamily members and share a core catalytic mechanism for the hydrolytic dehalogenation of the respective isomer of 3-haloacrylate. The observation that cis-CaaD requires two additional residues raises the question of how CaaD carries out a comparable reaction with fewer catalytic residues. As part of an effort to determine the basis for the apparently simpler CaaD-catalyzed reaction, the kinetic mechanism was determined by stopped-flow and chemical quench techniques using a fluorescent mutant form of the enzyme, αY60W-CaaD, and trans-3-bromoacrylate as the substrate. The data from these experiments as well as bromide inhibition studies are best accommodated by a six-step model that provides individual rate constants for substrate binding, chemistry, and a proposed conformational change occurring after chemistry followed by release of malonate semialdehyde and bromide. The conformational change and product release rates are comparable and together they limit the rate of turnover. The kinetic analysis and modeling studies validate the αY60W-CaaD mutant as an accurate reporter of active site events during the course of the enzyme-catalyzed reaction. The kinetic mechanism for the αY60W-CaaD-catalyzed reaction is comparable to that obtained for the cis-CaaD-catalyzed reaction. The kinetic model and the validated αY60W-CaaD mutant set the stage for an analysis of active site mutants to explore the contributions of individual catalytic residues and the basis for the simplicity of the reaction.
Stereocilia are actin-based protrusions on auditory sensory hair cells that are deflected by sound waves to initiate the conversion of mechanical energy to neuronal signals. Stereocilia maintenance is essential because auditory hair cells are not renewed in mammals. This process requires both β-actin and γ-actin as knockout mice lacking either isoform develop distinct stereocilia pathology during aging. In addition, stereocilia integrity may hinge on immobilizing actin, which outside of a small region at stereocilia tips turns over with a very slow, months-long half-life. Here, we establish that β-actin and the actin crosslinking protein fascin-2 cooperate to maintain stereocilia length and auditory function. We observed that mice expressing mutant fascin-2 (p.R109H) or mice lacking β-actin share a common phenotype including progressive, high-frequency hearing loss together with shortening of a defined subset of stereocilia in the hair cell bundle. Fascin-2 binds β-actin and γ-actin filaments with similar affinity in vitro and fascin-2 does not depend on β-actin for localization in vivo. Nevertheless, double mutant mice lacking β-actin and expressing fascin-2 p.R109H have a more severe phenotype suggesting that each protein has a different function in a common stereocilia maintenance pathway. Since the fascin-2 p.R109H mutant binds but fails to efficiently crosslink actin filaments, we propose that fascin-2 crosslinks function to slow actin depolymerization at stereocilia tips to maintain stereocilia length.
We previously mapped a locus (ahl4) on distal Chromosome 10 that contributes to the age-related hearing loss of A/J strain mice. Here, we report on a refined genetic map position for ahl4 and its association with a mutation in the citrate synthase gene (Cs). We mapped ahl4 to the distal-most 7 Mb of Chromosome 10 by analysis of a new linkage backcross and then further narrowed the interval to 5.5 Mb by analysis of eight C57BL/6J congenic lines with different A/J-derived segments of Chr 10. A nucleotide variant in exon 3 of Cs is the only known DNA difference within the ahl4 candidate gene interval that is unique to the A/J strain and that causes a nonsynonymous codon change. Multiple lines of evidence implicate this missense mutation (H55N) as the underlying cause of ahl4-related hearing loss, likely through its effects on mitochondrial ATP and free radical production in cochlear hair cells. The A/J mouse thus provides a new model system for in vivo studies of mitochondrial function and hearing loss.
mouse; genetics; inbred strain; A/J; age-related hearing loss; ahl4; citrate synthase; oxidative stress; mitochondria
Cycloserine (CS, 4-amino-3-isoxazolidone) is a cyclic amino acid mimic that is known to inhibit many essential pyridoxal 5′-phosphate (PLP)-dependent enzymes. Two CS enantiomers are known; d-cycloserine (DCS, also known as Seromycin), is a natural product that is used to treat resistant Mycobacterium tuberculosis infections as well as neurological disorders since it is a potent NMDA receptor agonist, and l-cycloserine (LCS), is a synthetic enantiomer whose usefulness as a drug has been hampered by its inherent toxicity arising through inhibition of sphingolipid metabolism. Previous studies on various PLP-dependent enzymes revealed a common mechanism of inhibition by both enantiomers of CS; the PLP cofactor is disabled by forming a stable 3-hydroxyisoxazole/pyridoxamine 5′-phosphate (PMP) adduct at the active site where the cycloserine ring remains intact. Here we describe a novel mechanism of CS inactivation of the PLP-dependent enzyme serine palmitoyltransferase (SPT) from Sphingomonas paucimobilis. SPT catalyses the condensation of l-serine and palmitoyl-CoA, the first step in the de novo sphingolipid biosynthetic pathway. We have used a range of kinetic, spectroscopic and structural techniques to postulate that both LCS and DCS inactivate SPT by transamination to form a free pyridoxamine 5′-phosphate (PMP) and β-aminooxyacetaldehyde that remain bound at the active site. We suggest this occurs by ring opening of the cycloserine ring followed by decarboxylation. Enzyme kinetics show that inhibition is reversed by incubation with excess PLP and that LCS is a more effective SPT inhibitor than DCS. UV-visible spectroscopic data, combined with site-directed mutagenesis, suggest that a mobile Arg378 residue is involved in cycloserine inactivation of SPT.
Nearly every enzyme undergoes a significant change in structure after binding it’s substrate. New experimental and theoretical analyses of the role of changes in HIV reverse transcriptase structure in selecting a correct substrate are presented. Atomically detailed simulations using the Milestoning method predict a rate and free energy profile of the conformational change commensurate with experimental data. A large conformational change occurring on a ms timescale locks the correct nucleotide at the active site, but promotes release of a mismatched nucleotide. The positions along the reaction coordinate that decide the yield of the reaction are not determined by the chemical step. Rather, the initial steps of weak substrate binding and protein conformational transition significantly enrich the yield of a reaction with a correct substrate, while the same steps diminish the reaction probability of an incorrect substrate.
cis -3-Chloroacrylic acid dehalogenase (cis-CaaD) catalyzes the hydrolytic dehalogenation of cis-3-haloacrylates to yield malonate semialdehyde. The enzyme processes other substrates including an allene (2,3-butadienoate) to produce acetoacetate. In the course of a stereochemical analysis of the cis-CaaD-catalyzed reaction using this allene, the enzyme was unexpectedly inactivated in the presence of NaBH4 by the reduction of a covalent enzyme-substrate bond. Covalent modification was surprising because the accumulated evidence for cis-CaaD dehalogenation favored a mechanism involving direct substrate hydration mediated by Pro-1. However, the results of subsequent mechanistic, pre-steady state and full progress kinetic experiments are consistent with a mechanism in which an enamine forms between Pro-1 and the allene. Hydrolysis of the enamine or an imine tautomer produces acetoacetate. Reduction of the imine species is likely responsible for the observed enzyme inactivation. This is the first reported observation of a tautomerase superfamily member functioning by covalent catalysis. The result may suggest that some fraction of the cis-CaaD-catalyzed dehalogenation of cis-3-haloacrylates also proceeds by covalent catalysis.
“Hairpatches” (Hpt) is a naturally occurring, autosomal semi-dominant mouse mutation. Hpt/Hpt homozygotes die in utero, while Hpt/+ heterozygotes exhibit progressive renal failure accompanied by patchy alopecia. This mutation is a model for the rare human disorder “glomerulonephritis with sparse hair and telangiectases" (OMIM 137940). Fine mapping localized the Hpt locus to a 6.7 Mb region of Chromosome 4 containing 62 known genes. Quantitative real time PCR revealed differential expression for only one gene in the interval, T-cell acute lymphocytic leukemia 1 (Tal1), which was highly upregulated in the kidney and skin of Hpt/+ mice. Southern blot analysis of Hpt mutant DNA indicated a new EcoRI site in the Tal1 gene. High throughput sequencing identified an endogenous retroviral class II intracisternal A particle insertion in Tal1 intron 4. Our data suggests that the IAP insertion in Tal1 underlies the histopathological changes in the kidney by three weeks of age, and that glomerulosclerosis is a consequence of an initial developmental defect, progressing in severity over time. The Hairpatches mouse model allows an investigation into the effects of Tal1, a transcription factor characterized by complex regulation patterns, and its effects on renal disease.
Inbred strain variants of the Cdh23 gene have been shown to influence the onset and progression of age-related hearing loss (AHL) in mice. In linkage backcrosses, the recessive Cdh23 allele (ahl) of the C57BL/6J strain, when homozygous, confers increased susceptibility to AHL, while the dominant allele (Ahl+) of the CBA/CaJ strain confers resistance. To determine the isolated effects of these alleles on different strain backgrounds, we produced the reciprocal congenic strains B6.CBACa-Cdh23Ahl+and CBACa.B6-Cdh23ahl and tested 15-30 mice from each for hearing loss progression. ABR thresholds for 8 kHz, 16 kHz, and 32 kHz pure-tone stimuli were measured at 3, 6, 9, 12, 15 and 18 months of age and compared with age-matched mice of the C57BL/6J and CBA/CaJ parental strains. Mice of the C57BL/6N strain, which is the source of embryonic stem cells for the large International Knockout Mouse Consortium, were also tested for comparisons with C57BL/6J mice. Mice of the C57BL/6J and C57BL/6N strains exhibited identical hearing loss profiles: their 32 kHz ABR thresholds were significantly higher than those of CBA/CaJ and congenic strain mice by 6 months of age, and their 16 kHz thresholds were significantly higher by 12 months. Thresholds of the CBA/CaJ, the B6.CBACa-Cdh23Ahl+, and the CBACa.B6-Cdh23ahl strain mice differed little from one another and only slightly increased throughout the 18-month test period. Hearing loss, which corresponded well with cochlear hair cell loss, was most profound in the C57BL/6J and C57BL/6NJ strains. These results indicate that the CBA/CaJ-derived Cdh23Ahl+ allele dramatically lessens hearing loss and hair cell death in an otherwise C57BL/6J genetic background, but that the C57BL/6J-derived Cdh23ahl allele has little effect on hearing loss in an otherwise CBA/CaJ background. We conclude that although Cdh23ahl homozygosity is necessary, it is not by itself sufficient to account for the accelerated hearing loss of C57BL/6J mice.
age-related hearing loss; Cdh23; ahl; C57BL/6J; C57BL/6N; CBA/CaJ; inbred mouse strains; congenic mouse strains; cochleograms; ABR thresholds; hair cells
Asbestos is classified as a human carcinogen, and studies have consistently demonstrated that workplace exposure to it increases the risk of developing lung cancer. Few studies have evaluated risks in population-based settings where there is a greater variety in the types of occupations, and exposures.
This was a population based case–control study with 1,681 incident cases of lung cancer, and 2,053 controls recruited from 8 Canadian provinces between 1994 and 1997. Self-reported questionnaires were used to elicit a lifetime occupational history, including general tasks, and information for other risk factors. Occupational hygienists, who were blinded to case–control status, assigned asbestos exposures to each job on the basis of (i) concentration (low, medium, high), (ii) frequency (<5%, 5-30%, and >30% of the time in a normal work week), and (iii) reliability (possible, probable, definite). Logistic regression was used to estimate odds ratios (ORs) and their corresponding 95% confidence intervals (CI).
Those occupationally exposed to (i) low, and (ii) medium or high concentrations of asbestos had ORs for lung cancer of 1.17 (95% CI=0.92 – 1.50) and 2.16 (95% CI=1.21-3.88), respectively, relative to those who were unexposed. Medium or high exposure to asbestos roughly doubled the risk for lung cancer across all three smoking pack-year categories. The joint relationship between smoking and asbestos was consistent with a multiplicative risk model.
Our findings provide further evidence that exposure to asbestos has contributed to an increased risk of lung cancer in Canadian workplaces, and suggests that nearly 3% of lung cancers among Canadian men are caused by occupational exposure to asbestos.
Lung cancer; Asbestos; Cigarette smoking; Case–control; Occupational epidemiology
Nearly 100 years ago Michaelis and Menten published their now classic paper (Michaelis, L., and Menten, M. L. (1913) Die Kinetik der Invertinwirkung, Biochemische Zeitschrift 49, 333–369), in which they show that the rate of an enzyme-catalyzed reaction is proportional to the concentration of enzyme-substrate complex predicted by the Michaelis-Menten equation. Because the original text was written in German, yet is often quoted by English speaking authors, we undertook a complete translation of the 1913 publication, which we provide as an online supplement (http://pubs.acs.org). Here we introduce the translation, describe the historical context of the work, and show a new analysis of the original data. In doing so, we uncovered several surprises that reveal an interesting glimpse into the early history of enzymology. In particular, our re-analysis of Michaelis and Menten’s data using modern computational methods revealed an unanticipated rigor and precision in the original publication and uncovered a sophisticated, comprehensive analysis that has been overlooked in the century since their work was published. Michaelis and Menten not only analyzed initial velocity measurements, but they also fit their full time course data to the integrated form of the rate equations, including product inhibition, and derived a single global constant to represent all of their data. That constant was not the Michaelis constant, but rather, Vmax/Km, the specificity constant times the enzyme concentration (kcat/Km*E0).
The human mitochondrial DNA polymerase (pol γ) is responsible for the replication of the mitochondrial genome. Mutation Y955C in the active site of pol γ results in early onset progressive external ophthalmoplegia, premature ovarian failure, and Parkinson’s disease. In single turnover kinetic studies, we show that the Y955C mutation resulted in a decrease in the maximum rate of polymerization and an increase in the Km for correct incorporation. The mutation decreased the specificity constant for correct incorporation of dGTP, TTP, and ATP to values of 1.5, 0.35, and 0.044 μM−1s−1, respectively, representing reductions of 30-, 110- and 1300-fold relative to wild-type enzyme. The fidelity of incorporation was reduced 6- to 130-fold; largely due the significant decrease in the specificity constant for correct dATP:T incorporation. For example, kcat/Km for forming a TTP:T mismatch was decreased tenfold from 0.0002 to 0.00002 μM−1s−1 by the Y955C mutant, but the 1300-fold slower incorporation of the correct dATP:T relative to wild-type led to a 130-fold lower fidelity. While correct incorporation of 8-oxo-dGTP was largely unchanged, incorporation of 8-oxo-dG with dA in the template strand was reduced 500-fold. These results support a role of Y955 in stabilizing A:T base pairs at the active site of pol γ and suggest that the severe clinical symptoms of patients with this mutation may be due, in part, to the reduced efficiency of dATP incorporation opposite T, and that the autosomal dominant phenotype may arise from the resulting higher mutation frequency.
The anomalous dispersion signal of the bromine-containing kinase inhibitor H-89 was used to characterize discrete binding modes of the compound when complexed with the catalytic subunit of protein kinase A.
With its ability to show the interactions between drug-target proteins and small-molecule ligands, X-ray crystallography is an essential tool in drug-discovery programmes. However, its usefulness can be limited by crystallization artifacts or by the data resolution, and in particular when assumptions of unimodal binding (and isotropic motion) do not apply. Discrepancies between the modelled crystal structure and the physiological range of structures generally prevent quantitative estimation of binding energies. Improved crystal structure resolution will often not aid energy estimation because the conditions which provide the highest rigidity and resolution are not likely to reflect physiological conditions. Instead, strategies must be employed to measure and model flexibility and multiple binding modes to supplement crystallographic information. One useful tool is the use of anomalous dispersion for small molecules that contain suitable atoms. Here, an analysis of the binding of the kinase inhibitor H-89 to protein kinase A (PKA) is presented. H-89 contains a bromobenzene moiety that apparently binds with multiple conformations in the kinase ATP pocket. Using anomalous dispersion methods, it was possible to resolve these conformations into two distinct binding geometries.
protein kinase A; kinases; ligands; inhibitors; flexibility; anomalous dispersion; SAD; bromine
DNA recombinases (RecA in bacteria, Rad51 in eukarya and RadA in archaea) catalyse strand-exchange between homologous DNA molecules, the central reaction of homologous recombination, and are among the most conserved DNA repair proteins known. In bacteria, RecA is the sole protein responsible for this reaction, whereas, in eukaryotes, there are several RAD51 paralogs that cooperate to catalyse strand exchange. All archaea have at least one (and as many as four) RadA paralogs, but their function remains unclear. Here we show the three RadA paralogs encoded by the Sulfolobus solfataricus genome are expressed under normal growth conditions, and are not UV-inducible. We demonstrate that one of these proteins, Sso2452, which is representative of the large aRadC sub-family of archaeal RadA paralogs, functions as an ATPase that binds tightly to ssDNA. However, Sso2452 is not an active recombinase in vitro, and inhibits D-loop formation by RadA. We present the high-resolution crystal structure of Sso2452, which reveals key structural differences from the canonical RecA family recombinases that may explain its functional properties. The possible roles of the archaeal RadA paralogs in vivo are discussed.
Archaea; Recombinase; RadA; Homologous Recombination; Strand Exchange