Prevalence of childhood lead poisoning and respiratory disease associated with lead smelter emissions.

BACKGROUND: The city of Port Pirie in South Australia has been a world leading centre for lead and zinc smelting and processing since 1889 that continues to cause contamination of its environment and resident population. This study quantifies the effect of lead and SO2 emissions from Nyrstar Port Pirie Pty Ltd's smelter on blood lead and respiratory health outcomes, respectively, and establishes what air quality values are required to better protect human health. METHOD: Blood lead and emergency department presentation data collected by South Australia Health (SA Health) and lead in air and SO2 data collected by the South Australian Environment Protection Authority (SAEPA) were obtained and analysed to quantify health outcomes due to smelter emissions in Port Pirie. Regression analysis was used to assess the relationship between the concentration of lead in air and children's blood lead levels between the years of available data: 2003 to 2017. Ambient SO2 concentrations (SAEPA) measured continuously between 2008 and 2018 were 24-hour averaged and compared to daily local emergency department respiratory presentation rates (available from July 2012 to October 2018). Rates of emergency department respiratory presentations at Port Pirie and regional comparators were calculated as age-standardised rates. RESULTS: The data show that increases in ambient SO2 concentrations are associated with increased rates of emergency department respiratory presentations of Port Pirie residents, in which children are over-represented. The 30-day rolling average of respiratory presentations was significantly associated (p < 0.05) with incremental increases in SO2. Analysis of the relationship between lead in air and blood lead shows that annual geometric mean air lead concentrations need to be <0.11 µg/m3 to ensure the geometric mean blood lead of Port Pirie children under 5 years is ≤5 µg/dL. For children aged 24 months, lead in air needs to be no greater than 0.082 µg/m3 (annual geometric mean) to ensure geometric mean blood lead does not exceed 5 µg/dL. CONCLUSION: Current smelting emissions continue to pose a clear risk of harm to Port Pirie children. Allowable emissions must be lowered significantly to limit adverse childhood health outcomes including respiratory illness and IQ, academic achievement and socio-behavioural problems that are associated with lead exposure at levels experienced by Port Pirie children. Current SO2 levels are likely to be responsible for increased rates of emergency department respiratory presentations in Port Pirie compared with other South Australian locations. As a minimum, Australian SO2 air quality standards need to be enforced in Port Pirie to better protect human health. Lead in air needs to be approximately 80% lower than the current national standard (0.5 µg/m3) to ensure that the geometric blood lead of children under 5 years is less than or equal to 5 µg/dL.

Ambient soil cation exchange capacity inversely associates with infectious and parasitic disease risk in regional Australia.

Human contact with soil may be important for building and maintaining normal healthy immune defence mechanisms, however this idea remains untested at the population-level. In this continent-wide, cross-sectional study we examine the possible public health benefit of ambient exposures to soil of high cation exchange capacity (CEC), a surrogate for potential immunomodulatory soil microbial diversity. We compare distributions of normalized mean 2011/12-2012/13 age-standardized public hospital admission rates (cumulative incidence) for infectious and parasitic diseases across regional Australia (representing an average of 29,516 patients/year in 228 local government areas), within tertiles of socioeconomic status and soil exposure. To test the significance of soil CEC, we use probabilistic individual-level environmental exposure data (with or without soil), and group-level variables, in robust non-parametric multilevel modelling to predict disease rates in unseen groups. Our results show that in socioeconomically-deprived areas with high CEC soils, rates of infectious and parasitic disease are significantly lower than areas with low CEC soils. Also, health inequality (relative risk) due to socioeconomic status is significantly lower in areas with high CEC soils compared to low CEC soils (Δ relative risk = 0.47; 95% CI: 0.13, 0.82). Including soil exposure when modelling rates of infectious and parasitic disease significantly improves prediction performance, explaining an additional 7.5% (Δ r2 = 0.075; 95% CI: 0.05, 0.10) of variation in disease risk, in local government areas that were not used for model building. Our findings suggest that exposure to high CEC soils (typically high soil biodiversity) associates with reduced risk of infectious and parasitic diseases, particularly in lower socioeconomic areas.

Landscape biodiversity correlates with respiratory health in Australia.

Megatrends of urbanisation and reducing contact with natural environments may pose a largely unappreciated risk to human health, particularly in children, through declining normal (healthy) immunomodulatory environmental exposures. On the other hand, building knowledge of connections between environments, biodiversity and human health may offer new integrated ways of addressing global challenges of rising population health costs and declining biodiversity. In this study we are motivated to build insight and provide context and priority for emerging research into potential protective (e.g. immunomodulatory) environmental exposures. We use respiratory health as a test case to explore whether some types and qualities of environment may be more beneficial than others, and how such exposures may compare to known respiratory health influences, via a cross-sectional ecological epidemiology study for the continent of Australia. Using Lasso penalized regression (to interpret key predictors from many candidate variables) and 10-fold cross-validation modelling (to indicate reproducibility and uncertainty), within different socio-geographic settings, our results show surrogate measures of landscape biodiversity correlate with respiratory health, and rank amongst known predictors. A range of possible drivers for this relationship are discussed. Perhaps most novel and interesting of these is the possibility of protective immunomodulatory influence from microbial diversity (suggested by the understudied 'biodiversity hypothesis') and other bioactive agents associated with biodiverse environments. If beneficial influences can be demonstrated from biodiverse environments on immunomodulation and human health, there may be potential to design new cost-effective nature-based health intervention programs to reduce the risk of immune-related disease at a population level. Our approach and findings are also likely to have use in the evaluation of environment and health associations elsewhere.

Frequency of counterstrain tender points in osteopathic medical students.

CONTEXT: Counterstrain is 1 osteopathic manipulative treatment technique taught to osteopathic medical students, but teaching all 300 counterstrain tender points is not feasible at most colleges of osteopathic medicine (COMs) because of time limitations. OBJECTIVE: To identify high-yield tender points in osteopathic medical students for teaching and to assess for correlations between tender points and demographic information, weight, and history of pain or trauma. METHODS: First- and second-year osteopathic medical students at 5 COMs were surveyed regarding the presence and absence of tender points found on themselves by fellow students. Demographic information, weight, and history of pain and trauma data were collected. The McNemar test was used to compare the frequency of positive tender points between the right and left sides. Multiple logistic regression models were fit to the data to determine if participant characteristics were related to having 1 or more positive tender points in a tender point group. Wilcoxon signed rank tests were used to compare the percentage of positive anterior vs posterior tender points. Multiple logistic regression models were used to test for differences between COMs after accounting for differences in participant characteristics. RESULTS: Frequency of 78 tender point groups was obtained. Forty tender point groups (51%) were positive for the presence of 1 or more tender points by 50% or more of the participants. Positive tender points were more common on the right side for 23 groups (all P<.001). Female participants were more likely to have tender points for 22 groups (all P<.001). The 20- to 25-year-olds had more tender points for 6 groups (all P≤.03). Tender points were more common in participants with a history of pain for 29 groups (all P<.001) and with a history of trauma for 4 groups (all P≤.05). Anterior tender points were more common for cervical, thoracic, rib, and lumbar body regions (P<.001). Differences were found between COMs for all tender point groups (P≤.02). CONCLUSION: Nearly half of the tender point groups surveyed were reported positive by 50% or more of participants, and high-yield tender points were found in each body region. Ultimately, these results may guide counterstrain curricula for COMs.

Impact of cost of medicines for chronic conditions on low income households in Australia.

OBJECTIVES: To determine the cost of medicines for selected chronic illnesses and the proportion of discretionary income this would potentially displace for households with different pharmaceutical subsidy entitlements and incomes. METHODS: We analysed household income and expenditure data for 9,774 households participating in two Australian surveys in 2009-10. The amount of 'discretionary' income available to households after basic living and health care expenditure was modelled for households with high pharmaceutical subsidies: pensioner and non-pensioner concessional (social security entitlements); and households with general pharmaceutical subsidies and low, middle or high incomes. We calculated the proportion of discretionary income that would be needed for medicines if one household member had diabetes or acute coronary syndrome, or if one member also had two co-existing illnesses (gastro-oesophageal reflux disease and depression, or asthma and osteoarthritis). RESULTS: Pensioner and low income households had little discretionary income after basic living and health care expenditure (AUD$92 and $164/week, respectively). Medicines for the specified illnesses ranged from $11-$42/month for high subsidy households and $34-$186/month for low subsidy households. Costs reduced substantially once patients reached the annual pharmaceutical cap (safety net), prior to which medicine costs would displace the equivalent of 1%-10% of discretionary income for most household types. However, low income households would have to forego the equivalent of between 5%-26% of their discretionary income for between 7 and 9 months of the year before receiving additional subsidies. CONCLUSIONS: Prescription medicines for chronic conditions pose a substantial financial burden to many households, particularly those with low incomes and general pharmaceutical subsidies. Policies are needed to minimize the cost burden of prescription medicines, particularly for low-income working households.

Unraveling the mechanism of protein disaggregation through a ClpB-DnaK interaction.

HSP-100 protein machines, such as ClpB, play an essential role in reactivating protein aggregates that can otherwise be lethal to cells. Although the players involved are known, including the DnaK/DnaJ/GrpE chaperone system in bacteria, details of the molecular interactions are not well understood. Using methyl-transverse relaxation-optimized nuclear magnetic resonance spectroscopy, we present an atomic-resolution model for the ClpB-DnaK complex, which we verified by mutagenesis and functional assays. ClpB and GrpE compete for binding to the DnaK nucleotide binding domain, with GrpE binding inhibiting disaggregation. DnaK, in turn, plays a dual role in both disaggregation and subsequent refolding of polypeptide chains as they emerge from the aggregate. On the basis of a combined structural-biochemical analysis, we propose a model for the mechanism of protein aggregate reactivation by ClpB.

From the city to the bush: increases in patient co-payments for medicines have impacted on medicine use across Australia.

AIM: To determine whether the national declines in prescription medicine use occurring after the 2005 21% increase in co-payments affected all areas of Australia or were specific to remote and disadvantaged areas. METHODS: Observed dispensing of proton pump inhibitors (PPIs) and statins were obtained for 1392 statistical local areas (SLA) of Australia in 2004 and 2006. Expected dispensing was based on national dispensing rates and was age standardised to each SLA. Expected dispensing for 2006 was based on pre-2005 prescription trends. Ratios of observed to expected dispensing (dispensing ratios) for each SLA were calculated. Mean dispensing ratios for each medicine and year were calculated for all remoteness and disadvantage groups. Generalised regression models compared the percentage change in dispensing ratios from 2004 to 2006. RESULTS: Between 2004 and 2006 PPI dispensing fell significantly in major cities (-13.7%, 95% CI=-17.3--9.8), inner regional (-14.0, 95%CI=-19.5--8.2), outer regional (-14.6%, 95%CI=-19.9--9.0) and remote areas (-9.4%, 95%CI=-16.4--1.8). Statin dispensing fell in all groups but the most remote (range 6-7%). When focussing on disadvantage, PPI dispensing fell significantly in all groups (range 12-15%). Statins dispensing did not fall significantly in the most disadvantaged areas (-2.9%, 95%CI=-8.6-3.2) but did in the least (-6.5%, -11.3--1.5) and second-least (-5.8, -10.5--0.9) disadvantaged areas. Dispensing of PPIs and statins in the most remote and disadvantaged areas remained substantially below levels expected for Australia after the 21% co-payments increase. CONCLUSIONS: The findings suggest that the 2005 21% in patient co-payments adversely affected prescription medicine use in all areas of Australia and was not specific to remote or disadvantaged areas. Indeed, dispensing of statins fell significantly in all but the most remote and disadvantaged areas, and the existing gap in dispensing of PPIs and statins was not widened by the co-payments increase. PPIs, which are used at above-prevalence rates in Australia and have cheaper over-the-counter substitutes available, were more sensitive to co-payment increases than were statins.

A new perspective on Hsp104-mediated propagation and curing of the yeast prion [PSI (+) ].

Most prions in yeast form amyloid fibrils that must be severed by the protein disaggregase Hsp104 to be propagated and transmitted efficiently to newly formed buds. Only one yeast prion, [PSI (+) ], is cured by Hsp104 overexpression. We investigated the interaction between Hsp104 and Sup35, the priongenic protein in yeast that forms the [PSI (+) ] prion.1 We found that a 20-amino acid segment within the highly-charged, unstructured middle domain of Sup35 contributes to the physical interaction between the middle domain and Hsp104. When this segment was deleted from Sup35, the efficiency of [PSI (+) ] severing was substantially reduced, resulting in larger Sup35 particles and weakening of the [PSI (+) ] phenotype. Furthermore, [PSI (+) ] in these cells was completely resistant to Hsp104 curing. The affinity of Hsp104 was considerably weaker than that of model Hsp104-binding proteins and peptides, implying that Sup35 prions are not ideal substrates for Hsp104-mediated remodeling. In light of this finding, we present a modified model of Hsp104-mediated [PSI (+) ] propagation and curing that requires only partial remodeling of Sup35 assembled into amyloid fibrils.

Insight into molecular basis of curing of [PSI+] prion by overexpression of 104-kDa heat shock protein (Hsp104).

Yeast prions are a powerful model for understanding the dynamics of protein aggregation associated with a number of human neurodegenerative disorders. The AAA+ protein disaggregase Hsp104 can sever the amyloid fibrils produced by yeast prions. This action results in the propagation of "seeds" that are transmitted to daughter cells during budding. Overexpression of Hsp104 eliminates the [PSI+] prion but not other prions. Using biochemical methods we identified Hsp104 binding sites in the highly charged middle domain of Sup35, the protein determinant of [PSI+]. Deletion of a short segment of the middle domain (amino acids 129-148) diminishes Hsp104 binding and strongly affects the ability of the middle domain to stimulate the ATPase activity of Hsp104. In yeast, [PSI+] maintained by Sup35 lacking this segment, like other prions, is propagated by Hsp104 but cannot be cured by Hsp104 overexpression. These results provide new insight into the enigmatic specificity of Hsp104-mediated curing of yeast prions and sheds light on the limitations of the ability of Hsp104 to eliminate aggregates produced by other aggregation-prone proteins.

How much do we spend on prescription medicines? Out-of-pocket costs for patients in Australia and other OECD countries.

OBJECTIVES: To determine changes in out-of-pocket expenditure on prescription medicines for Australian patients, and how patient expenditure compares with other Organisation for Economic Co-operation and Development (OECD) countries. METHODS: We examined out-of-pocket expenditure on prescription medicines by patients in Australia between 1970 and 2007, and between Australia and 15 other OECD countries (Canada, Czech Republic, Denmark, Finland, France, Germany, Japan, Republic of Korea (South Korea), Luxembourg, Poland, Slovak Republic, Spain, Sweden, Switzerland and the United States) in 2005. FINDINGS: Spending on publicly subsidised medicines by Australian patients increased from $16 per person in 1971 to $62 in 2007. Patient expenditure on all prescription medicines had risen to $134 per person in 2007. Out-of-pocket expenditure for Australian patients ranked 4th of 14 OCED countries with universal pharmaceutical subsidies. Australian patients pay 28% of national pharmaceutical expenditure; more than patients in South Korea (27%), Slovak Republic (26%), Sweden (22%), France, Luxembourg, Japan and Switzerland (17%), Germany (15%), Czech Republic (11%) and Spain (6%), but less than patients in Finland (36%), Denmark (33%) and Poland (34%). CONCLUSIONS: Compared to other OECD countries, Australian out-of-pocket costs are now in the mid to upper range. Further increases have the potential to significantly affect access to care.

At last, a national health measurement survey program for Australia!

Objective assessment of the health of the population is the key to policy and planning.

Determinants of self-reported medicine underuse due to cost: a comparison of seven countries.

OBJECTIVES: To compare the predictors of self-reported medicine underuse due to cost across countries with different pharmaceutical subsidy systems and co-payments. METHODS: We analysed data from a 2007 survey of adults in Australia, Canada, Germany, the Netherlands, New Zealand (NZ), the United Kingdom (UK) and the United States (US). The predictors of underuse were calculated separately for each country using multivariate poisson regression. RESULTS: Reports of underuse due to cost varied from 3% in the Netherlands to 20% in the US. In Australia, Canada, NZ, the UK and the US, cost-related underuse was predicted by high out-of-pocket costs (RR range 2.0-4.6), below average income (RR range 1.9-3.1), and younger age (RR range 3.9-16.4). In all countries except Australia and the UK, history of depression was associated with cost-related underuse (RR range 1.2-4.1). In Australia, Canada, Germany, the UK and the US lack of patient involvement in treatment decisions was associated with cost-related underuse (RR range 1.2-1.4). In Australia, Canada and NZ, indigenous persons more commonly reported underuse due to cost (RR range 2.1-2.9). CONCLUSIONS: Cost-related underuse of medicines was least commonly reported in countries with the lowest out-of-pocket costs, the Netherlands and the UK. Countries with reduced co-payments or cost ceilings for low income patients showed the least disparity in rates of underuse between income groups. Despite differences in health insurance systems in these countries, age, ethnicity, depression, and involvement with treatment decisions were consistently predictive of underuse. There are opportunities for policy makers and clinicians to support medicine use in vulnerable groups.

The geographic distribution of private health insurance in Australia in 2001.

BACKGROUND: Private health insurance has been a major focus of Commonwealth Government health policy for the last decade. Over this period, the Howard government introduced a number of policy changes which impacted on the take up of private health insurance. The most expensive of these was the introduction of the private health insurance rebate in 1997, which had an estimated cost of $3 billion per annum. METHODS: This article uses information on the geographic distribution of the population with private health insurance cover to identify associations between rates of private health insurance cover and socioeconomic status. The geographic analysis is repeated with survey data on expenditure on private health insurance, to provide an estimate of the rebate flowing to different socioeconomic groups. RESULTS: The analysis highlights the strong association between high rates of private health insurance cover and high socioeconomic status and shows the substantial transfer of funds, under the private health insurance rebate, to those living in areas of highest socioeconomic status, compared with those in areas of lower socioeconomic status, and in particular those in the most disadvantaged areas. The article also provides estimates of private health insurance cover by federal electorate, emphasising the substantial gaps in cover between Liberal Party and Australian Labor Party seats. CONCLUSION: The article concludes by discussing implications of the uneven distribution of private health insurance cover across Australia for policy formation. In particular, the study shows that the prevalence of private health insurance is unevenly distributed across Australia, with marked differences in prevalence in rural and urban areas, and substantial differences by socioeconomic status. Policy formation needs to take this into account. Evaluating the potential impact of changes in private health insurance requires more nuanced consideration than has been implied in the rhetoric about private health insurance over the last decade.

A multistage system for the automated detection of epileptic seizures in neonatal electroencephalography.

This paper describes the design and test results of a three-stage automated system for neonatal EEG seizure detection. Stage I of the system is the initial detection stage and identifies overlapping 5-second segments of suspected seizure activity in each EEG channel. In stage II, the detected segments from stage I are spatiotemporally clustered to produce multichannel candidate seizures. In stage III, the candidate seizures are processed further using measures of quality and context-based rules to eliminate false candidates. False candidates because of artifacts and commonly occurring EEG background patterns such as bifrontal delta activity are also rejected. Seizures at least 10 seconds in duration are considered for reporting results. The testing data consisted of recordings of 28 seizure subjects (34 hours of data) and 48 nonseizure subjects (87 hours of data) obtained in the neonatal intensive care unit. The data were not edited to remove artifacts and were identical in every way to data normally processed visually. The system was able to detect seizures of widely varying morphology with an average detection sensitivity of almost 80% and a subject sensitivity of 96%, in comparison with a team of clinical neurophysiologists who had scored the same recordings. The average false detection rate obtained in nonseizure subjects was 0.74 per hour.

Increased patient co-payments and changes in PBS-subsidised prescription medicines dispensed in Western Australia.

OBJECTIVE: To determine whether a 24% increase in patient co-payments in January 2005 and two related co-payment changes for medicines subsidised under the Australian Pharmaceutical Benefits Scheme (PBS) were associated with changes in dispensings in Western Australia (WA). METHOD: We analysed aggregate monthly prescription counts and defined daily dose per 1,000 population per day (DDD/1,000/day) for atypical antipsychotics, combination asthma medicines, HmgCoA reductase inhibitors (statins) and proton-pump inhibitors (PPIs). Trends pre and post the co-payment increase in January 2005 were compared. RESULTS: In three of the four categories examined, prescription counts were significantly lower following the increase in co-payment thresholds. Compared with dispensings prior to the co-payment increase, prescriptions fell by 8% for combination asthma medicines (p<0.001), 9% for PPIs (p<0.001) and 5% for statins (p<0.001). Following the rise in co-payments, DDD/1,000/day decreased for all four categories. Decreases in dispensings to concessional beneficiaries were between 4% and 5% larger than for general beneficiary patients. CONCLUSIONS AND IMPLICATIONS: The reduction in the both prescription counts and DDD/1,000/day observed for combination asthma medicines, PPIs and statins, which all remained above co-payment thresholds, suggests the increase in PBS co-payments has affected utilisation of these subsidised medicines. The results indicate that increases in patient contributions particularly impact on concessional patients' ability to afford prescription medicines.

Remodeling of protein aggregates by Hsp104.

Hsp104 is molecular chaperone in the AAA+ family of ATPases that specializes in the resolubilization and refolding of thermally denatured proteins in yeast. In addition to providing high levels of thermotolerance, Hsp104 plays a pivotal role in the propagation of yeast prions, self-replicating, amyloid-like aggregates that are inherited during mitosis and meiosis. In this review, the structure and function of Hsp104 is discussed, its functional interaction with other molecular chaperones, and a model for disaggregation and refolding is proposed.

The SmpB-tmRNA tagging system plays important roles in Streptomyces coelicolor growth and development.

The ssrA gene encodes tmRNA that, together with a specialized tmRNA-binding protein, SmpB, forms part of a ribonucleoprotein complex, provides a template for the resumption of translation elongation, subsequent termination and recycling of stalled ribosomes. In addition, the mRNA-like domain of tmRNA encodes a peptide that tags polypeptides derived from stalled ribosomes for degradation. Streptomyces are unique bacteria that undergo a developmental cycle culminating at sporulation that is at least partly controlled at the level of translation elongation by the abundance of a rare tRNA that decodes UUA codons found in a relatively small number of open reading frames prompting us to examine the role of tmRNA in S. coelicolor. Using a temperature sensitive replicon, we found that the ssrA gene could be disrupted only in cells with an extra-copy wild type gene but not in wild type cells or cells with an extra-copy mutant tmRNA (tmRNA(DD)) encoding a degradation-resistant tag. A cosmid-based gene replacement method that does not include a high temperature step enabled us to disrupt both the ssrA and smpB genes separately and at the same time suggesting that the tmRNA tagging system may be required for cell survival under high temperature. Indeed, mutant cells show growth and sporulation defects at high temperature and under optimal culture conditions. Interestingly, even though these defects can be completely restored by wild type genes, the DeltassrA strain was only partially corrected by tmRNA(DD). In addition, wildtype tmRNA can restore the hygromycin-resistance to DeltassrA cells while tmRNA(DD) failed to do so suggesting that degradation of aberrant peptides is important for antibiotic resistance. Overall, these results suggest that the tmRNA tagging system plays important roles during Streptomyces growth and sporulation under both normal and stress conditions.

The impact of co-payment increases on dispensings of government-subsidised medicines in Australia.

PURPOSE: Patient co-payments for medicines subsidised under the Australian Pharmaceutical Benefits Scheme (PBS) increased by 24% in January 2005. We investigated whether this increase and two related co-payment changes were associated with changes in dispensings of selected subsidised medicines in Australia. METHOD: We analysed national aggregate monthly prescription dispensings for 17 medicine categories, selected to represent a range of treatments (e.g. for diabetes, cardiovascular diseases, gout). Trends in medication dispensings from January 2000 to December 2004 were compared with those from January 2005 to September 2007 using segmented regression analysis. RESULTS: Following the January 2005 increase in PBS co-payments, significant decrease in dispensing volumes were observed in 12 of the 17 medicine categories (range: 3.2-10.9%), namely anti-epileptics, anti-Parkinson's treatments, combination asthma medicines, eye-drops, glaucoma treatments, HmgCoA reductase inhibitors, insulin, muscle relaxants, non-aspirin antiplatelets, osteoporosis treatments, proton-pump inhibitors (PPIs) and thyroxine. The largest decrease was observed for medicines used in treating asymptomatic conditions or those with over-the-counter (OTC) substitutes. Decrease in dispensings to social security beneficiaries was consistently greater than for general beneficiaries following the co-payment changes (range: 1.8-9.4% greater, p = 0.028). CONCLUSIONS: The study findings suggest that recent increase in Australian PBS co-payments have had a significant effect on dispensings of prescription medicines. The results suggest large increase in co-payments impact on patients' ability to afford essential medicines. Of major concern is that, despite special subsidies for social security beneficiaries in the Australian system, the recent co-payment increase has particularly impacted on utilisation for this group.

Peptide and protein binding in the axial channel of Hsp104. Insights into the mechanism of protein unfolding.

The AAA+ molecular chaperone Hsp104 mediates the extraction of proteins from aggregates by unfolding and threading them through its axial channel in an ATP-driven process. An Hsp104-binding peptide selected from solid phase arrays enhanced the refolding of a firefly luciferase-peptide fusion protein. Analysis of peptide binding using tryptophan fluorescence revealed two distinct binding sites, one in each AAA+ module of Hsp104. As a further indication of the relevance of peptide binding to the Hsp104 mechanism, we found that it competes with the binding of a model unfolded protein, reduced carboxymethylated alpha-lactalbumin. Inactivation of the pore loops in either AAA+ module prevented stable peptide and protein binding. However, when the loop in the first AAA+ was inactivated, stimulation of ATPase turnover in the second AAA+ module of this mutant was abolished. Drawing on these data, we propose a detailed mechanistic model of protein unfolding by Hsp104 in which an initial unstable interaction involving the loop in the first AAA+ module simultaneously promotes penetration of the substrate into the second axial channel binding site and activates ATP turnover in the second AAA+ module.