The four-helical immunity protein Im7 folds through an on-pathway intermediate that has a specific, but partially misfolded, hydrophobic core. In order to gain further insight into the structure of ...this species, we have identified the backbone hydrogen bonds formed in the ensemble by measuring the amide exchange rates (under EX2 conditions) of the wild-type protein and a variant, I72V. In this mutant the intermediate is significantly destabilised relative to the unfolded state (ΔΔGui=4.4kJ/mol), but the native state is only slightly destabilised (ΔΔGnu=1.8kJ/mol) at 10 °C in 2H2O, pH∗ 7.0 containing 0.4 M Na2SO4, consistent with the view that this residue forms significant non-native stabilising interactions in the intermediate state. Comparison of the hydrogen exchange rates of the two proteins, therefore, enables the state from which hydrogen exchange occurs to be identified. The data show that amides in helices I, II and IV in both proteins exchange slowly with a free energy similar to that associated with global unfolding, suggesting that these helices form highly protected hydrogen-bonded helical structure in the intermediate. By contrast, amides in helix III exchange rapidly in both proteins. Importantly, the rate of exchange of amides in helix III are slowed substantially in the Im7∗ variant, I72V, compared with the wild-type protein, whilst other amides exchange more rapidly in the mutant protein, in accord with the kinetics of folding/unfolding measured using chevron analysis. These data demonstrate, therefore, that local fluctuations do not dominate the exchange mechanism and confirm that helix III does not form stable secondary structure in the intermediate. By combining these results with previously obtained Φ-values, we show that the on-pathway folding intermediate of Im7 contains extensive, stable hydrogen-bonded structure in helices I, II and IV, and that this structure is stabilised by both native and non-native interactions involving amino acid side-chains in these helices.
Here we describe the LifeTime Initiative, which aims to track, understand and target human cells during the onset and progression of complex diseases, and to analyse their response to therapy at ...single-cell resolution. This mission will be implemented through the development, integration and application of single-cell multi-omics and imaging, artificial intelligence and patient-derived experimental disease models during the progression from health to disease. The analysis of large molecular and clinical datasets will identify molecular mechanisms, create predictive computational models of disease progression, and reveal new drug targets and therapies. The timely detection and interception of disease embedded in an ethical and patient-centred vision will be achieved through interactions across academia, hospitals, patient associations, health data management systems and industry. The application of this strategy to key medical challenges in cancer, neurological and neuropsychiatric disorders, and infectious, chronic inflammatory and cardiovascular diseases at the single-cell level will usher in cell-based interceptive medicine in Europe over the next decade.
The helical bacterial immunity proteins Im7 and Im9 have been shown to fold via kinetic mechanisms of differing complexity, despite having 60 % sequence identity. At pH 7.0 and 10 degrees C, Im7 ...folds in a three-state mechanism involving an on-pathway intermediate, while Im9 folds in an apparent two-state transition. In order to examine the folding mechanisms of these proteins in more detail, the folding kinetics of both Im7 and Im9 (at 10 degrees C in 0.4 M sodium sulphate) have been examined as a function of pH. Kinetic modelling of the folding and unfolding data for Im7 between pH 5.0 and 8.0 shows that the on-pathway intermediate is stabilised by more acidic conditions, whilst the native state is destabilised. The opposing effect of pH on the stability of these states results in a significant population of the intermediate at equilibrium at pH 6.0 and below. At pH 7.0, the folding and unfolding kinetics for Im9 can be fitted adequately by a two-state model, in accord with previous results. However, under acidic conditions there is a clear change of slope in the plot of the logarithm of the folding rate constant versus denaturant concentration, consistent with the population of one or more intermediate(s) early during folding. The kinetic data for Im9 at these pH values can be fitted to a three-state model, where the intermediate ensemble is stabilised and the native state destabilised as the pH is reduced, rationalising previous results that showed that an intermediate is not observed experimentally at pH 7.0. The data suggest that intermediate formation is a general step in immunity protein folding and demonstrate that it is necessary to explore a wide range of refolding conditions in order to show that intermediates do not form in the folding of other small, single-domain proteins.
DNA lesions interfere with DNA and RNA polymerase activity. Cyclobutane pyrimidine dimers and photoproducts generated by ultraviolet irradiation cause stalling of RNA polymerase II, activation of ...transcription-coupled repair enzymes, and inhibition of RNA synthesis. During the Sphase of the cell cycle, collision of replication forks with damaged DNA blocks ongoing DNA replication while also triggering a biochemical signal that suppresses the firing of distant origins of replication. Whether the transcription machinery is affected by the presence of DNA double-strand breaks remains a long-standing question. Here we monitor RNA polymerase I (PolI) activity in mouse cells exposed to genotoxic stress and show that induction of DNA breaks leads to a transient repression in PolI transcription. Surprisingly, we find PolI inhibition is not itself the direct result of DNA damage but is mediated by ATM kinase activity and the repair factor proteins NBS1 (also known as NLRP2) and MDC1. Using live-cell imaging, laser micro-irradiation, and photobleaching technology we demonstrate that DNA lesions interfere with PolI initiation complex assembly and lead to a premature displacement of elongating holoenzymes from ribosomal DNA. Our data reveal a novel ATM/NBS1/MDC1-dependent pathway that shuts down ribosomal gene transcription in response to chromosome breaks.
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Introduction. The sedentary lifestyle is defined as prolonged sitting both at work and during leisure time, with energy expenditures of below 600 MET · min/week. The sedentary lifestyle is a ...well-known predictor of obesity and other components of the metabolic syndrome. The influence of the sedentary lifestyle and associated factors on nsLBP is still being discussed. Aim. The aim of this study was to assess the influence of a sedentary lifestyle and its associated metabolic predictors on the prevalence of nsLBP in nurses and paramedics. Materials and Methods. The study included 609 participants, aged 30-60 years, who were residents of north-east Poland. Data was collected using a questionnaire (based, in part, on the Nordic Musculoskeletal Questionnaire), and included details of sociodemographic profile, chronic illnesses, and a short version of the International Physical Activity Questionnaire (IPAQ). Results. Nearly half (49.59%) of the respondents reported decreased physical activity, and in the group with recurring nsLBP this figure was 67.59%. Univariate logistic regression modelling found that leading a sedentary lifestyle caused a 3.5-fold increase in the incidence of recurring nsLBP (p<0.001). Excessive coffee consumption significantly increased the likelihood of recurring LBP (OR=16.44, 95% CI: 8.55-31.61), and cigarette smoking increased the likelihood of both recurrent and chronic LBP. The likelihood of chronic low back pain was significantly increased by components of metabolic syndrome such as high blood pressure (over 9-fold), type 2 diabetes (over 3-fold), and hyperlipidemia (over 2-fold) (p<0.001, p<0.001, and p<0.01, respectively). Conclusions. A sedentary lifestyle significantly increased the incidence of recurring low back pain, while increased physical activity had a significant effect on the presence of chronic low back pain. In the sedentary lifestyle group, conditions classified within metabolic syndrome were found to significantly increase the chances of developing nonspecific low back pain.
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