Pressure perturbation calorimetry of protein unfolding

Thermodynamic and structural characterisation of various interactions stabilising protein native folds is a key part of multidisciplinary research efforts to solve the problems of protein folding and binding. Amongst such interactions the hydrophobic effect has been considered for several years to be the major driving force behind protein folding and main non-covalent interaction in protein stability. The hydrophobic effect arises from solvation effects around non-polar residues that are buried inside protein interior upon protein folding. The change in heat capacity on protein unfolding (DeltaCp) is believed to be one of the "signatures" of the hydrophobic effect. The thermal expansion coefficient (alpha) and protein volume change upon unfolding (DeltaV) are the volumetric parameters sensitive to solvation changes and hence could be utilised in the studies of hydrophobic interactions. Both these parameters have recently become easily available with the development of the new calorimetric method called Pressure Perturbation Calorimetry (PPC). In this research, the aqueous methanol solutions have been used as a solvent system with a modified hydrogen bonded structure in relation to water. Protein thermal unfolding has been studied in these solutions, where the solvation effects associated with exposing protein buried residues upon unfolding are diminished (the hydrophobic effect is weakened). Three small, one domain model proteins used in this study were: ubiquitin, lysozyme and ribonuclease A. DeltaCp of unfolding of those proteins in aqueous methanol solutions has been obtained by Differential Scanning Calorimetry (DSC) and DeltaV of unfolding was obtained by PPC. DeltaCp is a temperature-related thermodynamic parameter, while DeltaV is a pressure-related parameter, the combined determination of both parameters allows a more comprehensive description of protein unfolding transitions to be built than merely the more routinely studied temperature-related characteristics. DeltaCp and DeltaV, taken together, provide complementary data on the solvation changes during the thermal unfolding process, therefore the effect of increasing methanol concentration on the changes in both DeltaCp and DeltaV of unfolding has been investigated here. Additionally, two other applications of PPC were investigated in this thesis. Firstly, PPC was shown to allow straightforward collection of data used to predict the structure making and structure breaking of solutes in water. Amino acids as small molecular model compound have been investigated here, and the determination of structure making and structure breaking of side-chains in amino acid model has been critically reviewed. Importantly, a serious apparent discrepancy in published data was been identified here. Secondly, the study of another small model protein, cytochrome c conformational transitions (native to unfolded, molten globule to unfolded) has been tested here by the PPC method and the results have been shown to be in a good agreement with ones obtained from high-pressure spectroscopic studies. In summary , PPC has been shown to be a useful technique for studying structure breaking and structure making properties of solutes, both small and macromolecular, and for obtaining volumetric properties of protein transitions that complete the picture of already abundantly available temperature-related calorimetric data such as enthalpy, entropy and heat capacity

Seladelpar efficacy and safety at 3 months in patients with primary biliary cholangitis: ENHANCE, a phase 3, randomized, placebo-controlled study

Background and Aims: ENHANCE was a phase 3 study that evaluated efficacy and safety of seladelpar, a selective peroxisome proliferator-activated receptor-δ (PPAR) agonist, versus placebo in patients with primary biliary cholangitis with inadequate response or intolerance to ursodeoxycholic acid (UDCA). Approach and Results: Patients were randomized 1:1:1 to oral seladelpar 5 mg (n=89), 10 mg (n=89), placebo (n=87) daily (with UDCA, as appropriate). Primary end point was a composite biochemical response [alkaline phosphatase (ALP) < 1.67×upper limit of normal (ULN), ≥15% ALP decrease from baseline, and total bilirubin ≤ ULN] at month 12. Key secondary end points were ALP normalization at month 12 and change in pruritus numerical rating scale (NRS) at month 6 in patients with baseline score ≥4. Aminotransferases were assessed. ENHANCE was terminated early following an erroneous safety signal in a concurrent, NASH trial. While blinded, primary and secondary efficacy end points were amended to month 3. Significantly more patients receiving seladelpar met the primary end point (seladelpar 5 mg: 57.1%, 10 mg: 78.2%) versus placebo (12.5%) (p < 0.0001). ALP normalization occurred in 5.4% (p=0.08) and 27.3% (p < 0.0001) of patients receiving 5 and 10 mg seladelpar, respectively, versus 0% receiving placebo. Seladelpar 10 mg significantly reduced mean pruritus NRS versus placebo [10 mg: −3.14 (p=0.02); placebo: −1.55]. Alanine aminotransferase decreased significantly with seladelpar versus placebo [5 mg: 23.4% (p=0.0008); 10 mg: 16.7% (p=0.03); placebo: 4%]. There were no serious treatment-related adverse events. Conclusions: Patients with primary biliary cholangitis (PBC) with inadequate response or intolerance to UDCA who were treated with seladelpar 10 mg had significant improvements in liver biochemistry and pruritus. Seladelpar appeared safe and well tolerated

Seladelpar efficacy and safety at 3 months in patients with primary biliary cholangitis: ENHANCE, a phase 3, randomized, placebo-controlled study

Background and Aims: ENHANCEwas a phase 3 study that evaluated efficacy and safety of seladelpar, a selective peroxisome proliferator-activated receptor-delta (PPAR) agonist, versus placebo in patients with primary biliary cholangitis with inadequate response or intolerance to ursodeoxycholic acid (UDCA).Approach and Results: Patients were randomized 1:1:1 to oral seladelpar 5 mg (n= 89), 10 mg (n= 89), placebo (n= 87) daily (with UDCA, as appropriate). Primary end point was a composite biochemical response [alkaline phosphatase (ALP) < 1.67xupper limit of normal (ULN), >= 15% ALP decrease from baseline, and total bilirubin <= ULN] at month 12. Key secondary end points were ALP normalization at month 12 and change in pruritus numerical rating scale (NRS) at month 6 in patients with baseline score >= 4. Aminotransferases were assessed. ENHANCE was terminated early following an erroneous safety signal in a concurrent, NASH trial. While blinded, primary and secondary efficacy end points were amended to month 3. Significantly more patients receiving seladelpar met the primary end point (seladelpar 5 mg: 57.1%, 10mg: 78.2%) versus placebo (12.5%) (p < 0.0001). ALP normalization occurred in 5.4% (p= 0.08) and 27.3% (p < 0.0001) of patients receiving 5 and 10 mg seladelpar, respectively, versus 0% receiving placebo. Seladelpar 10 mg significantly reduced mean pruritus NRS versus placebo [10 mg: -3.14 (p= 0.02); placebo: -1.55]. Alanine aminotransferase decreased significantly with seladelpar versus placebo [5 mg: 23.4% (p= 0.0008); 10 mg: 16.7% (p= 0.03); placebo: 4%]. There were no serious treatment-related adverse events.Conclusions: Patients with primary biliary cholangitis (PBC) with inadequate response or intolerance to UDCA who were treated with seladelpar 10 mg had significant improvements in liver biochemistry and pruritus. Seladelpar appeared safe and well tolerated

Mechanism of interaction of the mammalian cysteine protease inhibitors, cystatin A and B, with target proteases

Human cystatin A was shown to bind rapidly and strongly to papain and cathepsin L, with Åj of 0.2-20 pM and £ass of -3-5x10^ whereas the affinities for actinidin and cathepsins B, C and H were weaker (Kj 1-40 nM). The inhibition of cathepsin B was ~100-fold slower than that of papain, i. e. fcass was ~104 M’^-s'1. The binding to papain was consistent with a one-step binding mechanism. An N-terminally truncated cystatin A variant had an appreciably reduced affinity for papain, indicating the importance of this region for interaction with cysteine proteases.Mutations in the second hairpin loop of cystatin B, Leu-73—>Gly and His-75—>Gly, decreased the affinity for papain and cathepsins L, H and B to an extent suggesting that this region contributes 20-30 % of the binding energy of cystatin B to target enzymes. A mutation in the C-terminal end of cystatin B, Tyr-97-»Ala, similarly indicated that this end contributes 6-12 % of the binding energy to papain and cathepsins L and H but is of limited importance for cathepsin B binding. The increased fcjiss for the binding of the mutants to proteases suggests that the two regions are important for complex stability.Human and bovine wild-type cystatin B were shown to have indistinguishable inhibitory properties towards cysteine proteases, binding tightly to the endopeptidases, papain and cathepsin L, and more weakly to the exopeptidases, cathepsins H and B. Mutation of the single Cys residue, Cys-3, in cystatin B showed that this residue is involved in the binding of the inhibitor to target enzymes. Cys-3 is most important for cathepsin B binding and for the bovine inhibitor.Sequential truncation of four residues from the N-terminal end of cystatin B resulted in progressively impaired affinities for papain and cathepsins L, H and B. The highest affinity loss was caused by removal of Cys-3, showing that this residue is most important for the binding. The decreased affinities ofthe truncated cystatin B mutants for papain and cathepsin H were due mainly to an increased Åtfiss» indicating that the N-terminal region keeps the inhibitor anchored to these enzymes in the complexes

Stan zachowania drewnianej architektury mieszkaniowej okresu dwudziestolecia międzywojennego w miastach na Lubelszczyźnie. Możliwości ochrony

The article describes the condition of the wooden buildings created in the interwar period in the Lublin region and the method of its conservation, taking into account legal forms in force in Poland and in the world. This is a very substantial topic, because buildings of this type are increasingly disappearing from the landscape of a small city, in favor of catalog construction, thereby destroying the unique small-town landscapeArtykuł opisuje stan zachowania drewnianej zabudowy powstałej w dwudziestoleciu międzywojennym na terenie Lubelszczyzny oraz sposobu jej konserwacji z uwzględnieniem form prawnych obowiązujących w Polsce i na świecie. Jest to bardzo aktualny temat ponieważ zabudowa tego typu coraz częściej zanika z krajobrazu małego miasta na rzecz katalogowe budownictwa, tym samym niszcząc unikalny krajobraz małomiasteczkowy

Wpływ zaborcy na kształt architektury drewnianej końca XIX i początku XX w. na terenie Lubelszczyzny

The aim of the article was to present the influence of the Russian national architecture on the wooden architecture of the Lublin region, created at the turn of the 19th and 20th centuries, which was characterized by great distinctiveness. The subject of the work was to show the importance of increased Russification, which touched not only the spheres of social and cultural life, but also architecture.The article analyzes the preserved wooden architecture from the turn of the century in the present-day Lubelskie Region. The starting material used was the available archival materials and documentation prepared for the needs of the provincial conservator of monuments (the card for the records of architectural and construction monuments) and a local inspection was made to prepare photographic documentation of the current state. Characteristic wooden buildings were used as comparative material, built at the end of the 19th and the beginning of the 20th century in Samara, Russia. The buildings have a well-documented history and are well researched by local scientists. Additionally, they are kept almost intact. During the research, a comparative method was used for the existing wooden buildings in the Lublin region from the turn of the century with the Russian wooden buildings, mainly in terms of detail and façade decoration. It allowed us to notice the similarities between the details used in traditional wooden construction in the Lublin Province. It is especially visible in the details of the window decorations – richly decorated drip and window sills. It is an element characteristic only for the analyzed period, which confirms the thesis about the influence of the foreign invaders on the shape of architecture.Celem artykułu było przedstawienie wpływu architektury narodowej rosyjskiej na drewnianą architekturę Lubelszczyzny powstałą na przełomie XIX i XX w., która charakteryzowała się dużą odrębnością. Przedmiotem pracy było ukazanie znaczenia wzmożonej rusyfikacji, która dotykała nie tylko sfery życia społecznego, kulturalnego, ale także architektury.W artykule dokonano analizy zachowanej architektury drewnianej powstałej na przełomie wieków na terenie obecnego województwa lubelskiego. Jako materiał wyjściowy wykorzystano dostępne materiały archiwalne oraz dokumentację stworzoną na potrzeby wojewódzkiego konserwatora zabytków (karta ewidencji zabytków architektury i budownictwa) oraz dokonano wizji lokalnej w celu przygotowania dokumentacji fotograficznej stanu aktualnego. Jako materiał porównawczy posłużyła charakterystyczna zabudowa drewniana powstała pod koniec XIX i na początku XX w. w Samarze w Rosji. Zabudowa ta ma dobrze udokumentowaną historię oraz jest dobrze zbadana przez lokalnych naukowców. Dodatkowo przetrwała do dziś w stanie prawie nienaruszonym.Podczas badań porównano istniejącą zabudowę drewnianej Lubelszczyzny z przełomu wieków z zabudową drewnianą rosyjską przede wszystkim pod względem detalu i dekoracji fasady. Pozwoliło to na zauważenie podobieństw między detalami stosowanymi w tradycyjnym budownictwie drewnianym na terenie województwa lubelskiego. Szczególnie jest to widoczne w detalu stolarki okiennej – bogato dekorowane nad- i podokienniki. Jest to element charakterystyczny tylko dla badanego okresu, co potwierdza tezę o wpływie zaborcy na kształt architektury