Cryo-EM reveals the conformation of a substrate analogue in the human 20S proteasome core

The proteasome is a highly regulated protease complex fundamental for cell homeostasis and controlled cell cycle progression. It functions by removing a wide range of specifically tagged proteins, including key cellular regulators. Here we present the structure of the human 20S proteasome core bound to a substrate analogue inhibitor molecule, determined by electron cryo-microscopy (cryo-EM) and single-particle analysis at a resolution of around 3.5 Å. Our map allows the building of protein coordinates as well as defining the location and conformation of the inhibitor at the different active sites. These results open new prospects to tackle the proteasome functional mechanisms. Moreover, they also further demonstrate that cryo-EM is emerging as a realistic approach for general structural studies of protein–ligand interactions

Characterization of fully recombinant human 20S and 20S-PA200 proteasome complexes

Proteasomes are essential in all eukaryotic cells. However, their function and regulation remain considerably elusive, particularly those of less abundant variants. We demonstrate the human 20S proteasome recombinant assembly and confirmed the recombinant complex integrity biochemically and with a 2.6 Å resolution cryo-EM map. To assess its competence to form higher-order assemblies, we prepared and analyzed recombinant human 20S-PA200, a poorly characterized nuclear complex. Its 3.0 Å resolution cryo-EM structure reveals the PA200 unique architecture; the details of its intricate interactions with the proteasome, resulting in unparalleled proteasome α ring rearrangements; and the molecular basis for PA200 allosteric modulation of the proteasome active sites. Non-protein cryo-EM densities could be assigned to PA200-bound inositol phosphates, and we speculate regarding their functional role. Here we open extensive opportunities to study the fundamental properties of the diverse and distinct eukaryotic proteasome variants and to improve proteasome targeting under different therapeutic conditions

Investigation of the Integrity of aC:H Coatings on Stainless Steel Micro-Moulds during Thermal Cycling

Micro-injection moulding (µIM) is a key technology for scaling down larger geometry components and can include functional features at the micrometre scale and as far as the sub-micrometre length scale. Thermal cycling of amorphous hydrogenated carbon (aC:H) coated Stainless Steel (SS) has been investigated to simulate long-term micro-injection moulding (µIM) wearing and damage. Micro indentations and cracks were made into the mould and predictions of the crack behaviour were made using thermal expansion models. Validation of the results was performed with multiple heating and cooling cycles along with hardness measurements of the damage to the coating. The undamaged surfaces showed no major deformation but the cracks were shown to propagate and change in behaviour. The first two heat cycles of the testing had the most significant effect on the substrate with varying thermal expansions of materials being the main cause. The aC:H is shown to have excellent properties for mould tool applications but delamination could occur in areas susceptible to damaged and periodic surface inspection will be required preserve tool life

Characterization of the virulence, growth temperature and antibiotic resistance of the Campylobacter jejuni IAL 2383 strain isolated from humans

The objective of this study was to characterize the C. jejuni IAL2383 strain isolated from humans in Brazil. Transcripts for the racR, dnaJ and ciaB genes were found and flaA, plda and cadF genes were present in the genome and bacteria was sensitive to most of the important antimicrobials used to treat humans. C. jejuni IAL2383 is a good experimental model to analyze the interactions with cells

Investigation of the Integrity of aC:H Coatings on Stainless Steel Micro-Moulds during Thermal Cycling

Micro-injection moulding (µIM) is a key technology for scaling down larger geometry components and can include functional features at the micrometre scale and as far as the sub-micrometre length scale. Thermal cycling of amorphous hydrogenated carbon (aC:H) coated Stainless Steel (SS) has been investigated to simulate long-term micro-injection moulding (µIM) wearing and damage. Micro indentations and cracks were made into the mould and predictions of the crack behaviour were made using thermal expansion models. Validation of the results was performed with multiple heating and cooling cycles along with hardness measurements of the damage to the coating. The undamaged surfaces showed no major deformation but the cracks were shown to propagate and change in behaviour. The first two heat cycles of the testing had the most significant effect on the substrate with varying thermal expansions of materials being the main cause. The aC:H is shown to have excellent properties for mould tool applications but delamination could occur in areas susceptible to damaged and periodic surface inspection will be required preserve tool life

CONCENTRAÇÕES DE LACTATO SANGUÍNEO E DETERMINAÇÃO DO V4 DE CAVALOS DA RAÇA ÁRABE DURANTE TESTE DE EXERCÍCIO PROGRESSIVO EM ESTEIRA DE ALTA VELOCIDADE

Com este estudo objetivou-se avaliar as alterações da concentração de lactato sanguíneo e determinar o V4 de cavalos da raça Árabe submetidos ao teste de exercício progressivo em esteira de alta velocidade. Onze eqüinos adultos foram submetidos a um período de condicionamento e ao teste de exercício progressivo em esteira de alta velocidade. Nas condições em que foi realizado o experimento foi possível concluir que o protocolo de exercício proposto para aplicação do teste padrão de exercício progressivo, mostrou-se eficaz na indução de respostas metabólicas e fisiológicas para várias intensidades de exercício de cavalos da raça Árabe durante o trabalho físico em esteira de alta velocidade. As concentrações de lactato sanguíneo elevam-se exponencialmente a partir da velocidade de exercício de 8,0m/s, determinada como o V4 para o presente estudo. Blood concentration of lactate and determination of V4 in Arabian horses during a incremental exercise test performed at a high-speed treadmill Abstract The purpose of this study was to evaluate the changes in the blood lactate concentrations and to determine the V4 in Arabian horses, submitted to incremental exercise test performed on a high-speed treadmill. Eleven adult horses underwent a conditioning period as well as incremental exercise test performed on a high-speed treadmill. Under the circumstances that the experiment was developed, it was possible to conclude that the results obtained during the incremental exercise test were useful to assess the horses metabolic capability. The blood lactate levels exponentially increase beyond the speed of 8.0 m/s, determined as the V4 for this study

Covalent Plasmodium falciparum-selective proteasome inhibitors exhibit a low propensity for generating resistance in vitro and synergize with multiple antimalarial agents

Therapeutics with novel modes of action and a low risk of generating resistance are urgently needed to combat drug-resistant Plasmodium falciparum malaria. Here, we report that the peptide vinyl sulfones WLL-vs (WLL) and WLW-vs (WLW), highly selective covalent inhibitors of the P. falciparum proteasome, potently eliminate genetically diverse parasites, including K13-mutant, artemisinin-resistant lines, and are particularly active against ring-stage parasites. Selection studies reveal that parasites do not readily acquire resistance to WLL or WLW and that mutations in the β2, β5 or β6 subunits of the 20S proteasome core particle or in components of the 19S proteasome regulatory particle yield only <five-fold decreases in parasite susceptibility. This result compares favorably against previously published non-covalent inhibitors of the Plasmodium proteasome that can select for resistant parasites with >hundred-fold decreases in susceptibility. We observed no cross-resistance between WLL and WLW. Moreover, most mutations that conferred a modest loss of parasite susceptibility to one inhibitor significantly increased sensitivity to the other. These inhibitors potently synergized multiple chemically diverse classes of antimalarial agents, implicating a shared disruption of proteostasis in their modes of action. These results underscore the potential of targeting the Plasmodium proteasome with covalent small molecule inhibitors as a means of combating multidrug-resistant malaria