BioChem, Volume 4, Issue 2 (June 2024) – 3 articles

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a human pathogenic virus that encodes for a helicase (SC2Hel) that is essential for viral replication. SC2Hel has the ability to unravel dsRNA or dsDNA in an NTP-dependent manner from the 5′ to 3′ directionality. The standard helicase assay from studies involving SARS-CoV and SARS-CoV-2 have relied on the concept of fluorescence resonance energy transfer. Adding to the collection of helicase assays, herein, we have developed a novel tetrazolium-based colorimetric assay system for the detection of ADP that is produced via SC2Hel activity. This SC2Hel assay combines three enzyme-coupled steps involving the ADP-dependent Thermococcus litoralis glucokinase (TlGlcK), Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (LmG6PDH), and Clostridium kluyveri diaphorase (CkDIA). Iodonitrotetrazolium chloride (INT), a colorimetric tetrazolium reagent, was used in the final step of the assay that converted into INT-formazan during reduction. INT-formazan in the assay’s buffered solution at pH 7.6 exhibited an intense colorimetric response at a wavelength maximum of 505 nm. The assay exhibited excellent performance characteristics as it revealed a Z’ factor of 0.87 and it has the potential to be further adopted into high-throughput screening studies for therapeutic drug discovery research. Full article

Metabolites are at the end of the gene–transcript–protein–metabolism cascade. As such, metabolomics is the omics approach that offers the most direct correlation with phenotype. This allows, where genomics, transcriptomics and proteomics fail to explain a trait, metabolomics to possibly provide an answer. Complex phenotypes, which are determined by the influence of multiple small-effect alleles, are an example of these situations. Consequently, the interest in metabolomics has increased exponentially in recent years. As a newer discipline, metabolomic bioinformatic analysis pipelines are not as standardized as in the other omics approaches. In this review, we synthesized the different steps that need to be carried out to obtain biological insight from annotated metabolite abundance raw data. These steps were grouped into three different modules: preprocessing, statistical analysis, and metabolic pathway enrichment. We included within each one of them the different state-of-the-art procedures and tools that can be used depending on the characteristics of the study, providing details about each method’s characteristics and the issues the reader might encounter. Finally, we introduce genome-scale metabolic modeling as a tool for obtaining pseudo-metabolomic data in situations where their acquisition is difficult, enabling the analysis of the resulting data with the modules of the described workflow. Full article

Heat Shock Protein 90 (Hsp90) acts as a crucial molecular chaperone, playing an essential role in activating numerous signaling proteins. The intricate mechanism of Hsp90 involving ATPase-coupled conformational changes and interactions with cochaperone proteins has been elucidated through biochemical and structural analyses, revealing its activation mechanism and its diverse set of “client” proteins. Despite recent advancements, certain aspects of Hsp90’s ATPase-coupled mechanism remain contentious, and the specific nature of the alterations induced by Hsp90 in client proteins remains largely undiscovered. In this review, we explore the current understanding of Hsp90’s structure and function, drawing insights from single-particle cryoEM studies. Structural studies on Hsp90 using cryoEM have provided valuable insights into the structural dynamics and interactions of this molecular chaperone. CryoEM structures have been instrumental in understanding the ATPase-coupled conformational changes that Hsp90 undergoes during its chaperone cycle. We also highlight recent progress in elucidating the structure of the ATP-bound state of the complete dimeric chaperone. Furthermore, we delve into the roles played by the multitude of cochaperones that collaborate with Hsp90, providing a glimpse into their biochemical mechanisms through the newly obtained cryoEM structures of Hsp90 cochaperone complexes. Full article