Biology subjects

Bukau, B. G.

Publications and source records attributed to Bukau, B. G..

3 recordsLinked to original sources

bioRxiv2026

Small-molecule activators of the Staphylococcus aureus ClpC/ClpP AAA+ protease

Summary/AbstractThe central AAA+ ClpC/ClpP protease in Gram-positive bacteria is crucial for virulence and stress resistance and has been recognized as drug target. Natural cyclic peptides deregulate the essential Mycobacterium tuberculosis ClpC1 and cause cell death. Similarly, overactivated mutants of the non-essential Staphylococcus aureus ClpC homologue cause uncontrolled proteolysis and severe toxicity in vivo. However, no chemical modulators of S. aureus ClpC have been described. Here, using a biochemical high-throughput screen we identify eight chemically distinct bona fide small molecules that robustly stimulate ClpC ATPase and proteolytic activity in vitro. Structural, computational, and mutational analyses define two ligandable regulatory sites within the ClpC N-terminal domain (NTD) as compound targets: a conserved hydrophobic groove and an allosteric pArg1 pocket, both engaged in substrate recognition. These findings establish S. aureus ClpC as chemically targetable and provide mechanistic insight into its regulatory architecture, enabling future development and optimization of chemical probes to deregulate AAA+ protease control.

biochemistry↗

bioRxiv2025

Distinct cis-acting elements mediate co-localization of mRNAs encoding for co-translational interactors in cytoplasmic clusters

Many newly synthesized proteins assemble co-translationally, providing a vital mechanism to prevent subunit misfolding in the crowded cytoplasm. Initial evidence indicates that the spatial organization of mRNAs aids this assembly, but it is unclear how these mRNAs are organized and how common this mechanism is. We used single-molecule Fluorescence in situ Hybridization in Saccharomyces cerevisiae to examine the spatial organization of mRNAs encoding subunits of various cytosolic complexes involved in critical cellular functions, such as fatty acid synthesis, glycolysis, translation and various amino acid biosynthesis. We found that mRNAs of the same protein complex often co-localize in specific cytoplasmic clusters. Additionally, we observed that the mRNAs encoding enzymes of biosynthetic pathways are organized in cytosolic clusters. Focusing on mRNAs encoding fatty acid synthase complex subunits, we discovered that non-coding cis elements significantly influence mRNA localization in an additive manner. Specifically, 5 and 3 UTRs, together with further upstream and downstream regions, facilitate co-localization. Inhibiting mRNA co-localization impaired growth when complex activity was essential, highlighting the importance of mRNA spatial organization for cellular survival. Transiently disrupting mRNA translation also affected clustering, indicating that both the nascent chains and mRNA sequence targeting cues are combinatorically contributing to spatial organization. Proteomics analysis demonstrates the impact of cis-elements on the abundance of the encoded subunits, as well as the entire pathway. In summary, we provide evidence that mRNA co-localization in cytoplasmic foci is coordinated by complementary mechanisms crucial for co-translational assembly, allowing efficient regulation of protein complex formation and entire pathways.

cell biology↗

bioRxiv2024

Structural polymorphism of alpha-synuclein fibrils alters pathway of Hsc70 mediated disaggregation

The pathological aggregation of -synuclein into amyloid fibrils is a hallmark of synucleinopathies including Parkinsons disease. Despite this commonality, synucleinopathies display divergent disease phenotypes that have been attributed to disease specific three-dimensional structures of -synuclein fibrils, each with a unique toxic gain-of-function profile. The Hsc70 chaperone is remarkable in its ability to disassemble pre-existing amyloid fibrils of different proteins in an ATP and co-chaperone dependent manner. We find however, using six well-defined conformational polymorphs of -synuclein fibrils, that the activity of the Hsc70 disaggregase machinery is sensitive to differences in the amyloid conformation, confirming that fibril polymorphism directly affects interactions with the proteostasis network. Amyloid conformation influences not only how efficiently fibrils are cleared by the Hsc70 machinery but also the preferred pathway of disaggregation. We further show that, in vitro, the active processing of fibrils by the Hsc70 machinery inadvertently produces seeding competent species that further promote protein aggregation. Amyloid conformation thus is an important feature that can tilt the balance between beneficial or detrimental protein quality control activities in the context of disease.

biochemistry↗