The E3 ubiquitin ligase CHIP (C‐terminus of Hsc70‐interacting protein) is believed to be a central player in the cellular triage decision, as it links the molecular chaperones Hsp70/Hsc70 and Hsp90 to the ubiquitin proteasomal degradation pathway. To better understand the decision process, we determined the affinity of CHIP for Hsp70 and Hsp90 using isothermal titration calorimetry. We analyzed the influence of CHIP on the ATPase cycles of both chaperones in the presence of co‐chaperones and a substrate, and determined the ubiquitination efficacy of CHIP in the presence of the chaperones. We found that CHIP has a sixfold higher affinity for Hsp90 compared with Hsc70. CHIP had no influence on ADP dissociation or ATP association, but reduced the Hsp70 cochaperone Hdj1‐stimulated single‐turnover ATPase rates of Hsc70 and Hsp70. CHIP did not influence the ATPase cycle of Hsp90 in the absence of co‐chaperones or in the presence of the Hsp90 cochaperones Aha1 or p23. Polyubiquitination of heat‐denatured luciferase and the native substrate p53 was much more efficient in the presence of Hsc70 and Hdj1 than in the presence of Hsp90, indicating that CHIP preferentially ubiquitinates Hsp70‐bound substrates.Structured digital abstract MINT‐7904367: CHIP (uniprotkb:Q9UNE7) and HSP 90‐beta (uniprotkb:P08238) physically interact (MI:0915) by molecular sieving (MI:0071) MINT‐7904785: HSP 90‐beta (uniprotkb:P08238) and p23 (uniprotkb:Q15185) bind (MI:0407) by molecular sieving (MI:0071) MINT‐7904047: CHIP (uniprotkb:Q9UNE7), HSP 90‐beta (uniprotkb:P08238) and p23 (uniprotkb:Q15185) physically interact (MI:0915) by molecular sieving (MI:0071) MINT‐7903424: Alpha‐lactalbumin (uniprotkb:P00711), HSP70 (uniprotkb:P08107) and CHIP (uniprotkb:Q9UNE7) physically interact (MI:0915) by molecular sieving (MI:0071) MINT‐7903354: CHIP (uniprotkb:Q9UNE7) and HSC70 (uniprotkb:P11142) bind (MI:0407) by isothermal titration calorimetry (MI:0065) MINT‐7903373: CHIP (uniprotkb:Q9UNE7) and HSP90‐beta (uniprotkb:P08238) bind (MI:0407) by isothermal titration calorimetry (MI:0065)

In all kingdoms of life, proteins are synthesized by ribosomes in a process referred to as translation. The amplitude of translational regulation exceeds the sum of transcription, mRNA degradation and protein degradation. Therefore, it is essential to investigate translation in a global scale. Like the other “omics”-methods, translatomics investigates the totality of the components in the translation process, including but not limited to translating mRNAs, ribosomes, tRNAs, regulatory RNAs and nascent polypeptide chains. Technical advances in recent years have brought breakthroughs in the investigation of these components at global scale, both for their composition and dynamics. These methods have been applied in a rapidly increasing number of studies to reveal multifaceted aspects of translation control. The process of translation is not restricted to the conversion of mRNA coding sequences into polypeptide chains, it also controls the composition of the proteome in a delicate and responsive way. Therefore, translatomics has extended its unique and innovative power to many fields including proteomics, cancer research, bacterial stress response, biological rhythmicity and plant biology. Rational design in translation can enhance recombinant protein production for thousands of times. This brief review summarizes the main state-of-the-art methods of translatomics, highlights recent discoveries made in this field and introduces applications of translatomics on basic biological and biomedical research.

Ribosome biogenesis is a fundamental multi-step cellular process in all domains of life that involves the production, processing, folding, and modification of ribosomal RNAs (rRNAs) and ribosomal proteins. To obtain insights into the still unexplored early assembly phase of the bacterial 50S subunit, we exploited a minimal in vitro reconstitution system using purified ribosomal components and scalable reaction conditions. Time-limited assembly assays combined with cryo-EM analysis visualizes the structurally complex assembly pathway starting with a particle consisting of ordered density for only ~500 nucleotides of 23S rRNA domain I and three ribosomal proteins. In addition, our structural analysis reveals that early 50S assembly occurs in a domain-wise fashion, while late 50S assembly proceeds incrementally. Furthermore, we find that both ribosomal proteins and folded rRNA helices, occupying surface exposed regions on pre-50S particles, induce, or stabilize rRNA folds within adjacent regions, thereby creating cooperativity.

Proline-rich antimicrobial peptides (PrAMPs) inhibit bacterial protein biosynthesis by binding to the polypeptide exit tunnel (PET) near the peptidyl transferase center. Api137, an optimized derivative of honeybee PrAMP apidaecin, inhibits protein expression by trapping release factors (RFs), which interact with stop codons on ribosomes to terminate translation. This study uses cryo-EM, functional assays and molecular dynamic (MD) simulations to show that Api137 additionally occupies a second binding site near the exit of the PET and can repress translation independently of RF-trapping. Api88, a C-terminally amidated (-CONH₂) analog of Api137 (-COOH), binds to the same sites, occupies a third binding pocket and interferes with the translation process presumably without RF-trapping. In conclusion, apidaecin-derived PrAMPs inhibit bacterial ribosomes by multimodal mechanisms caused by minor structural changes and thus represent a promising pool for drug development efforts.

The confinement of macromolecules has profound implications for cellular biochemistry. It generates environments with specific physical properties affecting diffusion, macromolecular crowding, and reaction rates. Yet, it remains unknown how intracellular density distributions emerge and affect cellular physiology. Here, we show that the nucleus is less dense than the cytoplasm and that living systems establish a conserved density ratio between these compartments due to a pressure balance across the nuclear envelope. Nuclear transport establishes a specific nuclear proteome that exerts a colloid osmotic pressure, which, assisted by chromatin pressure, increases nuclear volume. During C. elegans development, the nuclear-to-cytoplasmic density ratio is robustly maintained even when nuclear-to-cytoplasmic volume ratios change. We show that loss of density homeostasis correlates with altered cell functions like senescence and propose density distributions as key markers in pathophysiology. In summary, this study reveals a homeostatic coupling of macromolecular densities that drives cellular organization and function.