The formation of the CBM (CARD11-BCL10-MALT1) complex is pivotal for antigen-receptor-mediated activation of the transcription factor NF-κB. Signaling is dependent on MALT1 (mucosa-associated lymphoid tissue lymphoma translocation protein 1), which not only acts as a scaffolding protein but also possesses proteolytic activity mediated by its caspase-like domain. It remained unclear how the CBM activates MALT1. Here, we provide biochemical and structural evidence that MALT1 activation is dependent on its dimerization and show that mutations at the dimer interface abrogate activity in cells. The unliganded protease presents itself in a dimeric yet inactive state and undergoes substantial conformational changes upon substrate binding. These structural changes also affect the conformation of the C-terminal Ig-like domain, a domain that is required for MALT1 activity. Binding to the active site is coupled to a relative movement of caspase and Ig-like domains. MALT1 binding partners thus may have the potential of tuning MALT1 protease activity without binding directly to the caspase domain.

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  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7904367: CHIP (uniprotkb:http://www.uniprot.org/uniprot/Q9UNE7) and HSP 90‐beta (uniprotkb:http://www.uniprot.org/uniprot/P08238) physically interact (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) by molecular sieving (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7904785: HSP 90‐beta (uniprotkb:http://www.uniprot.org/uniprot/P08238) and p23 (uniprotkb:http://www.uniprot.org/uniprot/Q15185) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by molecular sieving (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7904047: CHIP (uniprotkb:http://www.uniprot.org/uniprot/Q9UNE7), HSP 90‐beta (uniprotkb:http://www.uniprot.org/uniprot/P08238) and p23 (uniprotkb:http://www.uniprot.org/uniprot/Q15185) physically interact (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) by molecular sieving (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7903424: Alpha‐lactalbumin (uniprotkb:http://www.uniprot.org/uniprot/P00711), HSP70 (uniprotkb:http://www.uniprot.org/uniprot/P08107) and CHIP (uniprotkb:http://www.uniprot.org/uniprot/Q9UNE7) physically interact (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) by molecular sieving (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0071)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7903354: CHIP (uniprotkb:http://www.uniprot.org/uniprot/Q9UNE7) and HSC70 (uniprotkb:http://www.uniprot.org/uniprot/P11142) bind (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407) by isothermal titration calorimetry (h...

The assembly of ribosomal subunits is an essential prerequisite for protein biosynthesis in all domains of life. Although biochemical and biophysical approaches have advanced our understanding of ribosome assembly, our mechanistic comprehension of this process is still limited. Here, we perform an in vitro reconstitution of the Escherichia coli 50S ribosomal subunit. Late reconstitution products were subjected to high-resolution cryo-electron microscopy and multiparticle refinement analysis to reconstruct five distinct precursors of the 50S subunit with 4.3-3.8 Å resolution. These assembly intermediates define a progressive maturation pathway culminating in a late assembly particle, whose structure is more than 96% identical to a mature 50S subunit. Our structures monitor the formation and stabilization of structural elements in a nascent particle in unprecedented detail and identify the maturation of the rRNA-based peptidyl transferase center as the final critical step along the 50S assembly pathway.

The Hsp70-interacting E3-ubiquitin ligase CHIP has been implicated in the decision as to whether a target protein enters the refolding or the degradation pathway. To further characterize the activity of CHIP we purified untagged Homo sapiens and Drosophila melanogaster CHIP (hCHIP, dCHIP). In contrast to other E3-ubiquitin ligases, both hCHIP and dCHIP proteins formed homodimers at physiological concentrations. We identified a predicted coiled-coil region in a mixed charge segment of the hCHIP and dCHIP sequence and found it to be necessary and sufficient for dimer formation. A mutant of hCHIP lacking this segment (hCHIP⌬-(128 -229)) was incapable of dimer formation, but the segment by itself (hCHIP-(128 -229)) readily dimerized. Furthermore, we demonstrated that dimerization is a prerequisite for activity of hCHIP in the reconstituted ubiquitination assay. Control of dimerization may thus provide a mechanism for regulation of CHIP activity.

In eukaryotes, newly synthesized proteins interact co-translationally with a multitude of different ribosome-bound factors and chaperones including the conserved heterodimeric nascent polypeptide-associated complex (NAC) and a Hsp40/70-based chaperone system. These factors are thought to play an important role in protein folding and targeting, yet their specific ribosomal localizations, which are prerequisite for their functions, remain elusive. This study describes the ribosomal localization of NAC and the molecular details by which NAC is able to contact the ribosome and gain access to nascent polypeptides. We identified a conserved RRK(X) n KK ribosome binding motif within the ␤-subunit of NAC that is essential for the entire NAC complex to attach to ribosomes and allow for its interaction with nascent polypeptide chains. The motif localizes within a potential loop region between two predicted ␣-helices in the N terminus of ␤NAC. This N-terminal ␤NAC ribosome-binding domain was completely portable and sufficient to target an otherwise cytosolic protein to the ribosome. NAC modified with a UV-activatable cross-linker within its ribosome binding motif specifically cross-linked to L23 ribosomal protein family members at the exit site of the ribosome, providing the first evidence of NAC-L23 interaction in the context of the ribosome. Mutations of L23 reduced NAC ribosome binding in vivo and in vitro, whereas other eukaryotic ribosome-associated factors such as the Hsp70/40 chaperones Ssb or Zuotin were unaffected. We conclude that NAC employs a conserved ribosome binding domain to position itself on the L23 ribosomal protein adjacent to the nascent polypeptide exit site.

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.

The dimeric E3 ubiquitin ligase CHIP binds with its tetratricopeptide repeat (TPR) domain the C-terminus of molecular chaperones Hsp70 and Hsp90 and with its U-box region E2 ubiquitin-conjugating enzymes. By ubiquitinating chaperone-bound polypeptides, CHIP thus links the chaperone machinery to the proteasomal degradation pathway. The molecular mechanism of how CHIP discriminates between folding and destruction of chaperone substrates is not yet understood. Two recently published crystal structures of mouse and zebrafish CHIP truncation constructs differ substantially, showing either an asymmetric assembly or a symmetric assembly with a highly ordered middle domain. To characterize the conformational properties of the intact full-length protein in solution, we performed amide hydrogen exchange mass spectrometry (HX-MS) with human CHIP. In addition, we monitored conformational changes in CHIP upon binding of Hsp70, Hsp90, and their respective C-terminal EEVD peptides, and in complex with the different E2 ubiquitin-conjugating enzymes UbcH5a and Ubc13. Solution HX-MS data suggest a symmetric dimer assembly with highly flexible parts in the middle domain contrasting both the asymmetric and the symmetric crystal structure. CHIP exhibited an extraordinary flexibility with a largely unprotected N-terminal TPR domain. Formation of a complex with intact Hsp70 and Hsp90 or their respective C-terminal octapeptides induced folding of the TPR domain to a defined, highly stabilized structure with protected amide hydrogens. Interaction of CHIP with two different E2 ubiquitin-conjugating enzymes, UbcH5a and Ubc13, had distinct effects on the conformational dynamics of CHIP, suggesting different roles of the CHIP-E2 interaction in the ubiquitination of substrates and interaction with chaperones.

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