The biological functions of proteins are governed by their three-dimensional fold. Protein folding, maintenance of proteome integrity, and protein homeostasis (proteostasis) critically depend on a complex network of molecular chaperones. Disruption of proteostasis is implicated in aging and the pathogenesis of numerous degenerative diseases. In the cytosol, different classes of molecular chaperones cooperate in evolutionarily conserved folding pathways. Nascent polypeptides interact cotranslationally with a first set of chaperones, including trigger factor and the Hsp70 system, which prevent premature (mis)folding. Folding occurs upon controlled release of newly synthesized proteins from these factors or after transfer to downstream chaperones such as the chaperonins. Chaperonins are large, cylindrical complexes that provide a central compartment for a single protein chain to fold unimpaired by aggregation. This review focuses on recent advances in understanding the mechanisms of chaperone action in promoting and regulating protein folding and on the pathological consequences of protein misfolding and aggregation.

Molecular Chaperone Functions in Protein Folding and Proteostasis

The N-end rule relates the regulation of the in vivo half-life of a protein to the identity of its N-terminal residue. Degradation signals (degrons) that are targeted by the N-end rule pathway include a set called N-degrons. The main determinant of an N-degron is a destabilizing N-terminal residue of a protein. In eukaryotes, the N-end rule pathway is a part of the ubiquitin system and consists of two branches, the Ac/N-end rule and the Arg/N-end rule pathways. The Ac/N-end rule pathway targets proteins containing Nα-terminally acetylated (Nt-acetylated) residues. The Arg/N-end rule pathway recognizes unacetylated N-terminal residues and involves N-terminal arginylation. Together, these branches target for degradation a majority of cellular proteins. For example, more than 80% of human proteins are cotranslationally Nt-acetylated. Thus, most proteins harbor a specific degradation signal, termed AcN-degron, from the moment of their birth. Specific N-end rule pathways are also present in prokaryotes and in mitochondria. Enzymes that produce N-degrons include methionine-aminopeptidases, caspases, calpains, Nt-acetylases, Nt-amidases, arginyl-transferases, and leucyl-transferases. Regulated degradation of specific proteins by the N-end rule pathway mediates a legion of physiological functions, including the sensing of heme, oxygen, and nitric oxide; selective elimination of misfolded proteins; the regulation of DNA repair, segregation, and condensation; the signaling by G proteins; the regulation of peptide import, fat metabolism, viral and bacterial infections, apoptosis, meiosis, spermatogenesis, neurogenesis, and cardiovascular development; and the functioning of adult organs, including the pancreas and the brain. Discovered 25 years ago, this pathway continues to be a fount of biological insights.

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The N-end rule pathway and regulation by proteolysis

The early events in the life of newly synthesized proteins in the cellular environment are remarkably complex. Concurrently with their synthesis by the ribosome, nascent polypeptides are subjected to enzymatic processing, chaperone-assisted folding or targeting to translocation pores at membranes. The ribosome itself has a key role in these different tasks and governs the interplay between the various factors involved. Indeed, the ribosome serves as a platform for the spatially and temporally regulated association of enzymes, targeting factors and chaperones that act upon the nascent polypeptides emerging from the exit tunnel. Furthermore, the ribosome provides opportunities to coordinate the protein-synthesis activity of its peptidyl transferase center with the protein targeting and folding processes. Here we review the early co-translational events involving the ribosome that guide cytosolic proteins to their native state.

The ribosome as a platform for co-translational processing, folding and targeting of newly synthesized proteins

http://www.biochem.uni-frankfurt.de/fileadmin/user_upload/courses_seminars/BA_Methodenseminar/WiSe2014/Oh.2011.Cell.pdf

Selective Ribosome Profiling Reveals the Cotranslational Chaperone Action of Trigger Factor In Vivo

Protein synthesis in all organisms is catalyzed by ribosomes. In comparison to their prokaryotic counterparts, eukaryotic ribosomes are considerably larger and are subject to more complex regulation. The large ribosomal subunit (60S) catalyzes peptide bond formation and contains the nascent polypeptide exit tunnel. We present the structure of the 60S ribosomal subunit from Tetrahymena thermophila in complex with eukaryotic initiation factor 6 (eIF6), cocrystallized with the antibiotic cycloheximide (a eukaryotic-specific inhibitor of protein synthesis), at a resolution of 3.5 angstroms. The structure illustrates the complex functional architecture of the eukaryotic 60S subunit, which comprises an intricate network of interactions between eukaryotic-specific ribosomal protein features and RNA expansion segments. It reveals the roles of eukaryotic ribosomal protein elements in the stabilization of the active site and the extent of eukaryotic-specific differences in other functional regions of the subunit. Furthermore, it elucidates the molecular basis of the interaction with eIF6 and provides a structural framework for further studies of ribosome-associated diseases and the role of the 60S subunit in the initiation of protein synthesis.

https://science.sciencemag.org/content/sci/334/6058/941.full.pdf

Crystal Structure of the Eukaryotic 60S Ribosomal Subunit in Complex with Initiation Factor 6

In bacteria, the transcription of DNA into mRNA by RNA polymerase is coupled to the translation of that mRNA into protein by the ribosome. How this coupling is achieved has been unclear. Kohler et al. show that RNA polymerase and the ribosome from Escherichia coli can form a so-called expressome complex. Electron microscopy structural analysis, together with functional experiments, revealed details of the coupled complex. The coupling could allow translation to prevent transcriptional pausing, backtracking, and termination.

Science , this issue p. [194][1]

 [1]: /lookup/doi/10.1126/science.aal3059

/pdf/architecture-of-a-transcribing-translating-expressome-503xhxqyp9.pdf

Architecture of a transcribing-translating expressome.

YidC and Oxa1 form dimeric insertion pores on the translating ribosome.

Messenger-RNA-directed protein synthesis is accomplished by the ribosome. In eubacteria, this complex process is initiated by a specialized transfer RNA charged with formylmethionine (tRNA(fMet)). The amino-terminal formylated methionine of all bacterial nascent polypeptides blocks the reactive amino group to prevent unfavourable side-reactions and to enhance the efficiency of translation initiation. The first enzymatic factor that processes nascent chains is peptide deformylase (PDF); it removes this formyl group as polypeptides emerge from the ribosomal tunnel and before the newly synthesized proteins can adopt their native fold, which may bury the N terminus. Next, the N-terminal methionine is excised by methionine aminopeptidase. Bacterial PDFs are metalloproteases sharing a conserved N-terminal catalytic domain. All Gram-negative bacteria, including Escherichia coli, possess class-1 PDFs characterized by a carboxy-terminal alpha-helical extension. Studies focusing on PDF as a target for antibacterial drugs have not revealed the mechanism of its co-translational mode of action despite indications in early work that it co-purifies with ribosomes. Here we provide biochemical evidence that E. coli PDF interacts directly with the ribosome via its C-terminal extension. Crystallographic analysis of the complex between the ribosome-interacting helix of PDF and the ribosome at 3.7 A resolution reveals that the enzyme orients its active site towards the ribosomal tunnel exit for efficient co-translational processing of emerging nascent chains. Furthermore, we have found that the interaction of PDF with the ribosome enhances cell viability. These results provide the structural basis for understanding the coupling between protein synthesis and enzymatic processing of nascent chains, and offer insights into the interplay of PDF with the ribosome-associated chaperone trigger factor.

Rebecca Kohler

Papers

Architecture of a transcribing-translating expressome.

YidC and Oxa1 form dimeric insertion pores on the translating ribosome.

A peptide deformylase-ribosome complex reveals mechanism of nascent chain processing.