Phage display and selection of lanthipeptides on the carboxy-terminus of the gene-3 minor coat protein.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are an emerging class of natural products with drug-like properties. To fully exploit the potential of RiPPs as peptide drug candidates, tools for their systematic engineering are required. Here we report the engineering of lanthipeptides, a subclass of RiPPs characterized by multiple thioether cycles that are enzymatically introduced in a regio- and stereospecific manner, by phage display.

A fully synthetic human Fab antibody library based on fixed VH/VL framework pairings with favorable biophysical properties.

This report describes the design, generation and testing of Ylanthia, a fully synthetic human Fab antibody library with 1.3E+11 clones. Ylanthia comprises 36 fixed immunoglobulin (Ig) variable heavy (VH)/variable light (VL) chain pairs, which cover a broad range of canonical complementarity-determining region (CDR) structures.

TRiC/CCT cooperates with different upstream chaperones in the folding of distinct protein classes.

(2003) 5230–5240, doi:10.1093/emboj/cdg483

Structural basis for subunit assembly in UDP-glucose pyrophosphorylase from Saccharomyces cerevisiae.

UDP-glucose is the universal activated form of glucose, employed in all organisms for glucosyl transfer reactions and as precursor for various activated carbohydrates. In animal and fungal metabolism, UDP-glucose is required for utilization of galactose and for the synthesis of glycogen, the major carbohydrate storage polymer. The formation of UDP-glucose is catalyzed by UDP-glucose pyrophosphorylase (UGPase), which is highly conserved among eukaryotes.

Chaperonin TRiC promotes the assembly of polyQ expansion proteins into nontoxic oligomers.

Aberrant folding and fibrillar aggregation by polyglutamine (polyQ) expansion proteins are associated with cytotoxicity in Huntington's disease and other neurodegenerative disorders. Hsp70 chaperones have an inhibitory effect on fibril formation and can alleviate polyQ cytotoxicity. Here we show that the cytosolic chaperonin, TRiC, functions synergistically with Hsp70 in this process and is limiting in suppressing polyQ toxicity in a yeast model.

L25 functions as a conserved ribosomal docking site shared by nascent chain-associated complex and signal-recognition particle.

The nascent chain-associated complex (NAC) is a dimeric protein complex of archaea and eukarya that interacts with ribosomes and translating polypeptide chains. We show that, in yeast, NAC and the signal-recognition particle (SRP) share the universally conserved ribosomal protein L25 as a docking site, which is in close proximity to the ribosomal exit tunnel. The amino-terminal segment of beta-NAC was found to be required for L25 binding.

Pathways of chaperone-mediated protein folding in the cytosol.

Cells are faced with the task of folding thousands of different polypeptides into a wide range of conformations. For many proteins, the folding process requires the action of molecular chaperones. In the cytosol of prokaryotic and eukaryotic cells, molecular chaperones of different structural classes form a network of pathways that can handle substrate polypeptides from the point of initial synthesis on ribosomes to the final stages of folding.

Cellular toxicity of polyglutamine expansion proteins: mechanism of transcription factor deactivation.

The expression of polyglutamine-expanded mutant proteins in Huntington's disease and other neurodegenerative disorders is associated with the formation of intraneural inclusions. These aggregates could potentially cause cellular toxicity by sequestering essential proteins possessing normal polyQ repeats, including the transcription factors TBP and CBP. We show, in vitro and in cells, that monomers or small soluble oligomers of huntingtin exon1 accumulate in the nucleus and inhibit the function of TBP in a polyQ-dependent manner.

TRiC/CCT cooperates with different upstream chaperones in the folding of distinct protein classes.

The role in protein folding of the eukaryotic chaperonin TRiC/CCT is only partially understood. Here, we show that a group of WD40 beta-propeller proteins in the yeast cytosol interact transiently with TRiC upon synthesis and require the chaperonin to reach their native state. TRiC cooperates in the folding of these proteins with the ribosome-associated heat shock protein (Hsp)70 chaperones Ssb1/2p.

Prediction of novel Bag-1 homologs based on structure/function analysis identifies Snl1p as an Hsp70 co-chaperone in Saccharomyces cerevisiae.

Polypeptide binding by the chaperone Hsp70 is regulated by its ATPase activity, which is itself regulated by co-chaperones including the Bag domain nucleotide exchange factors. Here, we tested the functional contribution of residues in the Bag domain of Bag-1M that contact Hsp70. Two point mutations, E212A and E219A, partially reduced co-chaperone activity, whereas the point mutation R237A completely abolished activity in vitro.