Dual Binding Mode of the Nascent Polypeptide-associated Complex Reveals a Novel Universal Adapter Site on the Ribosome

Nascent polypeptide-associated complex (NAC) was identified in eukaryotes as the first cytosolic factor that contacts the nascent polypeptide chain emerging from the ribosome. NAC is present as a homodimer in archaea and as a highly conserved heterodimer in eukaryotes. Mutations in NAC cause severe embryonically lethal phenotypes in mice, Drosophila melanogaster, and Caenorhabditis elegans. In the yeast Saccharomyces cerevisiae NAC is quantitatively associated with ribosomes. Here we show that NAC contacts several ribosomal proteins. The N terminus of βNAC, however, specifically contacts near the tunnel exit ribosomal protein Rpl31, which is unique to eukaryotes and archaea. Moreover, the first 23 amino acids of βNAC are sufficient to direct an otherwise non-associated protein to the ribosome. In contrast, αNAC (Egd2p) contacts Rpl17, the direct neighbor of Rpl31 at the ribosomal tunnel exit site. Rpl31 was also recently identified as a contact site for the SRP receptor and the ribosome-associated complex. Furthermore, in Escherichia coli peptide deformylase (PDF) interacts with the corresponding surface area on the eubacterial ribosome. In addition to the previously identified universal adapter site represented by Rpl25/Rpl35, we therefore refer to Rpl31/Rpl17 as a novel universal docking site for ribosome-associated factors on the eukaryotic ribosome.     

Occurrence of Chaperonin 60 and Chaperonin 10 in primary and secondary bacterial symbionts of aphids: Implications for the evolution of an endosymbiotic system in aphids

Summary All aphids harbor symbiotrophic prokaryotes (primary symbionts) in a specialized-abdominal cell, the bacteriocyte. Chaperonin 60 (Cpn60, symbionin) and chaperonin 10 (Cpn10), which are high and low molecular weight heatshock proteins, were sought in tissues of more than 60 aphid species. The endosymbionts were compared immunologically and histologically. It was demonstrated that (1) there are two types of aphids in terms of the endosymbiotic system: some with only primary symbionts and others with, in addition, secondary symbionts; (2) the primary symbionts of various aphids are quite similar in morphology whereas the secondary symbionts vary; and (3) irrespective of the aphid species, Cpn60 is abundant in both the primary and secondary symbionts, while Cpn10 is abundant in the secondary symbionts but present in small amounts in the primary ones. Based on these results, we suggest that the primary symbionts have been derived from a prokaryote that was acquired by the common ancestor of aphids whereas the secondary symbionts have been acquired by various aphids independently after divergence of the aphid species. In addition, we point out the possibility that the prokaryotes under intracellular conditions have been subject to some common evolutionary pressures, and as a result, have come to resemble cell organelles.     

Molecular mimicry in Lyme disease: monoclonal antibody H9724 to b. burgdorferi flagellin specifically detects chaperonin-HSP60

A monoclonal antibody (H9724), specific for the 41-kDa flagellar protein of the Lyme disease pathogen Borrelia burgdorferi, cross-reacts with human axons and detects one major protein in human neuroblastoma cell extracts. The homologous cross-reacting protein has now been isolated from calf adrenal and identified as chaperonin-HSP60 by N-terminal sequencing.     

Chaperonin-mediated Protein Folding

We have been studying chaperonins these past twenty years through an initial discovery of an action in protein folding, analysis of structure, and elucidation of mechanism. Some of the highlights of these studies were presented recently upon sharing the honor of the 2013 Herbert Tabor Award with my early collaborator, Ulrich Hartl, at the annual meeting of the American Society for Biochemistry and Molecular Biology in Boston. Here, some of the major findings are recounted, particularly recognizing my collaborators, describing how I met them and how our great times together propelled our thinking and experiments.     

A Shared Endoplasmic Reticulum-associated Degradation Pathway Involving the EDEM1 Protein for Glycosylated and Nonglycosylated Proteins

Studies of misfolded protein targeting to endoplasmic reticulum-associated degradation (ERAD) have largely focused on glycoproteins, which include the bulk of the secretory proteins. Mechanisms of targeting of nonglycosylated proteins are less clear. Here, we studied three nonglycosylated proteins and analyzed their use of known glycoprotein quality control and ERAD components. Similar to an established glycosylated ERAD substrate, the uncleaved precursor of asialoglycoprotein receptor H2a, its nonglycosylated mutant, makes use of calnexin, EDEM1, and HRD1, but only glycosylated H2a is a substrate for the cytosolic SCF^Fbs2 E3 ubiquitin ligase with lectin activity. Two nonglycosylated BiP substrates, NS-1κ light chain and truncated Igγ heavy chain, interact with the ERAD complex lectins OS-9 and XTP3-B and require EDEM1 for degradation. EDEM1 associates through a region outside of its mannosidase-like domain with the nonglycosylated proteins. Similar to glycosylated substrates, proteasomal inhibition induced accumulation of the nonglycosylated proteins and ERAD machinery in the endoplasmic reticulum-derived quality control compartment. Our results suggest a shared ERAD pathway for glycosylated and nonglycosylated proteins composed of luminal lectin machinery components also capable of protein-protein interactions.     

The Functional Interaction of Mitochondrial Hsp70s with the Escort Protein Zim17 Is Critical for Fe/S Biogenesis and Substrate Interaction at the Inner Membrane Preprotein Translocase

The yeast protein Zim17 belongs to a unique class of co-chaperones that maintain the solubility of Hsp70 proteins in mitochondria and plastids of eukaryotic cells. However, little is known about the functional cooperation between Zim17 and mitochondrial Hsp70 proteins in vivo. To analyze the effects of a loss of Zim17 function in the authentic environment, we introduced novel conditional mutations within the ZIM17 gene of the model organism Saccharomyces cerevisiae that allowed a recovery of temperature-sensitive but respiratory competent zim17 mutant cells. On fermentable growth medium, the mutant cells were prone to acquire respiratory deficits and showed a strong aggregation of the mitochondrial Hsp70 Ssq1 together with a concomitant defect in Fe/S protein biogenesis. In contrast, under respiring conditions, the mitochondrial Hsp70s Ssc1 and Ssq1 exhibited only a partial aggregation. We show that the induction of the zim17 mutant phenotype leads to strong import defects for Ssc1-dependent matrix-targeted precursor proteins that correlate with a significantly reduced binding of newly imported substrate proteins to Ssc1. We conclude that Zim17 is not only required for the maintenance of mtHsp70 solubility but also directly assists the functional interaction of mtHsp70 with substrate proteins in a J-type co-chaperone-dependent manner.     

HSP60/10 chaperonin systems are inhibited by a variety of approved drugs,natural products,and known bioactive molecules

All living organisms contain a unique class of molecular chaperones called 60?kDa heat shock proteins (HSP60 – also known as GroEL in bacteria). While some organisms contain more than one HSP60 or GroEL isoform, at least one isoform has always proven to be essential. Because of this, we have been investigating targeting HSP60 and GroEL chaperonin systems as an antibiotic strategy. Our initial studies focused on applying this antibiotic strategy for treating African sleeping sickness (caused by Trypanosoma brucei parasites) and drug-resistant bacterial infections (in particular Methicillin-resistant Staphylococcus aureus – MRSA). Intriguingly, during our studies we found that three known antibiotics – suramin, closantel, and rafoxanide – were potent inhibitors of bacterial GroEL and human HSP60 chaperonin systems. These findings prompted us to explore what other approved drugs, natural products, and known bioactive molecules might also inhibit HSP60 and GroEL chaperonin systems. Initial high-throughput screening of 3680 approved drugs, natural products, and known bioactives identified 161 hit inhibitors of the Escherichia coli GroEL chaperonin system (4.3% hit rate). From a purchased subset of 60 hits, 29 compounds (48%) re-confirmed as selective GroEL inhibitors in our assays, all of which were nearly equipotent against human HSP60. These findings illuminate the notion that targeting chaperonin systems might be a more common occurrence than we previously appreciated. Future studies are needed to determine if the in vivo modes of action of these approved drugs, natural products, and known bioactive molecules are related to GroEL and HSP60 inhibition.     

The cytosolic chaperonin CCT associates to cytoplasmic microtubular structures during mammalian spermiogenesis and to heterochromatin in germline and somatic cells

Spermiogenesis, the haploid phase of spermatogenesis, is characterised by a dramatic cytodifferentiation of spermatids. The two major steps, nuclear shaping and cytoplasmic reorganisation of the organelles, rely on an extensive remodelling of the microtubule cytoskeleton. Folding of alpha- and beta-tubulin is mediated by the cytoplasmic chaperonin containing TCP-1 (CCT), highly expressed in testis. We studied CCT cellular distribution throughout spermatogenesis by immunofluorescence and immunoelectron microscopy. We unveil two main cytoplasmic localisations for CCT: at the centrosome and at the microtubules of the manchette, a structure unique to male germ cells. Both structures are essential for spermatid differentiation and may require CCT function. Although CCT is essentially cytoplasmic, a few reports suggest that a subset may have a nuclear localisation. We demonstrate that in the nucleus of germline and somatic cells, part of CCT associates to heterochromatin. In interphase cells, CCT seems generally confined to constitutive heterochromatin. Nevertheless, in condensing nucleus of future spermatozoon, it is also associated with chromatin undergoing compaction. Finally, in fully-condensed mitotic chromosomes, CCT is located all along the chromosomes. Our finding that CCT is associated with constitutive heterochromatin and to compacting chromatin raises the possibility that it may be implicated in maintenance and remodelling of heterochromatin.     

A phospho-proteomic screen identifies novel S6K1 and mTORC1 substrates revealing additional complexity in the signaling network regulating cell growth

S6K1, a critical downstream substrate of mTORC1, has been implicated in regulating protein synthesis and a variety of processes that impinge upon cell growth and proliferation. While the role of the cytoplasmic p70^S6K1 isoform in the regulation of translation has been intensively studied, the targets and function of the nuclear p85^S6K1 isoform remain unclear. Therefore, we carried out a phospho-proteomic screen to identify novel p85^S6K1 substrates. Four novel putative p85^S6K1 substrates, GRP75, CCTβ, PGK1 and RACK1, and two mTORC1 substrates, ANXA4 and PSMA6 were identified, with diverse roles in chaperone function, ribosome maturation, metabolism, vesicle trafficking and the proteasome, respectively. The chaperonin subunit CCTβ was further investigated and the site of phosphorylation mapped to serine 260, a site located in the chaperonin apical domain. Consistent with this domain being involved in folding substrate interactions, we found that phosphorylation of serine 260 modulates chaperonin folding activity.     

Molecular characterization of the group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3

The group II chaperonin from the hyperthermophilic archaeum Pyrococcus horikoshii OT3 (PhCPN) and its functional cooperation with the cognate prefoldin were investigated. PhCPN existed as a homo-oligomer in a double-ring structure, which protected the citrate synthase of a porcine heart from thermal aggregation at 45°C, and did the same on the isopropylmalate dehydrogenase (IPMDH) of a thermophilic bacterium, Thermus thermophilus HB8, at 90°C. PhCPN also enhanced the refolding of green fluorescent protein (GFP), which had been unfolded by low pH, in an ATP-dependent manner. Unexpectedly, functional cooperation between PhCPN and Pyrococcus prefoldin (PhPFD) in the refolding of GFP was not observed. Instead, cooperation between PhCPN and PhPFD was observed in the refolding of IPMDH unfolded with guanidine hydrochloride. Although PhCPN alone was not effective in the refolding of IPMDH, the refolding efficiency was enhanced by the cooperation of PhCPN with PhPFD.