Seong Il Choi

Protein Solubility and Folding Enhancement by Interaction with RNA

While basic mechanisms of several major molecular chaperones are well understood, this machinery has been known to be involved in folding of only limited number of proteins inside the cells. Here, we report a chaperone type of protein folding facilitated by interaction with RNA. When an RNA-binding module is placed at the N-terminus of aggregation-prone target proteins, this module, upon binding with RNA, further promotes the solubility of passenger proteins, potentially leading to enhancement of proper protein folding. Studies on in vitro refolding in the presence of RNA, coexpression of RNA molecules in vivo and the mutants with impaired RNA binding ability suggests that RNA can exert chaperoning effect on their bound proteins. The results suggest that RNA binding could affect the overall kinetic network of protein folding pathway in favor of productive folding over off-pathway aggregation. In addition, the RNA binding-mediated solubility enhancement is extremely robust for increasing soluble yield of passenger proteins and could be usefully implemented for high-throughput protein expression for functional and structural genomic research initiatives. The RNA-mediated chaperone type presented here would give new insights into de novo folding in vivo.

N-terminal domains of native multidomain proteins have the potential to assist de novo folding of their downstream domains in vivo by acting as solubility enhancers

The fusion of soluble partner to the N terminus of aggregation‐prone polypeptide has been popularly used to overcome the formation of inclusion bodies in the E. coli cytosol.

RNA-mediated chaperone type for de novo protein folding

Traditionally the principles of protein folding in vivo have been obtained largely from molecular chaperone studies. Through extensive studies on molecular chaperones, it becomes clear that most proteins can fold without their assistance in vivo, suggesting the existence of other chaperone types and mechanisms. Since all nascent polypeptides are linked to the ribosomes, protein folding in vivo should be understood in the context of vectorial protein synthesis and linkage of nascent chains to ribosome whose major components and basic structural frames are RNAs. Here we introduce a novel RNA-mediated chaperone type and a possible molecular basis for how RNAs can exert chaperoning effect on their linked aggregation-prone polypeptides. Extending potential chaperoning role of ribosome on the bound nascent polypeptide in a cis-acting manner, the findings further suggest a novel function of RNA molecules for protein folding inside cells. RNA interaction-mediated stabilization of folding intermediate against aggregation provides new insights into de novo protein folding in vivo and further extends the functional diversity of RNA molecules.

Novel Secretion System of Recombinant Saccharomyces cerevisiae Using an N-terminus Residue of Human IL-1β as Secretion Enhancer

An N‐terminus sequence of human interleukin 1β (hIL‐1β) was used as a fusion expression partner for the production of two recombinant therapeutic proteins, human granulocyte‐colony stimulating factor (hG‐CSF) and human growth hormone (hGH), using Saccharomyces cerevisiae as a host. The expression cassette comprised the leader sequence of killer toxin of Kluyveromyces lactis, the N‐terminus 24 amino acids (Ser5‐Ala28) of mature hIL‐1β, the KEX2 dibasic endopeptidase cleavage site, and the target protein (hG‐CSF or hGH). The gene expression was controlled by the inducible UASgal/MF‐α1 promoter. With the expression vector above, both recombinant proteins were well secreted into culture medium with high secretion efficiencies, and especially, the recombinant hGH was accumulated up to around 1.3 g/L in the culture broth. This is due presumably to the significant role of fused hIL‐1β as secretion enhancer in the yeast secretory pathway. In our recent report, various immunoblotting analyses have shown that the presence of a core N‐glycosylation resident in the hIL‐1β fragment is likely to be of crucial importance in the high‐level secretion of hG‐CSF from the recombinantS. cerevisiae. When the N‐glycosylation was completely blocked with the addition of tunicamycin to the culture, the secretion of hG‐CSF and hGH was decreased to a negligible level although the other host‐derived proteins were well secreted to the culture broth regardless of the presence of tunicamycin. The N‐terminal sequencing of the purified hG‐CSF verified that the hIL‐1β fusion peptide was correctly removed by in vivo KEX2 protease upon the exit of fusion protein from Golgi complex. From the results presented in this article, it is strongly suggested that the N‐terminus fusion of the hIL‐1β peptide could be utilized as a potent secretion enhancer in the expression systems designed for the secretory production of other heterologous proteins from S. cerevisiae.

Identification of novel inhibitors of HCV RNA-dependent RNA polymerase by pharmacophore-based virtual screening and in vitro evaluation

Hepatitis C virus (HCV) is the major etiological agent of non-A, non-B hepatitis where no effective treatment is available. The HCV NS5B with RNA-dependent RNA polymerase (RdRp) activity is a key target for the treatment of HCV infection. Here we report novel NS5B polymerase inhibitors identified by virtual screening and in vitro evaluation of their inhibitory activities. On the basis of a newly identified binding pocket of NS5B, distinct from the nucleotide binding site but highly conserved among various HCV isolates, we performed virtual screening of compounds that fit this binding pocket from the available chemical database of 3.5 million compounds. The inhibitory activities of the in silico selected 119 compounds were estimated with in vitro RdRp assay. Three compounds with IC50 values of about 20 microM were identified, and their kinetic analyses suggest that these compounds are noncompetitive inhibitors with respect to the ribonucleotide substrate. Furthermore, the single-point mutations of the conserved residues in the binding pocket of NS5B resulted in the significant decrease of the RdRp activity, indicating that the binding pocket presented here might be important for the therapeutic intervention of HCV. These novel inhibitors would be useful for the development of effective anti-HCV agents.

RPS3a Over-Expressed in HBV-Associated Hepatocellular Carcinoma Enhances the HBx-Induced NF-κB Signaling via Its Novel Chaperoning Function

Hepatitis B virus (HBV) infection is one of the major causes of hepatocellular carcinoma (HCC) development. Hepatitis B virus X protein (HBx) is known to play a key role in the development of hepatocellular carcinoma (HCC). Several cellular proteins have been reported to be over-expressed in HBV-associated HCC tissues, but their role in the HBV-mediated oncogenesis remains largely unknown. Here, we explored the effect of the over-expressed cellular protein, a ribosomal protein S3a (RPS3a), on the HBx-induced NF-κB signaling as a critical step for HCC development. The enhancement of HBx-induced NF-κB signaling by RPS3a was investigated by its ability to translocate NF-κB (p65) into the nucleus and the knock-down analysis of RPS3a. Notably, further study revealed that the enhancement of NF-κB by RPS3a is mediated by its novel chaperoning activity toward physiological HBx. The over-expression of RPS3a significantly increased the solubility of highly aggregation-prone HBx. This chaperoning function of RPS3a for HBx is closely correlated with the enhanced NF-κB activity by RPS3a. In addition, the mutational study of RPS3a showed that its N-terminal domain (1–50 amino acids) is important for the chaperoning function and interaction with HBx. The results suggest that RPS3a, via extra-ribosomal chaperoning function for HBx, contributes to virally induced oncogenesis by enhancing HBx-induced NF-κB signaling pathway.

Protein Folding In Vivo Revisited

Protein folding in vivo is extremely intricate and challenging to examine or predict because the conformational changes, including folding, misfolding, and aggregation, are largely influenced by the cellular environment. Traditionally, cellular protein folding has been considered predominantly in the context of the Anfinsen postulate and molecular chaperones. However, accumulating evidence reveals that these models have limitations. In this review we revisit these models, and discuss co-translational folding, binding partner-mediated folding, and RNA-mediated folding as alternative or supplementary folding helpers. In addition, we discuss the folding helper systems mediated by macromolecules (e.g., ribosomes, membranes, and prefolded domains in multidomain proteins) that are tightly linked to newly synthesized polypeptides during protein biogenesis. These cis-acting folding helper systems, conceptually different from the trans-acting molecular chaperones, could play a crucial role in protein folding in vivo. Importantly, there is increasing evidence that the surface charges and excluded volume of macromolecules are important factors for stabilizing their connected polypeptides against aggregation. This stabilizing mechanism suggests that macromolecules including RNAs and proteins, let alone molecular chaperones, have an intrinsic ability to exert chaperoning function on their connected polypeptides independent of the linkage type between them. As an effective way to overcome the adverse effect of macromolecular crowding on protein folding, here we suggest that nascent polypeptide chains utilize the crowded environment in favor of productive folding by interacting with macromolecules.

5S rRNA-assisted DnaK refolding

Although accumulating evidence has revealed that most proteins can fold without the assistance of molecular chaperones, little attention has been paid to other types of chaperoning macromolecules. A variety of proteins interact with diverse RNA molecules in vivo, suggesting a potential role of RNAs for folding of their interacting proteins. Here we show that the in vitro refolding of a representative molecular chaperone, DnaK, an Escherichia coli homolog of Hsp70, could be assisted by its interacting 5S rRNA. The folding enhancement occurred in RNA concentration and its size dependent manner whereas neither the RNA with the reverse sequence of 5S rRNA nor the RNase pretreated 5S rRNA stimulated the folding in vitro. Based on our results, we propose that 5S rRNA could exert the chaperoning activity on DnaK during the folding process. The results suggest an interesting possibility that the folding of RNA-interacting proteins could be assisted by their cognate RNA ligands.

Chaperoning roles of macromolecules interacting with proteins in vivo.

The principles obtained from studies on molecular chaperones have provided explanations for the assisted protein folding in vivo. However, the majority of proteins can fold without the assistance of the known molecular chaperones, and little attention has been paid to the potential chaperoning roles of other macromolecules. During protein biogenesis and folding, newly synthesized polypeptide chains interact with a variety of macromolecules, including ribosomes, RNAs, cytoskeleton, lipid bilayer, proteolytic system, etc. In general, the hydrophobic interactions between molecular chaperones and their substrates have been widely believed to be mainly responsible for the substrate stabilization against aggregation. Emerging evidence now indicates that other features of macromolecules such as their surface charges, probably resulting in electrostatic repulsions, and steric hindrance, could play a key role in the stabilization of their linked proteins against aggregation. Such stabilizing mechanisms are expected to give new insights into our understanding of the chaperoning functions for de novo protein folding. In this review, we will discuss the possible chaperoning roles of these macromolecules in de novo folding, based on their charge and steric features.

M1 RNA is important for the in-cell solubility of its cognate C5 protein: Implications for RNA-mediated protein folding.

It is one of the fundamental questions in biology how proteins efficiently fold into their native conformations despite off-pathway events such as misfolding and aggregation in living cells. Although molecular chaperones have been known to assist the de novo folding of certain types of proteins, the role of a binding partner (or a ligand) in the folding and in-cell solubility of its interacting protein still remains poorly defined. RNase P is responsible for the maturation of tRNAs as adaptor molecules of amino acids in ribosomal protein synthesis. The RNase P from Escherichia coli, composed of M1 RNA and C5 protein, is a prototypical ribozyme in which the RNA subunit contains the catalytic activity. Using E. coli RNase P, we demonstrate that M1 RNA plays a pivotal role in the in-cell solubility of C5 protein both in vitro and in vivo. Mutations in either the C5 protein or M1 RNA that affect their interactions significantly abolished the folding of C5 protein. Moreover, we find that M1 RNA provides quality insurance of interacting C5 protein, either by promoting the degradation of C5 mutants in the presence of functional proteolytic machinery, or by abolishing their solubility if the machinery is non-functional. Our results describe a crucial role of M1 RNA in the folding, in-cell solubility, and, consequently, the proteostasis of the client C5 protein, giving new insight into the biological role of RNAs as chaperones and mediators that ensure the quality of interacting proteins.