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

Macromolecule-Assisted<em> de novo</em> Protein Folding

In the processes of protein synthesis and folding, newly synthesized polypeptides are tightly connected to the macromolecules, such as ribosomes, lipid bilayers, or cotranslationally folded domains in multidomain proteins, representing a hallmark of <em>de novo</em> protein folding environments <em>in vivo</em>. Such linkage effects on the aggregation of endogenous polypeptides have been largely neglected, although all these macromolecules have been known to effectively and robustly solubilize their linked heterologous proteins in fusion or display technology. Thus, their roles in the aggregation of linked endogenous polypeptides need to be elucidated and incorporated into the mechanisms of <em>de novo</em> protein folding <em>in vivo</em>. In the classic hydrophobic interaction-based stabilizing mechanism underlying the molecular chaperone-assisted protein folding, it has been assumed that the macromolecules connected through a simple linkage without hydrophobic interactions and conformational changes would make no effect on the aggregation of their linked polypeptide chains. However, an increasing line of evidence indicates that the intrinsic properties of soluble macromolecules, especially their surface charges and excluded volume, could be important and universal factors for stabilizing their linked polypeptides against aggregation. Taken together, these macromolecules could act as folding helpers by keeping their linked nascent chains in a folding-competent state. The folding assistance provided by these macromolecules in the linkage context would give new insights into <em>de novo</em> protein<em> </em>folding inside the cell

HBx-induced NF-κB signaling in liver cells is potentially mediated by the ternary complex of HBx with p22-FLIP and NEMO.

Sustained activation of NF-κB is one of the causative factors for various liver diseases, including liver inflammation and hepatocellular carcinoma (HCC). It has been known that activating the NF-κB signal by hepatitis B virus X protein (HBx) is implicated in the development of HCC. However, despite numerous studies on HBx-induced NF-κB activation, the detailed mechanisms still remain unsolved. Recently, p22-FLIP, a cleavage product of c-FLIPL, has been reported to induce NF-κB activation through interaction with the IκB kinase (IKK) complex in primary immune cells. Since our previous report on the interaction of HBx with c-FLIPL, we explored whether p22-FLIP is involved in the modulation of HBx function. First, we identified the expression of endogenous p22-FLIP in liver cells. NF-κB reporter assay and electrophoretic mobility shift assay (EMSA) revealed that the expression of p22-FLIP synergistically enhances HBx-induced NF-κB activation. Moreover, we found that HBx physically interacts with p22-FLIP and NEMO and potentially forms a ternary complex. Knock-down of c-FLIP leading to the downregulation of p22-FLIP showed that endogenous p22-FLIP is involved in HBx-induced NF-κB activation, and the formation of a ternary complex is necessary to activate NF-κB signaling. In conclusion, we showed a novel mechanism of HBx-induced NF-κB activation in which ternary complex formation is involved among HBx, p22-FLIP and NEMO. Our findings will extend the understanding of HBx-induced NF-κB activation and provide a new target for intervention in HBV-associated liver diseases and in the development of HCC

The treatment of siFLIP abolished the synergistic effect of p22-FLIP on HBx-mediated NF-κB activation.

<p>(<b>A</b>) A schematic representation of the siFLIP design. (<b>B</b>) The knock-down effect of siFLIP on p22-FLIP. The plasmid for p22-FLIP was co-transfected with siFLIP in Huh7 (left panel) and 293T cells (right panel). At 48 hours post-transfection, the expression level of p22-FLIP was analyzed by Western blot. (<b>C–D</b>) The effect of p22-FLIP knock-down on NF-κB activity. The indicated plasmids and pNF-κB-Luc (0.25 µg) were co-transfected with siFLIP (20 nM) or control siRNA in Huh7 cells(C) and 293T cells (D), respectively. Relative NF-κB activity was determined as described above. The expression levels of p22-FLIP and HBx were analyzed by western blot.</p

p22-FLIP synergistically up-regulates HBx-mediated NF-κB signaling.

<p>(<b>A–B</b>) Relative NF-κB activity after co-transfection of pNF-κB-Luc and p22-FLIP plasmid with/without HBx-HA plasmid in Huh7 and 293T cells, respectively. pEGFP were transfected for the monitoring of transfection efficiency and negative control. (<b>C</b>) Dose-dependent activation of HBx-mediated NF-κB by p22-FLIP. pNF-κB-Luc (0.25 µg) and HBx-HA plasmid (0.4 µg) were co-transfected with increasing amounts of p22-FLIP (0∼1.2 µg) in 293T cells. Total transfected DNA amounts were adjusted using the empty vector (pCMV). The expression levels of p22-FLIP and HBx were determined by Western blot. (<b>D</b>) The level of phospho-IκB (P-IκB) was determined by western blot. The plasmids of p22-FLIP (1 µg) and HBx-HA (1 µg) were co-transfected in Huh7 cells. After 48 hours, the levels of P-IκB and total IκB were analyzed by western blot. (<b>E</b>) NF-κB ELISA was measured by p50 ELISA using nuclear extracts. The interaction of plate-bound NF-κB consensus DNA oligomer and NF-κB subunit (p50) in nuclear extracts was measured by chemiluminescence. (<b>F</b>) NF-κB electrophoretic mobility shift assay (EMSA). The [P³²]-labeled NF-κB consensus DNA oligomer probe was reacted with nuclear extracts (3 µg) <i>in vitro</i>. Non-labeled NF-κB consensus oligomer (30 fold) was used for cold competition. Relative binding affinity was calculated by densitometry.</p

p22-FLIP synergistically enhances HBx-induced NF-κB signaling in the context of replication-competent HBV through interaction with HBx.

<p>(A) p22-FLIP synergistically enhances HBx-mediated NF-κB signaling in the context of HBV full genome. Relative NF-κB activity (left panel) was measured at 48 hours post-transfection of pNF-κB-Luc(0.25μg) and the indicated plasmids (0.4μg) in Huh7 cells. Total amounts of DNA were adjusted by pCMV vector. Results were obtained by at least four independent experiments (*, P = 0.011; **, P<0.001). Expression levels of p22-FLIP and HBV genome-driven HBx were determined by Western blot analysis using the indicated antibodies (right panel). (B) p22-FLIP physically interacts with HBV genome-driven HBx in liver cells. At 72 hours post-transfection of wt HBV1.2mer (2μg) and p22-FLIP (2μg) plasmids, Huh7 cell lysates were immunoprecipitated with anti-Flip (left panel) or anti-HBx (right panel) antibodies, respectively. Total amounts of transfected DNA were adjusted by pcDNA3.1 vector. Western blot analysis was carried out using the indicated antibodies. Cell lysates were used as a positive control. (C) Potential formation of ternary complex among endogenous p22-FLIP, NEMO, and genome-driven HBx in Huh7 cells. Approximately 7.8×10⁶ Huh7 cells were transfected with wt HBV1.2mer plasmids and cultured for 72 hours. Thereafter, cell lysates were immunoprecipitated using anti-NEMO antibody and blotted by anti-NEMO, anti-Flip, and anti-HBx antibodies.</p

Detection of endogenous p22-FLIP in human liver cells.

<p>(<b>A</b>) Schematic illustrations of c-FLIP_L, c-FLIP_S, and p22-FLIP. (<b>B</b>) Detection of endogenous p22-FLIP in Huh7 cells. Approximately 9.5×10⁶ Huh7 cells were used for immunoprecipitation and Western blot. As positive controls, plasmids for p22-FLIP (1 µg) and c-FLIP_L (1 µg) were co-transfected in Huh7 cells and subjected to immunoprecipitation.</p

Endogenous p22-FLIP is involved in HBx-mediated NF-κB signal.

<p>(<b>A</b>) The effect of endogenous p22-FLIP on NF-κB activation. NF-κB activity was determined after co-transfection with pNF-κB-Luc and siFLIP (20 nM) with/without HBx-HA in Huh7 cells (left panel). The levels of mRNAs after treatment of siFLIP (right panel). HBx-HA was co-transfected with siFLIP with the indicated concentration. The expression levels were analyzed by semi-qRT-PCR using specific primers, respectively. GAPDH was used as a loading control. (B) The effect of c-FLIP_L expression on HBx-mediated NF-κB activation. NF-κB activity was measured after co-transfection of pNF-κB-Luc and the indicated plasmids in Huh7 cells.</p