Lon and ClpP proteases participate in the physiological disintegration of bacterial inclusion bodies

Abstract

Aggregated protein is solubilized by the combined activity of chaperones ClpB, DnaK and small heat-shock proteins, and this could account, at least partially, for the physiological disintegration of bacterial inclusion bodies. In vivo, the involvement of proteases in this process had been suspected but not investigated. By using an aggregation prone β-galactosidase fusion protein produced in Escherichia coli, we show in this study that the main ATP-dependent proteases Lon and ClpP participate in the physiological disintegration of cytoplasmic inclusion bodies, their absence minimizing the protein removal up to 40%. However, the role of these proteases is clearly distinguishable especially regarding the fate of solubilized protein. While Lon appears as a minor contributor in the disintegration process, ClpP directs an important attack on the released or releasable protein even not being irreversibly misfolded. ClpP is then observed as a wide-spectrum, main processor of aggregation-prone proteins and also of polypeptides physiologically released from inclusion bodies, even when occurring as soluble versions with a conformation compatible with their enzymatic activity.

Introduction

Bacterial inclusion bodies are amorphous aggregates resulting from the deposition of insoluble polypeptide chains (Villaverde and Carrió, 2003). They are commonly occurring during the overexpression of foreign genes, whose products are kinetically trapped as stable intermediates of deficient folding processes. In bacteria, thermal stress but also the overproduction or misfolding-prone proteins trigger the expression of heat-shock genes, whose products, chaperones and proteases, act coordinately to minimize the occurrence of conformationally aberrant protein forms (Yura et al., 1993, Arsene et al., 2000, Feldman and Frydman, 2000). Bacterial chaperones DnaK and GroEL (and their cochaperones DnaJ-GrpE and GroES, respectively) address folding of folding reluctant protein forms in a sequential way (Buchberger et al., 1996, Goloubinoff et al., 1999), while small heat-shock proteins such as IbpA/B protect misfolded proteins from irreversible aggregation (Thomas and Baneyx, 1998, Shearstone and Baneyx, 1999, Schlieker et al., 2002). Also, chaperones belonging to the AAA+ family such as ClpB, in cooperation with DnaK and small heat-shock proteins, remove polypeptides from aggregates for their eventual refolding (Thomas and Baneyx, 2000, Mogk et al., 2003a, Mogk et al., 2003b, Mogk and Bukau, 2004, Weibezahn et al., 2004). The activity of these and other heat-shock elements might account for an almost complete in vivo disintegration of inclusion bodies in absence of the novo protein synthesis (Carrió and Villaverde, 2001). Therefore, the formation and dissolution of protein aggregates in bacteria results from unbalanced kinetics of protein deposition and removal, both processes being simultaneous and thus producing a continuous physiological reorganization of inclusion body composition even during their volumetric growth (Carrió and Villaverde, 2002).

On the other hand, ATP-dependent proteases degrade misfolded polypeptides that are reluctant to chaperone-mediated folding. How substrates are selected for either further folding attempts or digestion is still a matter of discussion (Tomoyasu et al., 2001, Dougan et al., 2002). It is currently accepted that proteolysis is mostly restricted to soluble targets, aggregation resulting then in proteolytic stabilization by limiting the accessibility of protease target sites. However, inclusion body protein is efficiently digested in vitro by trypsin (Carrió et al., 2000) in a cascade process that renders progressively shorter products (Cubarsi et al., 2001). In vivo, aggregated polypeptides are cleaved in a site-limited process that is linked to their transfer from the insoluble to the soluble cell fraction (Corchero et al., 1997, Carrió et al., 1999). This observation indicates that proteolysis, apart from the activity of disaggregating chaperones, might be also involved in the physiological dissolution of bacterial protein aggregates, although this possibility has not been further analysed. In this work we have explored the involvement of Lon and ClpP in the formation and disintegration of inclusion bodies. These proteases are responsible for more of 70% of the cellular ATP-dependent proteolysis (Maurizi, 1992) and are in charge of the degradation of aggregation-prone proteins (Tomoyasu et al., 2001, Rozkov and Enfors, 2004), a condition shared by most of bacterially produced foreign polypeptides. The obtained results prove a dramatic role of these proteases in the dissolution process as well as a dissimilar substrate selection in their activity. These data allow the conceptual integration of the activities of chaperones and proteases for the surveillance of protein aggregation and the cellular managing of protein aggregates.