Protein Unfolding by ATP-dependent Proteases

ATP-dependent proteases degrade regulatory proteins and thereby control processes such as cell cycle, gene transcription and signal transduction. In addition, the proteases degrade misfolded or damaged polypeptides and produce many of the antigenic peptides displayed at the cell surface for immune response. In eukaryotes, this activity is performed mainly by the proteasome. In prokaryotes and eukaryotic organelles, similar functions are performed by analogues of the proteasome such as the ClpAP, ClpXP, HslUV, Lon, and FtsH proteases.

The proteasome is highly selective for its substrates despite the fact that the active sites of proteolysis themselves show very little sequence specificity. Discrimination is achieved by sequestering the proteolytic sites within the structure of these proteases and tightly controlling access.

Substrate proteins are specifically targeted to the proteasome, most commonly by the covalent attachment of multiple ubiquitin moieties to lysine residues in the substrate. This modification mediates the association of substrates with the protease. Some proteins are targeted for degradation through adaptors that bind both substrate and protease simultaneously. A small number of proteasome substrates are recognized by the protease directly through specific targeting signals encoded in the primary sequence of the protein. These latter two recognition mechanisms are similar to the way in which most prokaryotic substrates bind their proteases.

Native proteins must be in an unfolded conformation during degradation and stabilizing a substrate protein against unfolding can prevent its proteolysis. ATP-dependent proteases can accelerate the denaturation of their substrates. It has been shown, for example, ClpAP can unfold green fluorescent protein (GFP) at a rate that is six-orders of magnitude faster than the rate at which the protein would unfold spontaneously.

Subsequent studies have found that the proteasome, ClpXP, Lon and the archebacterial proteasome can also actively unfold their substrates. We have found that the proteasome catalyzes unfolding by engaging the substrates through their unstructured initiation sites and sequentially unraveling them from their degradation initiation sites.

In a multidomain protein, the domain closest to the initiation site is degraded first, whereas domains located further away are degraded subsequently. Stabilizing a domain close to the initiation site protects downstream domains from degradation but not vice versa. Experiments using fluorescence resonance energy transfer (FRET) confirms that the bacterial protease ClpAP translocates its substrate sequentially to its proteolytic sites. ClpXP and FtsH have also been shown to degrade their substrates sequentially along the polypeptide chain.

Our lab focusses on characterizing the mechanism of protein unfolding by the ATP-dependent proteases and determining the role of protein structure, stability & sequence in modulating protease activity and to affect the specificity of these processes