The Cleavage Profile of Protein Substrates by ClpXP Reveals Deliberate Starts and Pauses

Abstract

Cells rely on protein degradation by AAA+ proteases. A well-known example is the hexameric ClpX unfoldase, which captures ATP hydrolysis to feed substrates into the oligomeric ClpP peptidase. Recent studies show that an asymmetric ClpX spiral cycles protein translocation upon ATP hydrolysis. However, how this cycle affects peptide products is less explored in part because ClpP cleavage is thought to be solely defined by sequence constraints. Here, we comprehensively characterize peptides from Caulobacter crescentus ClpXP degradation of three different substrates using high-resolution mass spectrometry and find that cleavage of translocated substrates is driven by factors other than sequence. We report that defined locations in a translocated protein are especially sensitive to cleavage spaced on average every 10−13 residues. These sites are not exclusively controlled by sequence and are independent of bulk changes in catalytic peptidase sites, ATP hydrolysis, or the efficiency of initial recognition. These results fit a model in which processive translocation through ClpX starts at a specific location in a polypeptide and pauses during reset of the ClpX hexamer after a cycle of translocation. Our work suggests that defined peptides, which could be used as signaling molecules, can be generated from a given substrate by a nonspecific peptidase.

Caspase-9 Activation of Procaspase-3 but Not Procaspase-6 Is Based on the Local Context of Cleavage Site Motifs and on Sequence

“…7 In fact, it was later discovered that proteolysis by ClpP protease (from Caulobacter crescentus) complexed with ClpX (an unfoldase that feeds ClpP) is mainly governed by local context (substrate cleavage occurred after every 10−13 residues) and not dependent on sequence. 8 Upon examination of the cleavage preferences for any protease and its suite of cleaved substrates, it becomes clear that (i) no single peptide sequence can fully account for the cleavage properties observed for a natural protease and (ii) the sequence specificity alone cannot completely account for the selection of substrates that are cleaved. Thus, we hypothesized at the outset of this work that both the sequence and the local context of the substrate cleavage site may be important factors governing recognition by a protease.…”

“…Another example is the family of ClpP proteases (from Escherichia coli , Staphylococcus aureus , and human mitochondria) that favor peptide substrates composed of specific residues at the P3–P1 positions (e.g., natural amino acids preferred by human mitochondria ClpP at P3–P1 positions are F/W-M/T/L-M/L); nonetheless, proteomics studies of these proteases revealed a very weak cleavage preference . In fact, it was later discovered that proteolysis by ClpP protease (from Caulobacter crescentus ) complexed with ClpX (an unfoldase that feeds ClpP) is mainly governed by local context (substrate cleavage occurred after every 10–13 residues) and not dependent on sequence . Upon examination of the cleavage preferences for any protease and its suite of cleaved substrates, it becomes clear that (i) no single peptide sequence can fully account for the cleavage properties observed for a natural protease and (ii) the sequence specificity alone cannot completely account for the selection of substrates that are cleaved.…”

Mitochondrial quality control proteases and their modulation for cancer therapy

“…33 To achieve sustained activity, ClpP develops a heterodimer with its partner molecule ClpX, which is usually called a ClpXP complex. 34 ClpX (NCBI GeneID: 10845), a member of the nuclear-encoded AAA+ protein superfamily with 633 amino acid residues in length, is the only known ATPase component for mammalian ClpP. 30 Similar to ClpP, ClpX owns a 56-residue N-terminal MTS that exists as a hexamer under physiological conditions, and binds with ATP to sustain the stability 35 (Figure 2A).…”

“…Due to the lack of ATPase activity, it only hydrolyzes short peptides of six or fewer amino acids 33 . To achieve sustained activity, ClpP develops a heterodimer with its partner molecule ClpX, which is usually called a ClpXP complex 34 . ClpX (NCBI GeneID: 10845), a member of the nuclear‐encoded AAA+ protein superfamily with 633 amino acid residues in length, is the only known ATPase component for mammalian ClpP 30 .…”

A closed translocation channel in the substrate-free AAA+ ClpXP protease diminishes rogue degradation

“…A related question is how the peptide products of ClpXP cleavage, which typically range from 5 to 20 residues long [30][31] , exit the degradation chamber following cleavage of a substrate polypeptide? It has been proposed that structural fluctuations at the equatorial ring-ring interface of ClpP create transient gaps or windows that allow product egress 32 .…”