The Bacterial ClpXP-ClpB Family Is Enriched with RNA-Binding Protein Complexes

Abstract

In the matrix of bacteria/mitochondria/chloroplasts, Lon acts as the degradation machine for soluble proteins. In stress periods, however, proteostasis and survival depend on the strongly conserved Clp/Hsp100 family. Currently, the targets of ATP-powered unfoldases/disaggregases ClpB and ClpX and of peptidase ClpP heptameric rings are still unclear. Trapping experiments and proteome profiling in multiple organisms triggered confusion, so we analyzed the consistency of ClpP-trap targets in bacteria. We also provide meta-analyses of protein interactions in humans, to elucidate where Clp family members are enriched. Furthermore, meta-analyses of mouse complexomics are provided. Genotype–phenotype correlations confirmed our concept. Trapping, proteome, and complexome data retrieved consistent coaccumulation of CLPXP with GFM1 and TUFM orthologs. CLPX shows broad interaction selectivity encompassing mitochondrial translation elongation, RNA granules, and nucleoids. CLPB preferentially attaches to mitochondrial RNA granules and translation initiation components; CLPP is enriched with them all and associates with release/recycling factors. Mutations in CLPP cause Perrault syndrome, with phenotypes similar to defects in mtDNA/mtRNA. Thus, we propose that CLPB and CLPXP are crucial to counteract misfolded insoluble protein assemblies that contain nucleotides. This insight is relevant to improve ClpP-modulating drugs that block bacterial growth and for the treatment of human infertility, deafness, and neurodegeneration.

Translation fidelity and respiration deficits in CLPP-deficient tissues: Mechanistic insights from mitochondrial complexome

“…The targets of CLPXP proteolytic activity remain controversial, despite analyses over decades in numerous organisms with diverse technical approaches, most of which involved recombinant overexpression [49,55]. As a novel approach, we now focused on endogenous proteins in their stable interactions within three mouse tissues, trying to define potential CLPX targets based on four criteria:…”

“…Similarly, a close comigration with CLPX was documented for GFM1 from its monomeric size (84 kDa) to positions around 250 kDa, in addition to very strong accumulation (average 10-fold). A recent review of putative CLPXP substrates in different species concluded that the translation elongation GTPase GFM1/GFM2 orthologs are exceptionally consistent candidates [49]. Curiously, the extra-mitochondrial translation initiation complex core factor EIF3C (precursor size 106 kDa) also appeared in CLPP-null mitochondria greatly accumulated (average 5-fold) and showed comigration across the entire CLPX distribution range.…”

“…We concentrated the validation work on GFM1 as translation elongation GTPase, the MRPL18/MRPL38 cluster as mtLSU-CP components that are folded around tRNA-valine or 5S-rRNA, and the amino acid homeostasis enzyme OAT which is soluble in the mitochondrial matrix. All of them were reported as targets of PLP binding [126][127][128][129] As shown in Figure 7, we prioritized GFM1 which was the most consistent CLPXP target among all surveys in various organisms [49]. Anti-CLPX antibodies (Invitrogen) coimmunoprecipitated GFM1…”

“…In addition, the progressive sensorineural deafness due to the chronic administration of aminoglycoside antibiotics is also caused by neurotoxic interference and genetic variance at the mtSSU [193][194][195]. A detailed review of mitoribosomal pathology underlying progressive deafness as in PRLTS was recently compiled [49]. It also should be noted that the principal target of the CLPX cofactor PLP at the bacterial SSU is the inhibition of mRNA-dependent aminoacyl-tRNA binding [126].…”

“…It was demonstrated that CLPP deficiency in mice causes impaired translation fidelity at mitoribosomes, claiming that this occurs due to the accumulation and inappropriate interactions of the rRNA chaperone ERAL1, which acts mainly in the assembly of mtSSU, the small mitoribosomal subunit [45][46][47]. However, the crucial role of ERAL1 for mitoribosome pathology in CLPP-null mice has since been disputed [48,49]. Problems of translation fidelity in mitoribosomes would affect primarily the 13 proteins encoded by mitochondrial DNA to function within respiratory chain complexes [50].…”