Recognition of enzymes lacking bound cofactor by protein quality control

Abstract

Protein biogenesis is tightly linked to protein quality control (PQC). The role of PQC machinery in recognizing faulty polypeptides is becoming increasingly understood. Molecular chaperones and cytosolic and vacuolar degradation systems collaborate to detect, repair, or hydrolyze mutant, damaged, and mislocalized proteins. On the other hand, the contribution of PQC to cofactor bindingrelated enzyme maturation remains largely unexplored, although the loading of a cofactor represents an all-or-nothing transition in regard to the enzymatic function and thus must be surveyed carefully. Combining proteomics and biochemical analysis, we demonstrate here that cells are able to detect functionally immature wild-type enzymes. We show that PQC-dedicated ubiquitin ligase C-terminal Hsp70-interacting protein (CHIP) recognizes and marks for degradation not only a mutant protein but also its wild-type variant as long as the latter remains cofactor free. A distinct structural feature, the protruding C-terminal tail, which appears in both the mutant and wild-type polypeptides, contributes to recognition by CHIP. Our data suggest that relative insufficiency of apoprotein degradation caused by cofactor shortage can increase amyloidogenesis and aggravate protein aggregation disorders.apoprotein | ubiquitin ligase | protein aggregation

Targeting HIF-1α Function in Cancer through the Chaperone Action of NQO1

“…Thus effects on the intracellular levels of NQO1 or in the strength of its interaction with partners may translate into altered stability of the nodes in the interactomic network. For instance, reducing the availability of the FAD precursor riboflavin leads to increased population of the flavin-free NQO1apo state, which is very sensitive to proteasomal degradation through ubiquitin-dependent and -independent mechanisms [115,116]. This is in part caused by the destabilization of the CTD of NQO1 in the absence of bound FAD that promotes its ubiquitination and degradation [116].…”

“…For instance, reducing the availability of the FAD precursor riboflavin leads to increased population of the flavin-free NQO1apo state, which is very sensitive to proteasomal degradation through ubiquitin-dependent and -independent mechanisms [115,116]. This is in part caused by the destabilization of the CTD of NQO1 in the absence of bound FAD that promotes its ubiquitination and degradation [116]. Remarkably, addition of the inhibitor dicoumarol does not stabilize the NQO1 protein intracellularly but seems to reduce its ability to interact with and stabilize these protein partners (see Table A2).…”

“…Although no high resolution structural model is available for this state, recent kinetic studies using hydrogen-deuterium exchange mass spectrometry (HDXMS) have identified a minimal stable core that holds the protein dimer while most of the protein exists forming a highly dynamic structural ensemble, including the FAD and substrate binding sites in non-competent states for binding [129]. This remarkable conformational flexibility makes NQO1apo likely the most relevant state to understand the intracellular stability of NQO1, since the flexible CTD acts as initiation site for rapid degradation through ubiquitin-dependent and -independent proteasomal pathways [90,116]. It is plausible that this unstable and highly dynamic NQO1apo state is not capable of interacting with NQO1 protein partners, or at least, does so with lower affinity [78].…”

“…This NQO1holo state is amenable for high-resolution structural studies [81]( Figure 6A). This state is also known to be intracellularly stable and likely to interact with protein partners quite efficiently [115,116,117,119,123].…”

“…The association of NQO1 activity with several human diseases with a huge social impact, particularly cancer, HIV infection and neurological and cardiovascular diseases, has attracted the attention towards the effects of naturally-occurring single amino acid changes on NQO1 and the predisposition provided by these changes towards disease [76,79,116,119,122,123,130,133,136,137,138,139,140,141]. There are 106 missense variants described in human population (ExAC and gnomAD databases; https://gnomad.broadinstitute.org/gene/ENSG00000181019?dataset=gnomad_r2_1) and 47 missense variants in the COSMIC database (https://cancer.sanger.ac.uk/cosmic/gene/analysis?ln=NQO1#variants).…”