Defective mismatch repair leads to increased mutation rates, and germline loss-of-function variants in the repair component MLH1 cause the hereditary cancer predisposition disorder known as Lynch syndrome. Early diagnosis is important, but complicated by many variants being of unknown significance. Here we show that a majority of the disease-linked MLH1 variants we studied are present at reduced cellular levels. We show that destabilized MLH1 variants are targeted for chaperone-assisted proteasomal degradation, resulting also in degradation of co-factors PMS1 and PMS2. In silico saturation mutagenesis and computational predictions of thermodynamic stability of MLH1 missense variants revealed a correlation between structural destabilization, reduced steady-state levels and loss-of-function. Thus, we suggest that loss of stability and cellular degradation is an important mechanism underlying many MLH1 variants in Lynch syndrome. Combined with analyses of conservation, the thermodynamic stability predictions separate disease-linked from benign MLH1 variants, and therefore hold potential for Lynch syndrome diagnostics.

25Defective mismatch repair leads to increased mutation rates, and germline loss-of-function variants 26 in the repair component MLH1 cause the hereditary cancer predisposition disorder known as Lynch 27 syndrome. Early diagnosis is important, but complicated by many variants being of unknown 28 significance. Here we show that a majority of the disease-linked MLH1 variants we studied are 29 present at reduced cellular levels. We show that destabilized MLH1 variants are targeted for 30 chaperone-assisted proteasomal degradation, resulting also in degradation of co-factors PMS1 and 31 PMS2. In silico saturation mutagenesis and computational predictions of thermodynamic stability of 32 MLH1 missense variants revealed a correlation between structural destabilization, reduced steady-33 state levels and loss-of-function. Thus, we suggest that loss of stability and cellular degradation is an 34 important mechanism underlying many MLH1 variants in Lynch syndrome. Combined with analyses 35 of conservation, the thermodynamic stability predictions separate disease-linked from benign MLH1 36 variants, and therefore hold potential for Lynch syndrome diagnostics. 37 38 39 77 mechanistic origin for many LS-causing MLH1 missense variants our studies provide a starting point 78 for development of novel therapies. 79 80 81 5 Results 82 83 In silico saturation mutagenesis and thermodynamic stability predictions 84 Most missense proteins are less structurally stable than the wild-type protein (Tokuriki & Tawfik, 85 2009), and individual missense variants may thus lead to increased degradation and insufficient 86 amounts of protein. To comprehensively assess this effect for MLH1, we performed energy 87 calculations based on crystal structures of MLH1 to predict the consequences of missense mutations 88 in MLH1 on the thermodynamic stability of the MLH1 protein structure. Full-length human MLH1 89 is a 756 residue protein which forms two folded units, an N-terminal domain (residues 7-315) and a 90 C-terminal domain (residues 502-756) (Mitchell et al., 2019) separated by a flexible and intrinsically 91 disordered linker (Fig. 1A). Using the structures (Wu et al., 2015) of the two domains (PDB IDs 4P7A 92 and 3RBN) (Fig. 1A), we performed in silico saturation mutagenesis, introducing all possible single 93 site amino acid substitutions into the wild-type MLH1 sequence at the 564 structurally resolved 94 residues. We then applied the FoldX energy function (Schymkowitz et al., 2005) to estimate the 95 change in thermodynamic folding stability compared to the wild-type MLH1 protein (ΔΔG) (Fig. 96 1BC). Negative values indicate mutations that are predicted to stabilize MLH1, while positive values 97 indicate that the mutations may destabilize the protein. Thus, those variants with ΔΔG predictions > 98 0 kcal/mol are expected to have a larger population of fully or partially unfolded structures that, in 99 turn, may be prone to protein quality control (PQC)-mediated degradation. Our saturation 100 mutagenesis dataset comprises 19 (amino a...

Protein homeostasis (proteostasis) is essential for the cell and is maintained by a highly conserved protein quality control (PQC) system, which triages newly synthesized, mislocalized and misfolded proteins. The ubiquitin-proteasome system (UPS), molecular chaperones, and co-chaperones are vital PQC elements that work together to facilitate degradation of misfolded and toxic protein species through the 26S proteasome. However, the underlying mechanisms are complex and remain partly unclear. Here, we provide an overview of the current knowledge on the co-chaperones that directly take part in targeting and delivery of PQC substrates for degradation. While J-domain proteins (JDPs) target substrates for the heat shock protein 70 (HSP70) chaperones, nucleotide-exchange factors (NEFs) deliver HSP70-bound substrates to the proteasome. So far, three NEFs have been established in proteasomal delivery: HSP110 and the ubiquitin-like (UBL) domain proteins BAG-1 and BAG-6, the latter acting as a chaperone itself and carrying its substrates directly to the proteasome. A better understanding of the individual delivery pathways will improve our ability to regulate the triage, and thus regulate the fate of aberrant proteins involved in cell stress and disease, examples of which are given throughout the review.

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Exocytosis involves fusion of secretory vesicles with the plasma membrane, thereby delivering membrane proteins to the cell surface and releasing material into the extracellular space. The tethering of the secretory vesicles before membrane fusion is mediated by the exocyst, an essential phylogenetically conserved octameric protein complex. Exocyst biogenesis is regulated by several processes, but the mechanisms by which the exocyst is degraded are unknown. Here, to unravel the components of the exocyst degradation pathway, we screened for extragenic suppressors of a temperature-sensitive fission yeast strain mutated in the exocyst subunit Sec3 (). One of the suppressing DNAs encoded a truncated dominant-negative variant of the 26S proteasome subunit, Rpt2, indicating that exocyst degradation is controlled by the ubiquitin-proteasome system. The temperature-dependent growth defect of the strain was gene dosage-dependent and suppressed by blocking the proteasome, Hsp70-type molecular chaperones, the Pib1 E3 ubiquitin-protein ligase, and the deubiquitylating enzyme Ubp3. Moreover, defects in cell septation, exocytosis, and endocytosis in mutant strains were similarly alleviated by mutation of components in this pathway. We also found that, particularly under stress conditions, wild-type Sec3 degradation is regulated by Pib1 and the 26S proteasome. In conclusion, our results suggest that a cytosolic protein quality control pathway monitors folding and proteasome-dependent turnover of an exocyst subunit and, thereby, controls exocytosis in fission yeast.