Lysine deserts prevent adventitious ubiquitylation of ubiquitin-proteasome components.

In terms of its relative frequency, lysine is a common amino acid in the human proteome. However, by bioinformatics we find hundreds of proteins that contain long and evolutionarily conserved stretches completely devoid of lysine residues. These so-called lysine deserts show a high prevalence in intrinsically disordered proteins with known or predicted functions within the ubiquitin-proteasome system (UPS), including many E3 ubiquitin-protein ligases and UBL domain proteasome substrate shuttles, such as BAG6, RAD23A, UBQLN1 and UBQLN2. We show that introduction of lysine residues into the deserts leads to a striking increase in ubiquitylation of some of these proteins. In case of BAG6, we show that ubiquitylation is catalyzed by the E3 RNF126, while RAD23A is ubiquitylated by E6AP. Despite the elevated ubiquitylation, mutant RAD23A appears stable, but displays a partial loss of function phenotype in fission yeast. In case of UBQLN1 and BAG6, introducing lysine leads to a reduced abundance due to proteasomal degradation of the proteins. For UBQLN1 we show that arginine residues within the lysine depleted region are critical for its ability to form cytosolic speckles/inclusions. We propose that selective pressure to avoid lysine residues may be a common evolutionary mechanism to prevent unwarranted ubiquitylation and/or perhaps other lysine post-translational modifications. This may be particularly relevant for UPS components as they closely and frequently encounter the ubiquitylation machinery and are thus more susceptible to nonspecific ubiquitylation.

HSP70-binding motifs function as protein quality control degrons.

Protein quality control (PQC) degrons are short protein segments that target misfolded proteins for proteasomal degradation, and thus protect cells against the accumulation of potentially toxic non-native proteins. Studies have shown that PQC degrons are hydrophobic and rarely contain negatively charged residues, features which are shared with chaperone-binding regions. Here we explore the notion that chaperone-binding regions may function as PQC degrons. When directly tested, we found that a canonical Hsp70-binding motif (the APPY peptide) functioned as a dose-dependent PQC degron both in yeast and in human cells. In yeast, Hsp70, Hsp110, Fes1, and the E3 Ubr1 target the APPY degron. Screening revealed that the sequence space within the chaperone-binding region of APPY that is compatible with degron function is vast. We find that the number of exposed Hsp70-binding sites in the yeast proteome correlates with a reduced protein abundance and half-life. Our results suggest that when protein folding fails, chaperone-binding sites may operate as PQC degrons, and that the sequence properties leading to PQC-linked degradation therefore overlap with those of chaperone binding.

Mapping the degradation pathway of a disease-linked aspartoacylase variant.

Canavan disease is a severe progressive neurodegenerative disorder that is characterized by swelling and spongy degeneration of brain white matter. The disease is genetically linked to polymorphisms in the aspartoacylase (ASPA) gene, including the substitution C152W. ASPA C152W is associated with greatly reduced protein levels in cells, yet biophysical experiments suggest a wild-type like thermal stability. Here, we use ASPA C152W as a model to investigate the degradation pathway of a disease-causing protein variant. When we expressed ASPA C152W in Saccharomyces cerevisiae, we found a decreased steady state compared to wild-type ASPA as a result of increased proteasomal degradation. However, molecular dynamics simulations of ASPA C152W did not substantially deviate from wild-type ASPA, indicating that the native state is structurally preserved. Instead, we suggest that the C152W substitution interferes with the de novo folding pathway resulting in increased proteasomal degradation before reaching its stable conformation. Systematic mapping of the protein quality control components acting on misfolded and aggregation-prone species of C152W, revealed that the degradation is highly dependent on the molecular chaperone Hsp70, its co-chaperone Hsp110 as well as several quality control E3 ubiquitin-protein ligases, including Ubr1. In addition, the disaggregase Hsp104 facilitated refolding of aggregated ASPA C152W, while Cdc48 mediated degradation of insoluble ASPA protein. In human cells, ASPA C152W displayed increased proteasomal turnover that was similarly dependent on Hsp70 and Hsp110. Our findings underscore the use of yeast to determine the protein quality control components involved in the degradation of human pathogenic variants in order to identify potential therapeutic targets.

Random Mutagenesis Analysis of the Influenza A M2 Proton Channel Reveals Novel Resistance Mutants.

The influenza M2 proton channel is a major drug target, but unfortunately, the acquisition of resistance mutations greatly reduces the functional life span of a drug in influenza treatment. New M2 inhibitors that inhibit mutant M2 channels otherwise resistant to the early adamantine-based drugs have been reported, but it remains unclear whether and how easy resistance could arise to such inhibitors. We have combined a newly developed proton conduction assay with an established method for selection and screening, both Escherichia coli-based, to enable the study of M2 function and inhibition. Combining this platform with two groups of structurally different M2 inhibitors allowed us to isolate drug resistant M2 channels from a mutant library. Two groups of M2 variants emerged from this analysis. A first group appeared almost unaffected by the inhibitor, M_089 (N13I, I35L, and F47L) and M_272 (G16C and D44H), and the single-substitution variants derived from these (I35L, L43P, D44H, and L46P). Functionally, these resemble the known drug resistant M2 channels V27A, S31N, and swine flu. In addition, a second group of tested M2 variants were all still inhibited by drugs but to a lesser extent than wild type M2. Molecular dynamics simulations aided in distinguishing the two groups where drug binding to the wild type and the less resistant M2 group showed a stable positioning of the ligand in the canonical binding pose, as opposed to the drug resistant group in which the ligand rapidly dissociated from the complex during the simulations.

The exocyst subunit Sec3 is regulated by a protein quality control pathway.

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 (sec3-913). 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 sec3-913 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 sec3 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.

Blocking protein quality control to counter hereditary cancers.

Inhibitors of molecular chaperones and the ubiquitin-proteasome system have already been clinically implemented to counter certain cancers, including multiple myeloma and mantle cell lymphoma. The efficacy of this treatment relies on genomic alterations in cancer cells causing a proteostatic imbalance, which makes them more dependent on protein quality control (PQC) mechanisms than normal cells. Accordingly, blocking PQC, e.g. by proteasome inhibitors, may cause a lethal proteotoxic crisis in cancer cells, while leaving normal cells unaffected. Evidence, however, suggests that the PQC system operates by following a better-safe-than-sorry principle and is thus prone to target proteins that are only slightly structurally perturbed, but still functional. Accordingly, implementing PQC inhibitors may also, through an entirely different mechanism, hold potential for other cancers. Several inherited cancer susceptibility syndromes, such as Lynch syndrome and von Hippel-Lindau disease, are caused by missense mutations in tumor suppressor genes, and in some cases, the resulting amino acid substitutions in the encoded proteins cause the cellular PQC system to target them for degradation, although they may still retain function. As a consequence of this over-meticulous PQC mechanism, the cell may end up with an insufficient amount of the abnormal, but functional, protein, which in turn leads to a loss-of-function phenotype and manifestation of the disease. Increasing the amounts of such proteins by stabilizing with chemical chaperones, or by targeting molecular chaperones or the ubiquitin-proteasome system, may thus avert or delay the disease onset. Here, we review the potential of targeting the PQC system in hereditary cancer susceptibility syndromes.

The moonlighting of RAD23 in DNA repair and protein degradation.

A moonlighting protein is one, which carries out multiple, often wholly unrelated, functions. The RAD23 protein is a fascinating example of this, where the same polypeptide and the embedded domains function independently in both nucleotide excision repair (NER) and protein degradation via the ubiquitin-proteasome system (UPS). Hence, through direct binding to the central NER component XPC, RAD23 stabilizes XPC and contributes to DNA damage recognition. Conversely, RAD23 also interacts directly with the 26S proteasome and ubiquitylated substrates to mediate proteasomal substrate recognition. In this function, RAD23 activates the proteolytic activity of the proteasome and engages specifically in well-characterized degradation pathways through direct interactions with E3 ubiquitin-protein ligases and other UPS components. Here, we summarize the past 40 years of research into the roles of RAD23 in NER and the UPS.

Disease-linked mutations cause exposure of a protein quality control degron.

More than half of disease-causing missense variants are thought to lead to protein degradation, but the molecular mechanism of how these variants are recognized by the cell remains enigmatic. Degrons are stretches of amino acids that help mediate recognition by E3 ligases and thus confer protein degradation via the ubiquitin-proteasome system. While degrons that mediate controlled degradation of, for example, signaling components and cell-cycle regulators are well described, so-called protein-quality-control degrons that mediate the degradation of destabilized proteins are poorly understood. Here, we show that disease-linked dihydrofolate reductase (DHFR) missense variants are structurally destabilized and chaperone-dependent proteasome targets. We find two regions in DHFR that act as degrons, and the proteasomal turnover of one of these was dependent on the molecular chaperone Hsp70. Structural analyses by nuclear magnetic resonance (NMR) and hydrogen/deuterium exchange revealed that this degron is buried in wild-type DHFR but becomes transiently exposed in the disease-linked missense variants.

Mutations in a Single Signaling Pathway Allow Cell Growth in Heavy Water.

Life is completely dependent on water. To analyze the role of water as a solvent in biology, we replaced water with heavy water (D2O) and investigated the biological effects by a wide range of techniques, using Schizosaccharomyces pombe as model organism. We show that high concentrations of D2O lead to altered glucose metabolism and growth retardation. After prolonged incubation in D2O, cells displayed gross morphological changes, thickened cell walls, and aberrant cytoskeletal organization. By transcriptomics and genetic screens, we show that the solvent replacement activates two signaling pathways: (1) the heat-shock response pathway and (2) the cell integrity pathway. Although the heat-shock response system upregulates various chaperones and other stress-relieving enzymes, we find that the activation of this pathway does not offer any fitness advantage to the cells under the solvent-replaced conditions. However, limiting the D2O-triggered activation of the cell integrity pathway allows cell growth when H2O is completely replaced with D2O. The isolated D2O-tolerant strains may aid biological production of deuterated biomolecules.

UBL/BAG-domain co-chaperones cause cellular stress upon overexpression through constitutive activation of Hsf1.

As a result of exposure to stress conditions, mutations, or defects during synthesis, cellular proteins are prone to misfold. To cope with such partially denatured proteins, cells mount a regulated transcriptional response involving the Hsf1 transcription factor, which drives the synthesis of molecular chaperones and other stress-relieving proteins. Here, we show that the fission yeast Schizosaccharomyces pombe orthologues of human BAG-1, Bag101, and Bag102, are Hsp70 co-chaperones that associate with 26S proteasomes. Only a subgroup of Hsp70-type chaperones, including Ssa1, Ssa2, and Sks2, binds Bag101 and Bag102 and key residues in the Hsp70 ATPase domains, required for interaction with Bag101 and Bag102, were identified. In humans, BAG-1 overexpression is typically observed in cancers. Overexpression of bag101 and bag102 in fission yeast leads to a strong growth defect caused by triggering Hsp70 to release and activate the Hsf1 transcription factor. Accordingly, the bag101-linked growth defect is alleviated in strains containing a reduced amount of Hsf1 but aggravated in hsp70 deletion strains. In conclusion, we propose that the fission yeast UBL/BAG proteins release Hsf1 from Hsp70, leading to constitutive Hsf1 activation and growth defects.