Destabilization and Degradation of a Disease-Linked PGM1 Protein Variant.

PGM1-linked congenital disorder of glycosylation (PGM1-CDG) is an autosomal recessive disease characterized by several phenotypes, some of which are life-threatening. Research focusing on the disease-related variants of the α-D-phosphoglucomutase 1 (PGM1) protein has shown that several are insoluble in vitro and expressed at low levels in patient fibroblasts. Due to these observations, we hypothesized that some disease-linked PGM1 protein variants are structurally destabilized and subject to protein quality control (PQC) and rapid intracellular degradation. Employing yeast-based assays, we show that a disease-associated human variant, PGM1 L516P, is insoluble, inactive, and highly susceptible to ubiquitylation and rapid degradation by the proteasome. In addition, we show that PGM1 L516P forms aggregates in S. cerevisiae and that both the aggregation pattern and the abundance of PGM1 L516P are chaperone-dependent. Finally, using computational methods, we perform saturation mutagenesis to assess the impact of all possible single residue substitutions in the PGM1 protein. These analyses identify numerous missense variants with predicted detrimental effects on protein function and stability. We suggest that many disease-linked PGM1 variants are subject to PQC-linked degradation and that our in silico site-saturated data set may assist in the mechanistic interpretation of PGM1 variants.

Deep mutational scanning reveals a correlation between degradation and toxicity of thousands of aspartoacylase variants.

Unstable proteins are prone to form non-native interactions with other proteins and thereby may become toxic. To mitigate this, destabilized proteins are targeted by the protein quality control network. Here we present systematic studies of the cytosolic aspartoacylase, ASPA, where variants are linked to Canavan disease, a lethal neurological disorder. We determine the abundance of 6152 of the 6260 ( ~ 98%) possible single amino acid substitutions and nonsense ASPA variants in human cells. Most low abundance variants are degraded through the ubiquitin-proteasome pathway and become toxic upon prolonged expression. The data correlates with predicted changes in thermodynamic stability, evolutionary conservation, and separate disease-linked variants from benign variants. Mapping of degradation signals (degrons) shows that these are often buried and the C-terminal region functions as a degron. The data can be used to interpret Canavan disease variants and provide insight into the relationship between protein stability, degradation and cell fitness.

PRKN-linked familial Parkinson's disease: cellular and molecular mechanisms of disease-linked variants.

Parkinson's disease (PD) is a common and incurable neurodegenerative disorder that arises from the loss of dopaminergic neurons in the substantia nigra and is mainly characterized by progressive loss of motor function. Monogenic familial PD is associated with highly penetrant variants in specific genes, notably the PRKN gene, where homozygous or compound heterozygous loss-of-function variants predominate. PRKN encodes Parkin, an E3 ubiquitin-protein ligase important for protein ubiquitination and mitophagy of damaged mitochondria. Accordingly, Parkin plays a central role in mitochondrial quality control but is itself also subject to a strict protein quality control system that rapidly eliminates certain disease-linked Parkin variants. Here, we summarize the cellular and molecular functions of Parkin, highlighting the various mechanisms by which PRKN gene variants result in loss-of-function. We emphasize the importance of high-throughput assays and computational tools for the clinical classification of PRKN gene variants and how detailed insights into the pathogenic mechanisms of PRKN gene variants may impact the development of personalized therapeutics.

Characterizing glucokinase variant mechanisms using a multiplexed abundance assay.

BACKGROUND: Amino acid substitutions can perturb protein activity in multiple ways. Understanding their mechanistic basis may pinpoint how residues contribute to protein function. Here, we characterize the mechanisms underlying variant effects in human glucokinase (GCK) variants, building on our previous comprehensive study on GCK variant activity. RESULTS: Using a yeast growth-based assay, we score the abundance of 95% of GCK missense and nonsense variants. When combining the abundance scores with our previously determined activity scores, we find that 43% of hypoactive variants also decrease cellular protein abundance. The low-abundance variants are enriched in the large domain, while residues in the small domain are tolerant to mutations with respect to abundance. Instead, many variants in the small domain perturb GCK conformational dynamics which are essential for appropriate activity. CONCLUSIONS: In this study, we identify residues important for GCK metabolic stability and conformational dynamics. These residues could be targeted to modulate GCK activity, and thereby affect glucose homeostasis.

Cellular and molecular mechanisms of aspartoacylase and its role in Canavan disease.

Canavan disease is an autosomal recessive and lethal neurological disorder, characterized by the spongy degeneration of the white matter in the brain. The disease is caused by a deficiency of the cytosolic aspartoacylase (ASPA) enzyme, which catalyzes the hydrolysis of N-acetyl-aspartate (NAA), an abundant brain metabolite, into aspartate and acetate. On the physiological level, the mechanism of pathogenicity remains somewhat obscure, with multiple, not mutually exclusive, suggested hypotheses. At the molecular level, recent studies have shown that most disease linked ASPA gene variants lead to a structural destabilization and subsequent proteasomal degradation of the ASPA protein variants, and accordingly Canavan disease should in general be considered a protein misfolding disorder. Here, we comprehensively summarize the molecular and cell biology of ASPA, with a particular focus on disease-linked gene variants and the pathophysiology of Canavan disease. We highlight the importance of high-throughput technologies and computational prediction tools for making genotype-phenotype predictions as we await the results of ongoing trials with gene therapy for Canavan disease.

A mutational atlas for Parkin proteostasis.

Proteostasis can be disturbed by mutations affecting folding and stability of the encoded protein. An example is the ubiquitin ligase Parkin, where gene variants result in autosomal recessive Parkinsonism. To uncover the pathological mechanism and provide comprehensive genotype-phenotype information, variant abundance by massively parallel sequencing (VAMP-seq) is leveraged to quantify the abundance of Parkin variants in cultured human cells. The resulting mutational map, covering 9219 out of the 9300 possible single-site amino acid substitutions and nonsense Parkin variants, shows that most low abundance variants are proteasome targets and are located within the structured domains of the protein. Half of the known disease-linked variants are found at low abundance. Systematic mapping of degradation signals (degrons) reveals an exposed degron region proximal to the so-called "activation element". This work provides examples of how missense variants may cause degradation either via destabilization of the native protein, or by introducing local signals for degradation.

Discovering functionally important sites in proteins.

Proteins play important roles in biology, biotechnology and pharmacology, and missense variants are a common cause of disease. Discovering functionally important sites in proteins is a central but difficult problem because of the lack of large, systematic data sets. Sequence conservation can highlight residues that are functionally important but is often convoluted with a signal for preserving structural stability. We here present a machine learning method to predict functional sites by combining statistical models for protein sequences with biophysical models of stability. We train the model using multiplexed experimental data on variant effects and validate it broadly. We show how the model can be used to discover active sites, as well as regulatory and binding sites. We illustrate the utility of the model by prospective prediction and subsequent experimental validation on the functional consequences of missense variants in HPRT1 which may cause Lesch-Nyhan syndrome, and pinpoint the molecular mechanisms by which they cause disease.

Phosphorylation of Schizosaccharomyces pombe Dss1 mediates direct binding to the ubiquitin-ligase Dma1 in vitro.

Intrinsically disordered proteins (IDPs) are often multifunctional and frequently posttranslationally modified. Deleted in split hand/split foot 1 (Dss1-Sem1 in budding yeast) is a highly multifunctional IDP associated with a range of protein complexes. However, it remains unknown if the different functions relate to different modified states. In this work, we show that Schizosaccharomyces pombe Dss1 is a substrate for casein kinase 2 in vitro, and we identify three phosphorylated threonines in its linker region separating two known disordered ubiquitin-binding motifs. Phosphorylations of the threonines had no effect on ubiquitin-binding but caused a slight destabilization of the C-terminal α-helix and mediated a direct interaction with the forkhead-associated (FHA) domain of the RING-FHA E3-ubiquitin ligase defective in mitosis 1 (Dma1). The phosphorylation sites are not conserved and are absent in human Dss1. Sequence analyses revealed that the Txx(E/D) motif, which is important for phosphorylation and Dma1 binding, is not linked to certain branches of the evolutionary tree. Instead, we find that the motif appears randomly, supporting the mechanism of ex nihilo evolution of novel motifs. In support of this, other threonine-based motifs, although frequent, are nonconserved in the linker, pointing to additional functions connected to this region. We suggest that Dss1 acts as an adaptor protein that docks to Dma1 via the phosphorylated FHA-binding motifs, while the C-terminal α-helix is free to bind mitotic septins, thereby stabilizing the complex. The presence of Txx(D/E) motifs in the disordered regions of certain septin subunits may be of further relevance to the formation and stabilization of these complexes.

Characterizing glucokinase variant mechanisms using a multiplexed abundance assay.

Amino acid substitutions can perturb protein activity in multiple ways. Understanding their mechanistic basis may pinpoint how residues contribute to protein function. Here, we characterize the mechanisms of human glucokinase (GCK) variants, building on our previous comprehensive study on GCK variant activity. We assayed the abundance of 95% of GCK missense and nonsense variants, and found that 43% of hypoactive variants have a decreased cellular abundance. By combining our abundance scores with predictions of protein thermodynamic stability, we identify residues important for GCK metabolic stability and conformational dynamics. These residues could be targeted to modulate GCK activity, and thereby affect glucose homeostasis.

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.

A comprehensive map of human glucokinase variant activity.

BACKGROUND: Glucokinase (GCK) regulates insulin secretion to maintain appropriate blood glucose levels. Sequence variants can alter GCK activity to cause hyperinsulinemic hypoglycemia or hyperglycemia associated with GCK-maturity-onset diabetes of the young (GCK-MODY), collectively affecting up to 10 million people worldwide. Patients with GCK-MODY are frequently misdiagnosed and treated unnecessarily. Genetic testing can prevent this but is hampered by the challenge of interpreting novel missense variants. RESULT: Here, we exploit a multiplexed yeast complementation assay to measure both hyper- and hypoactive GCK variation, capturing 97% of all possible missense and nonsense variants. Activity scores correlate with in vitro catalytic efficiency, fasting glucose levels in carriers of GCK variants and with evolutionary conservation. Hypoactive variants are concentrated at buried positions, near the active site, and at a region of known importance for GCK conformational dynamics. Some hyperactive variants shift the conformational equilibrium towards the active state through a relative destabilization of the inactive conformation. CONCLUSION: Our comprehensive assessment of GCK variant activity promises to facilitate variant interpretation and diagnosis, expand our mechanistic understanding of hyperactive variants, and inform development of therapeutics targeting GCK.

Rare Catechol-O-methyltransferase Missense Variants Are Structurally Unstable Proteasome Targets.

Catechol-O-methyltransferase (COMT) is a key enzyme in the metabolism of catecholamines. Substrates of the enzyme include neurotransmitters such as dopamine and epinephrine, and therefore, COMT plays a central role in neurobiology. Since COMT also metabolizes catecholamine drugs such as L-DOPA, variation in COMT activity could affect pharmacokinetics and drug availability. Certain COMT missense variants have been shown to display decreased enzymatic activity. Additionally, studies have shown that such missense variants may lead to loss of function induced by impaired structural stability, which results in activation of the protein quality control system and degradation by the ubiquitin-proteasome system. Here, we demonstrate that two rare missense variants of COMT are ubiquitylated and targeted for proteasomal degradation as a result of structural destabilization and misfolding. This results in strongly reduced intracellular steady-state levels of the enzyme, which for the L135P variant is rescued upon binding to the COMT inhibitors entacapone and tolcapone. Our results reveal that the degradation is independent of the COMT isoform as both soluble (S-COMT) and ER membrane-bound (MB-COMT) variants are degraded. In silico structural stability predictions identify regions within the protein that are critical for stability overlapping with evolutionarily conserved residues, pointing toward other variants that are likely destabilized and degraded.

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.

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.

Lynch syndrome, molecular mechanisms and variant classification.

Patients with the heritable cancer disease, Lynch syndrome, carry germline variants in the MLH1, MSH2, MSH6 and PMS2 genes, encoding the central components of the DNA mismatch repair system. Loss-of-function variants disrupt the DNA mismatch repair system and give rise to a detrimental increase in the cellular mutational burden and cancer development. The treatment prospects for Lynch syndrome rely heavily on early diagnosis; however, accurate diagnosis is inextricably linked to correct clinical interpretation of individual variants. Protein variant classification traditionally relies on cumulative information from occurrence in patients, as well as experimental testing of the individual variants. The complexity of variant classification is due to (1) that variants of unknown significance are rare in the population and phenotypic information on the specific variants is missing, and (2) that individual variant testing is challenging, costly and slow. Here, we summarise recent developments in high-throughput technologies and computational prediction tools for the assessment of variants of unknown significance in Lynch syndrome. These approaches may vastly increase the number of interpretable variants and could also provide important mechanistic insights into the disease. These insights may in turn pave the road towards developing personalised treatment approaches for Lynch syndrome.

Prediction of Quality-control Degradation Signals in Yeast Proteins.

Effective proteome homeostasis is key to cellular and organismal survival, and cells therefore contain efficient quality control systems to monitor and remove potentially toxic misfolded proteins. Such general protein quality control to a large extent relies on the efficient and robust delivery of misfolded or unfolded proteins to the ubiquitin-proteasome system. This is achieved via recognition of so-called degradation motifs-degrons-that are assumed to become exposed as a result of protein misfolding. Despite their importance, the nature and sequence properties of quality-control degrons remain elusive. Here, we have used data from a yeast-based screen of 23,600 17-residue peptides to build a predictor of quality-control degrons. The resulting model, QCDPred (Quality Control Degron Prediction), achieves good accuracy using only the sequence composition of the peptides as input. Our analysis reveals that strong degrons are enriched in hydrophobic amino acids and depleted in negatively charged amino acids, in line with the expectation that they are buried in natively folded proteins. We applied QCDPred to the yeast proteome, enabling us to analyse more widely the potential effects of degrons. As an example, we show a correlation between cellular abundance and degron potential in disordered regions of proteins. Together with recent results on membrane proteins, our work suggest that the recognition of exposed hydrophobic residues is a key and generic mechanism for proteome homeostasis. QCDPred is freely available as open source code and via a web interface.

Conserved degronome features governing quality control associated proteolysis.

The eukaryotic proteome undergoes constant surveillance by quality control systems that either sequester, refold, or eliminate aberrant proteins by ubiquitin-dependent mechanisms. Ubiquitin-conjugation necessitates the recognition of degradation determinants, termed degrons, by their cognate E3 ubiquitin-protein ligases. To learn about the distinctive properties of quality control degrons, we performed an unbiased peptidome stability screen in yeast. The search identify a large cohort of proteome-derived degrons, some of which exhibited broad E3 ligase specificity. Consequent application of a machine-learning algorithm establishes constraints governing degron potency, including the amino acid composition and secondary structure propensities. According to the set criteria, degrons with transmembrane domain-like characteristics are the most probable sequences to act as degrons. Similar quality control degrons are present in viral and human proteins, suggesting conserved degradation mechanisms. Altogether, the emerging data indicate that transmembrane domain-like degron features have been preserved in evolution as key quality control determinants of protein half-life.

14-fold increased prevalence of rare glucokinase gene variant carriers in unselected Danish patients with newly diagnosed type 2 diabetes.

AIMS: Rare variants in the glucokinase gene (GCK) cause Maturity-Onset Diabetes of the Young (MODY2/GCK-MODY). We investigated the prevalence of GCK variants, phenotypic characteristics, micro- and macrovascular disease at baseline and follow-up, and treatment among individuals with and without pathogenic GCK variants. METHODS: This is a cross-sectional study in a population-based cohort of 5,433 individuals without diabetes (Inter99 cohort) and in 2,855 patients with a new clinical diagnosis of type 2 diabetes (DD2 cohort) with sequencing of GCK. Phenotypic characteristics, presence of micro- and macrovascular disease and treatment information were available for patients in the DD2 cohort at baseline and after an average follow-up of 7.4 years. RESULTS: Twenty-two carriers of potentially deleterious GCK variants were found among patients with type 2 diabetes compared to three among 5,433 nondiabetic individuals [OR = 14.1 (95 % CI 4.2; 47.0), p = 8.9*10-6]. Patients with type 2 diabetes carrying GCK variants had significantly lower waist circumference, hip circumference and BMI, compared to non-carriers. Three GCK variant carriers with diabetes had microvascular complications during follow-up. CONCLUSIONS: Approximately 0.8% of Danish patients with newly diagnosed type 2 diabetes carry non-synonymous variants in GCK and resemble patients with GCK-MODY. Glucose-lowering treatment cessation should be considered in this subset of diabetes patients.

How phosphorylation impacts intrinsically disordered proteins and their function.

Phosphorylation is the most common post-translational modification (PTM) in eukaryotes, occurring particularly frequently in intrinsically disordered proteins (IDPs). These proteins are highly flexible and dynamic by nature. Thus, it is intriguing that the addition of a single phosphoryl group to a disordered chain can impact its function so dramatically. Furthermore, as many IDPs carry multiple phosphorylation sites, the number of possible states increases, enabling larger complexities and novel mechanisms. Although a chemically simple and well-understood process, the impact of phosphorylation on the conformational ensemble and molecular function of IDPs, not to mention biological output, is highly complex and diverse. Since the discovery of the first phosphorylation site in proteins 75 years ago, we have come to a much better understanding of how this PTM works, but with the diversity of IDPs and their capacity for carrying multiple phosphoryl groups, the complexity grows. In this Essay, we highlight some of the basic effects of IDP phosphorylation, allowing it to serve as starting point when embarking on studies into this topic. We further describe how recent complex cases of multisite phosphorylation of IDPs have been instrumental in widening our view on the effect of protein phosphorylation. Finally, we put forward perspectives on the phosphorylation of IDPs, both in relation to disease and in context of other PTMs; areas where deep insight remains to be uncovered.

Deciphering the Alphabet of Disorder-Glu and Asp Act Differently on Local but Not Global Properties.

Compared to folded proteins, the sequences of intrinsically disordered proteins (IDPs) are enriched in polar and charged amino acids. Glutamate is one of the most enriched amino acids in IDPs, while the chemically similar amino acid aspartate is less enriched. So far, the underlying functional differences between glutamates and aspartates in IDPs remain poorly understood. In this study, we examine the differential effects of aspartate and glutamates in IDPs by comparing the function and conformational ensemble of glutamate and aspartate variants of the disordered protein Dss1, using a range of assays, including interaction studies, nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and molecular dynamics simulation. First, we analyze the sequences of the rapidly growing database of experimentally verified IDPs (DisProt) and show that glutamate enrichment is not caused by a taxonomy bias in IDPs. From analyses of local and global structural properties as well as cell growth and protein-protein interactions using a model acidic IDP from yeast and three Glu/Asp variants, we find that while the Glu/Asp variants support similar function and global dimensions, the variants differ in their binding affinities and population of local transient structural elements. We speculate that these local structural differences may play roles in functional diversity, where glutamates can support increased helicity, important for folding and binding, while aspartates support extended structures and form helical caps, as well as playing more relevant roles in, e.g., transactivation domains and ion-binding.