The Eukaryotic Proteome Is Shaped by E3 Ubiquitin Ligases Targeting C-Terminal Degrons.

Degrons are minimal elements that mediate the interaction of proteins with degradation machineries to promote proteolysis. Despite their central role in proteostasis, the number of known degrons remains small, and a facile technology to characterize them is lacking. Using a strategy combining global protein stability (GPS) profiling with a synthetic human peptidome, we identify thousands of peptides containing degron activity. Employing CRISPR screening, we establish that the stability of many proteins is regulated through degrons located at their C terminus. We characterize eight Cullin-RING E3 ubiquitin ligase (CRL) complex adaptors that regulate C-terminal degrons, including six CRL2 and two CRL4 complexes, and computationally implicate multiple non-CRLs in end recognition. Proteome analysis revealed that the C termini of eukaryotic proteins are depleted for C-terminal degrons, suggesting an E3-ligase-dependent modulation of proteome composition. Thus, we propose that a series of "C-end rules" operate to govern protein stability and shape the eukaryotic proteome.

A germline point mutation in the MYC-FBW7 phosphodegron initiates hematopoietic malignancies.

Oncogenic activation of MYC in cancers predominantly involves increased transcription rather than coding region mutations. However, MYC-dependent lymphomas frequently acquire point mutations in the MYC phosphodegron, including at threonine 58 (T58), where phosphorylation permits binding via the FBW7 ubiquitin ligase triggering MYC degradation. To understand how T58 phosphorylation functions in normal cell physiology, we introduced an alanine mutation at T58 (T58A) into the endogenous c-Myc locus in the mouse germline. While MYC-T58A mice develop normally, lymphomas and myeloid leukemias emerge in ∼60% of adult homozygous T58A mice. We found that primitive hematopoietic progenitor cells from MYC-T58A mice exhibit aberrant self-renewal normally associated with hematopoietic stem cells (HSCs) and up-regulate a subset of MYC target genes important in maintaining stem/progenitor cell balance. In lymphocytes, genomic occupancy by MYC-T58A was increased at all promoters compared with WT MYC, while genes differentially expressed in a T58A-dependent manner were significantly more proximal to MYC-bound enhancers. MYC-T58A lymphocyte progenitors exhibited metabolic alterations and decreased activation of inflammatory and apoptotic pathways. Our data demonstrate that a single point mutation stabilizing MYC is sufficient to skew target gene expression, producing a profound gain of function in multipotential hematopoietic progenitors associated with self-renewal and initiation of lymphomas and leukemias.

Ubiquitin-independent proteasomal degradation driven by C-degron pathways.

Although most eukaryotic proteins are targeted for proteasomal degradation by ubiquitination, a subset have been demonstrated to undergo ubiquitin-independent proteasomal degradation (UbInPD). However, little is known about the molecular mechanisms driving UbInPD and the degrons involved. Utilizing the GPS-peptidome approach, a systematic method for degron discovery, we found thousands of sequences that promote UbInPD; thus, UbInPD is more prevalent than currently appreciated. Furthermore, mutagenesis experiments revealed specific C-terminal degrons required for UbInPD. Stability profiling of a genome-wide collection of human open reading frames identified 69 full-length proteins subject to UbInPD. These included REC8 and CDCA4, proteins which control proliferation and survival, as well as mislocalized secretory proteins, suggesting that UbInPD performs both regulatory and protein quality control functions. In the context of full-length proteins, C termini also play a role in promoting UbInPD. Finally, we found that Ubiquilin family proteins mediate the proteasomal targeting of a subset of UbInPD substrates.

Elucidation of E3 ubiquitin ligase specificity through proteome-wide internal degron mapping.

The ubiquitin-proteasome system plays a critical role in biology by regulating protein degradation. Despite their importance, precise recognition specificity is known for a few of the 600 E3s. Here, we establish a two-pronged strategy for identifying and mapping critical residues of internal degrons on a proteome-scale in HEK-293T cells. We employ global protein stability profiling combined with machine learning to identify 15,800 peptides likely to contain sequence-dependent degrons. We combine this with scanning mutagenesis to define critical residues for over 5,000 predicted degrons. Focusing on Cullin-RING ligase degrons, we generated mutational fingerprints for 219 degrons and developed DegronID, a computational algorithm enabling the clustering of degron peptides with similar motifs. CRISPR analysis enabled the discovery of E3-degron pairs, of which we uncovered 16 pairs that revealed extensive degron variability and structural determinants. We provide the visualization of these data on the public DegronID data browser as a resource for future exploration.

Noncanonical mono(ADP-ribosyl)ation of zinc finger SZF proteins counteracts ubiquitination for protein homeostasis in plant immunity.

Protein ADP-ribosylation is a reversible post-translational modification that transfers ADP-ribose from NAD+ onto acceptor proteins. Poly(ADP-ribosyl)ation (PARylation), catalyzed by poly(ADP-ribose) polymerases (PARPs) and poly(ADP-ribose) glycohydrolases (PARGs), which remove the modification, regulates diverse cellular processes. However, the chemistry and physiological functions of mono(ADP-ribosyl)ation (MARylation) remain elusive. Here, we report that Arabidopsis zinc finger proteins SZF1 and SZF2, key regulators of immune gene expression, are MARylated by the noncanonical ADP-ribosyltransferase SRO2. Immune elicitation promotes MARylation of SZF1/SZF2 via dissociation from PARG1, which has an unconventional activity in hydrolyzing both poly(ADP-ribose) and mono(ADP-ribose) from acceptor proteins. MARylation antagonizes polyubiquitination of SZF1 mediated by the SH3 domain-containing proteins SH3P1/SH3P2, thereby stabilizing SZF1 proteins. Our study uncovers a noncanonical ADP-ribosyltransferase mediating MARylation of immune regulators and underpins the molecular mechanism of maintaining protein homeostasis by the counter-regulation of ADP-ribosylation and polyubiquitination to ensure proper immune responses.

Transfer learning to leverage larger datasets for improved prediction of protein stability changes.

Amino acid mutations that lower a protein's thermodynamic stability are implicated in numerous diseases, and engineered proteins with enhanced stability can be important in research and medicine. Computational methods for predicting how mutations perturb protein stability are, therefore, of great interest. Despite recent advancements in protein design using deep learning, in silico prediction of stability changes has remained challenging, in part due to a lack of large, high-quality training datasets for model development. Here, we describe ThermoMPNN, a deep neural network trained to predict stability changes for protein point mutations given an initial structure. In doing so, we demonstrate the utility of a recently released megascale stability dataset for training a robust stability model. We also employ transfer learning to leverage a second, larger dataset by using learned features extracted from ProteinMPNN, a deep neural network trained to predict a protein's amino acid sequence given its three-dimensional structure. We show that our method achieves state-of-the-art performance on established benchmark datasets using a lightweight model architecture that allows for rapid, scalable predictions. Finally, we make ThermoMPNN readily available as a tool for stability prediction and design.

Trimethylamine-N-oxide depletes urea in a peptide solvation shell.

Trimethylamine-N-oxide (TMAO) and urea are metabolites that are used by some marine animals to maintain their cell volume in a saline environment. Urea is a well-known denaturant, and TMAO is a protective osmolyte that counteracts urea-induced protein denaturation. TMAO also has a general protein-protective effect, for example, it counters pressure-induced protein denaturation in deep-sea fish. These opposing effects on protein stability have been linked to the spatial relationship of TMAO, urea, and protein molecules. It is generally accepted that urea-induced denaturation proceeds through the accumulation of urea at the protein surface and their subsequent interaction. In contrast, it has been suggested that TMAO's protein-stabilizing effects stem from its exclusion from the protein surface, and its ability to deplete urea from protein surfaces; however, these spatial relationships are uncertain. We used neutron diffraction, coupled with structural refinement modeling, to study the spatial associations of TMAO and urea with the tripeptide derivative glycine-proline-glycinamide in aqueous urea, aqueous TMAO, and aqueous urea-TMAO (in the mole ratio 1:2 TMAO:urea). We found that TMAO depleted urea from the peptide's surface and that while TMAO was not excluded from the tripeptide's surface, strong atomic interactions between the peptide and TMAO were limited to hydrogen bond donating peptide groups. We found that the repartition of urea, by TMAO, was associated with preferential TMAO-urea bonding and enhanced urea-water hydrogen bonding, thereby anchoring urea in the bulk solution and depleting urea from the peptide surface.

The CRL3KCTD10 ubiquitin ligase-USP18 axis coordinately regulates cystine uptake and ferroptosis by modulating SLC7A11.

SLC7A11 is a cystine transporter and ferroptosis inhibitor. How the stability of SLC7A11 is coordinately regulated in response to environmental cystine by which E3 ligase and deubiquitylase (DUB) remains elusive. Here, we report that neddylation inhibitor MLN4924 increases cystine uptake by causing SLC7A11 accumulation, via inactivating Cullin-RING ligase-3 (CRL-3). We identified KCTD10 as the substrate-recognizing subunit of CRL-3 for SLC7A11 ubiquitylation, and USP18 as SLC7A11 deubiquitylase. Upon cystine deprivation, the protein levels of KCTD10 or USP18 are decreased or increased, respectively, contributing to SLC7A11 accumulation. By destabilizing or stabilizing SLC7A11, KCTD10, or USP18 inversely regulates the cystine uptake and ferroptosis. Biologically, MLN4924 combination with SLC7A11 inhibitor Imidazole Ketone Erastin (IKE) enhanced suppression of tumor growth. In human breast tumor tissues, SLC7A11 levels were negatively or positively correlated with KCTD10 or USP18, respectively. Collectively, our study defines how SLC7A11 and ferroptosis is coordinately regulated by the CRL3KCTD10/E3-USP18/DUB axis, and provides a sound rationale of drug combination to enhance anticancer efficacy.

Atomic resolution map of the solvent interactions driving SOD1 unfolding in CAPRIN1 condensates.

Biomolecules can be sequestered into membrane-less compartments, referred to as biomolecular condensates. Experimental and computational methods have helped define the physical-chemical properties of condensates. Less is known about how the high macromolecule concentrations in condensed phases contribute "solvent" interactions that can remodel the free-energy landscape of other condensate-resident proteins, altering thermally accessible conformations and, in turn, modulating function. Here, we use solution NMR spectroscopy to obtain atomic resolution insights into the interactions between the immature form of superoxide dismutase 1 (SOD1), which can mislocalize and aggregate in stress granules, and the RNA-binding protein CAPRIN1, a component of stress granules. NMR studies of CAPRIN1:SOD1 interactions, focused on both unfolded and folded SOD1 states in mixed phase and demixed CAPRIN1-based condensates, establish that CAPRIN1 shifts the SOD1 folding equilibrium toward the unfolded state through preferential interactions with the unfolded ensemble, with little change to the structure of the folded conformation. Key contacts between CAPRIN1 and the H80-H120 region of unfolded SOD1 are identified, as well as SOD1 interaction sites near both the arginine-rich and aromatic-rich regions of CAPRIN1. Unfolding of immature SOD1 in the CAPRIN1 condensed phase is shown to be coupled to aggregation, while a more stable zinc-bound, dimeric form of SOD1 is less susceptible to unfolding when solvated by CAPRIN1. Our work underscores the impact of the condensate solvent environment on the conformational states of resident proteins and supports the hypothesis that ALS mutations that decrease metal binding or dimerization function as drivers of aggregation in condensates.

KLF4 protein stability regulated by interaction with pluripotency transcription factors overrides transcriptional control.

Embryonic stem (ES) cells are regulated by a network of transcription factors that maintain the pluripotent state. Differentiation relies on down-regulation of pluripotency transcription factors disrupting this network. While investigating transcriptional regulation of the pluripotency transcription factor Kruppel-like factor 4 (Klf4), we observed that homozygous deletion of distal enhancers caused a 17-fold decrease in Klf4 transcript but surprisingly decreased protein levels by less than twofold, indicating that posttranscriptional control of KLF4 protein overrides transcriptional control. The lack of sensitivity of KLF4 to transcription is due to high protein stability (half-life >24 h). This stability is context-dependent and is disrupted during differentiation, as evidenced by a shift to a half-life of <2 h. KLF4 protein stability is maintained through interaction with other pluripotency transcription factors (NANOG, SOX2, and STAT3) that together facilitate association of KLF4 with RNA polymerase II. In addition, the KLF4 DNA-binding and transactivation domains are required for optimal KLF4 protein stability. Posttranslational modification of KLF4 destabilizes the protein as cells exit the pluripotent state, and mutations that prevent this destabilization also prevent differentiation. These data indicate that the core pluripotency transcription factors are integrated by posttranslational mechanisms to maintain the pluripotent state and identify mutations that increase KLF4 protein stability while maintaining transcription factor function.

Regulation of MLL/COMPASS stability through its proteolytic cleavage by taspase1 as a possible approach for clinical therapy of leukemia.

Chromosomal translocations of the Mixed-lineage leukemia 1 (MLL1) gene generate MLL chimeras that drive the pathogenesis of acute myeloid and lymphoid leukemia. The untranslocated MLL1 is a substrate for proteolytic cleavage by the endopeptidase threonine aspartase 1 (taspase1); however, the biological significance of MLL1 cleavage by this endopeptidase remains unclear. Here, we demonstrate that taspase1-dependent cleavage of MLL1 results in the destabilization of MLL. Upon loss of taspase1, MLL1 association with chromatin is markedly increased due to the stabilization of its unprocessed version, and this stabilization of the uncleaved MLL1 can result in the displacement of MLL chimeras from chromatin in leukemic cells. Casein kinase II (CKII) phosphorylates MLL1 proximal to the taspase1 cleavage site, facilitating its cleavage, and pharmacological inhibition of CKII blocks taspase1-dependent MLL1 processing, increases MLL1 stability, and results in the displacement of the MLL chimeras from chromatin. Accordingly, inhibition of CKII in a MLL-AF9 mouse model of leukemia delayed leukemic progression in vivo. This study provides insights into the direct regulation of the stability of MLL1 through its cleavage by taspase1, which can be harnessed for targeted therapeutic approaches for the treatment of aggressive leukemia as the result of MLL translocations.

Crowding revisited: Open questions and future perspectives.

Although biophysical studies have traditionally been performed in diluted solutions, it was pointed out in the late 1990s that the cellular milieu contains several other macromolecules, creating a condition of molecular crowding. How crowding affects protein stability is an important question heatedly discussed over the past 20 years. Theoretical estimations have suggested a 5-20°C effect of fold stabilisation. This estimate, however, is at variance with what has been verified experimentally that proposes only a limited increase of stability, opening the question whether some of the assumptions taken for granted should be reconsidered. The present review critically analyses the causes of this discrepancy and discusses the limitations and implications of the current concept of crowding.

Protein structure and aggregation: a marriage of necessity ruled by aggregation gatekeepers.

Protein aggregation propensity is a pervasive and seemingly inescapable property of proteomes. Strikingly, a significant fraction of the proteome is supersaturated, meaning that, for these proteins, their native conformation is less stable than the aggregated state. Maintaining the integrity of a proteome under such conditions is precarious and requires energy-consuming proteostatic regulation. Why then is aggregation propensity maintained at such high levels over long evolutionary timescales? Here, we argue that the conformational stability of the native and aggregated states are correlated thermodynamically and that codon usage strengthens this correlation. As a result, the folding of stable proteins requires kinetic control to avoid aggregation, provided by aggregation gatekeepers. These unique residues are evolutionarily selected to kinetically favor native folding, either on their own or by coopting chaperones.

Novel mechanisms of MITF regulation identified in a mouse suppressor screen.

MITF, a basic Helix-Loop-Helix Zipper (bHLHZip) transcription factor, plays vital roles in melanocyte development and functions as an oncogene. We perform a genetic screen for suppressors of the Mitf-associated pigmentation phenotype in mice and identify an intragenic Mitf mutation that terminates MITF at the K316 SUMOylation site, leading to loss of the C-end intrinsically disordered region (IDR). The resulting protein is more nuclear but less stable than wild-type MITF and retains DNA-binding ability. As a dimer, it can translocate wild-type and mutant MITF partners into the nucleus, improving its own stability thus ensuring nuclear MITF supply. smFRET analysis shows interactions between K316 SUMOylation and S409 phosphorylation sites across monomers; these interactions largely explain the observed effects. The recurrent melanoma-associated E318K mutation in MITF, which affects K316 SUMOylation, also alters protein regulation in concert with S409. This suggests that residues K316 and S409 of MITF are impacted by SUMOylation and phosphorylation, respectively, mediating effects on nuclear localization and stability through conformational changes. Our work provides a novel mechanism of genetic suppression, and an example of how apparently deleterious mutations lead to normal phenotypes.

Mapping Degradation Signals and Pathways in a Eukaryotic N-terminome.

Most eukaryotic proteins are N-terminally acetylated. This modification can be recognized as a signal for selective protein degradation (degron) by the N-end rule pathways. However, the prevalence and specificity of such degrons in the proteome are unclear. Here, by systematically examining how protein turnover is affected by N-terminal sequences, we perform a comprehensive survey of degrons in the yeast N-terminome. We find that approximately 26% of nascent protein N termini encode cryptic degrons. These degrons exhibit high hydrophobicity and are frequently recognized by the E3 ubiquitin ligase Doa10, suggesting a role in protein quality control. In contrast, N-terminal acetylation rarely functions as a degron. Surprisingly, we identify two pathways where N-terminal acetylation has the opposite function and blocks protein degradation through the E3 ubiquitin ligase Ubr1. Our analysis highlights the complexity of N-terminal degrons and argues that hydrophobicity, not N-terminal acetylation, is the predominant feature of N-terminal degrons in nascent proteins.

Folding stabilities of ribosome-bound nascent polypeptides probed by mass spectrometry.

The folding of most proteins occurs during the course of their translation while their tRNA-bound C termini are embedded in the ribosome. How the close proximity of nascent proteins to the ribosome influences their folding thermodynamics remains poorly understood. Here, we have developed a mass spectrometry-based approach for determining the stabilities of nascent polypeptide chains using methionine oxidation as a folding probe. This approach enables quantitative measurement subglobal folding stabilities of ribosome nascent chains within complex protein mixtures and extracts. To validate the methodology, we analyzed the folding thermodynamics of three model proteins (dihydrofolate reductase, chemotaxis protein Y, and DNA polymerase IV) in soluble and ribosome-bound states. The data indicate that the ribosome can significantly alter the stability of nascent polypeptides. Ribosome-induced stability modulations were highly variable among different folding domains and were dependent on localized charge distributions within nascent polypeptides. The results implicated electrostatic interactions between the ribosome surface and nascent polypeptides as the cause of ribosome-induced stability modulations. The study establishes a robust proteomic methodology for analyzing localized stabilities within ribosome-bound nascent polypeptides and sheds light on how the ribosome influences the thermodynamics of protein folding.

Evolution of a novel regulatory mechanism of hypoxia inducible factor in hypoxia-tolerant electric fishes.

Hypoxia is a significant source of metabolic stress that activates many cellular pathways involved in cellular differentiation, proliferation, and cell death. Hypoxia is also a major component in many human diseases and a known driver of many cancers. Despite the challenges posed by hypoxia, there are animals that display impressive capacity to withstand lethal levels of hypoxia for prolonged periods of time and thus offer a gateway to a more comprehensive understanding of the hypoxic response in vertebrates. The weakly electric fish genus Brachyhypopomus inhabits some of the most challenging aquatic ecosystems in the world, with some species experiencing seasonal anoxia, thus providing a unique system to study the cellular and molecular mechanisms of hypoxia tolerance. In this study, we use closely related species of Brachyhypopomus that display a range of hypoxia tolerances to probe for the underlying molecular mechanisms via hypoxia inducible factors (HIFs)-transcription factors known to coordinate the cellular response to hypoxia in vertebrates. We find that HIF1⍺ from hypoxia tolerant Brachyhypopomus species displays higher transactivation in response to hypoxia than that of intolerant species, when overexpressed in live cells. Moreover, we identified two SUMO-interacting motifs near the oxygen-dependent degradation and transactivation domains of the HIF1⍺ protein that appear to boost transactivation of HIF1, regardless of the genetic background. Together with computational analyses of selection, this shows that evolution of HIF1⍺ are likely to underlie adaptations to hypoxia tolerance in Brachyhypopomus electric fishes, with changes in two SUMO-interacting motifs facilitating the mechanism of this tolerance.

HSP90AB1 is a host factor that promotes porcine deltacoronavirus replication.

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus. It causes mortality in neonatal piglets and is of growing concern because of its broad host range, including humans. To date, the mechanism of PDCoV infection remains poorly understood. Here, based on a genome-wide CRISPR screen of PDCoV-infected cells, we found that HSP90AB1 (heat shock protein 90 alpha family class B1) promotes PDCoV infection. Knockdown or KO of HSP90AB1 in LLC-PK cells resulted in a significantly suppressed PDCoV infection. Infected cells treated with HSP90 inhibitors 17-AAG and VER-82576 also showed a significantly suppressed PDCoV infection, although KW-2478, which does not affect the ATPase activity of HSP90AB1, had no effect on PDCoV infection. We found that HSP90AB1 interacts with the N, NS7, and NSP10 proteins of PDCoV. We further evaluated the interaction between N and HSP90AB1 and found that the C-tail domain of the N protein is the HSP90AB1-interacting domain. Further studies showed that HSP90AB1 protects N protein from degradation via the proteasome pathway. In summary, our results reveal a key role for HSP90AB1 in the mechanism of PDCoV infection and contribute to provide new host targets for PDCoV antiviral research.

A facile approach for incorporating tyrosine esters to probe ion-binding sites and backbone hydrogen bonds.

Amide-to-ester substitutions are used to study the role of the amide bonds of the protein backbone in protein structure, function, and folding. An amber suppressor tRNA/synthetase pair has been reported for incorporation of p-hydroxy-phenyl-L-lactic acid (HPLA), thereby introducing ester substitution at tyrosine residues. However, the application of this approach was limited due to the low yields of the modified proteins and the high cost of HPLA. Here we report the in vivo generation of HPLA from the significantly cheaper phenyl-L-lactic acid. We also construct an optimized plasmid with the HPLA suppressor tRNA/synthetase pair that provides higher yields of the modified proteins. The combination of the new plasmid and the in-situ generation of HPLA provides a facile and economical approach for introducing tyrosine ester substitutions. We demonstrate the utility of this approach by introducing tyrosine ester substitutions into the K+ channel KcsA and the integral membrane enzyme GlpG. We introduce the tyrosine ester in the selectivity filter of the M96V mutant of the KcsA to probe the role of the second ion binding site in the conformation of the selectivity filter and the process of inactivation. We use tyrosine ester substitutions in GlpG to perturb backbone H-bonds to investigate the contribution of these H-bonds to membrane protein stability. We anticipate that the approach developed in this study will facilitate further investigations using tyrosine ester substitutions.

Missense mutations in the central domains of cardiac myosin binding protein-C and their potential contribution to hypertrophic cardiomyopathy.

Myosin binding protein-C (MyBP-C) is a multidomain protein that regulates muscle contraction. Mutations in MYBPC3, the gene encoding for the cardiac variant (henceforth called cMyBP-C), are amongst the most frequent causes of hypertrophic cardiomyopathy. Most mutations lead to a truncated version of cMyBP-C, which is most likely unstable. However, missense mutations have also been reported, which tend to cluster in the central domains of the cMyBP-C molecule. This suggests that these central domains are more than just a passive spacer between the better characterized N- and C-terminal domains. Here, we investigated the potential impact of four different missense mutations, E542Q, G596R, N755K, and R820Q, which are spread over the domains C3 to C6, on the function of MyBP-C on both the isolated protein level and in cardiomyocytes in vitro. Effect on domain stability, interaction with thin filaments, binding to myosin, and subcellular localization behavior were assessed. Our studies show that these missense mutations result in slightly different phenotypes at the molecular level, which are mutation specific. The expected functional readout of each mutation provides a valid explanation for why cMyBP-C fails to work as a brake in the regulation of muscle contraction, which eventually results in a hypertrophic cardiomyopathy phenotype. We conclude that missense mutations in cMyBP-C must be evaluated in context of their domain localization, their effect on interaction with thin filaments and myosin, and their effect on protein stability to explain how they lead to disease.