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Targeted protein degradation (TPD) strategies exploit bivalent small molecules to bridge substrate proteins to an E3 ubiquitin ligase to induce substrate degradation. Few E3s have been explored as degradation effectors due to a dearth of E3-binding small molecules. We show that genetically induced recruitment to the GID4 subunit of the CTLH E3 complex induces protein degradation. An NMR-based fragment screen followed by structure-guided analog elaboration identified two binders of GID4

A comprehensive analysis of the phosphoproteome is essential for understanding molecular mechanisms of human diseases. However, current tools used to enrich phosphotyrosine (pTyr) are limited in their applicability and scope. Here, we engineered new superbinder Src-Homology 2 (SH2) domains that enrich diverse sets of pTyr-peptides. We used phage display to select a Fes-SH2 domain variant (superFes; sFes(1)) with high affinity for pTyr and solved its structure bound to a pTyr-peptide. We performed systematic structure–function analyses of the superbinding mechanisms of sFes(1) and superSrc-SH2 (sSrc(1)), another SH2 superbinder. We grafted the superbinder motifs from sFes(1) and sSrc(1) into 17 additional SH2 domains and confirmed increased binding affinity for specific pTyr-peptides. Using mass spectrometry (MS), we demonstrated that SH2 superbinders have distinct specificity profiles and superior capabilities to enrich pTyr-peptides. Finally, using combinations of SH2 superbinders as affinity purification (AP) tools we showed that unique subsets of pTyr-peptides can be enriched with unparalleled depth and coverage.

KEOPS (Kinase, Endopeptidase and Other Proteins of Small size) is a five-subunit protein complex that is highly conserved in eukaryotes and archaea and is essential for the fitness of cells and for animal development. In humans, mutations in KEOPS genes underlie Galloway–Mowat syndrome, which manifests in severe microcephaly and renal dysfunction that lead to childhood death. The Kae1 subunit of KEOPS catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine (t6A), while the auxiliary subunits Cgi121, the kinase/ATPase Bud32, Pcc1 and Gon7 play a supporting role. Kae1 orthologs are also present in bacteria and mitochondria but function in distinct complexes with proteins that are not related in structure or function to the auxiliary subunits of KEOPS. Over the past 15 years since its discovery, extensive study in the KEOPS field has provided many answers towards understanding the roles that KEOPS plays in cells and in human disease and how KEOPS carries out these functions. In this review, we provide an overview into recent advances in the study of KEOPS and illuminate exciting future directions.

FBXW7, which encodes a substrate-specific receptor of an SCF E3 ligase complex, is a frequently mutated human tumor suppressor gene known to regulate the post-translational stability of various proteins involved in cellular proliferation. Here, using genome-wide CRISPR screens, we report a novel synthetic lethal genetic interaction between FBXW7 and CCNL1 and describe CCNL1 as a new substrate of the SCF-FBXW7 E3 ligase. Further analysis showed that the CCNL1-CDK11 complex is critical at the G2-M phase of the cell cycle since defective CCNL1 accumulation, resulting from FBXW7 mutation, leads to shorter mitotic time. Cells harboring FBXW7 loss-of-function mutations are hypersensitive to treatment with a CDK11 inhibitor, highlighting a genetic vulnerability that could be leveraged for cancer treatment.

FBXW7, which encodes a substrate specific receptor of an SCF E3 ligase complex, is a frequently mutated human tumor suppressor gene known to regulate the post-translational stability of various proteins involved in cellular proliferation. Here, using genome-wide CRISPR screens we report a novel synthetic lethal genetic interaction between FBXW7 and CCNL1 and describe CCNL1 as a new substrate of the SCF-FBXW7 E3 ligase. Further analysis showed that the CCNL1-CDK11 complex is critical at the G2-M phase of the cell cycle since defective CCNL1 accumulation, resulting from FBXW7 mutation, leads to shorter mitotic time. Cells harboring FBXW7 loss-of-function mutations are hypersensitive to treatment with a CDK11 inhibitor, highlighting a genetic vulnerability that could be leveraged for cancer treatment.

DNA polymerase theta (Polθ) is an attractive synthetic lethal target for drug discovery, predicted to be efficacious against breast and ovarian cancers harboring BRCA-mutant alleles. Here, we describe our hit-to-lead efforts in search of a selective inhibitor of human Polθ (encoded by POLQ). A high-throughput screening campaign of 350,000 compounds identified an 11 micromolar hit, giving rise to the N2-substituted fused pyrazolo series, which was validated by biophysical methods. Structure-based drug design efforts along with optimization of cellular potency and ADME ultimately led to the identification of

Bud32 is a member of the protein kinase superfamily that is invariably conserved in all eukaryotic and archaeal organisms. In both of these kingdoms, Bud32 forms part of the KEOPS (Kinase, Endopeptidase and Other Proteins of Small size) complex together with the three other core subunits Kae1, Cgi121 and Pcc1. KEOPS functions to generate the universal and essential tRNA post-transcriptional modification N

Significance The OAS–RNase L system is an innate immunity pathway activated by viral infection. Viral dsRNA stimulates OAS enzymes to produce short 2′,5′-oligoadenylates (2-5A) that activate RNase L, resulting in cleavage of single-stranded (ss) RNA. We discovered a small-molecule inhibitor of RNase L that rescues the toxic phenotype of cells deficient in the dsRNA-editing enzyme ADAR1. ADAR1 destabilizes dsRNA to prevent OAS activity. ADAR1 mutations are responsible for a subset of cases of Aicardi-Goutières syndrome (AGS), a severe neurodevelopmental and inflammatory genetic disease of children with no effective medical therapy. We posit that an RNase L inhibitor may have utility against cases of AGS in which RNase L is activated and other indications where overactivation of RNase L is harmful., The oligoadenylate synthetase (OAS)–RNase L system is an IFN-inducible antiviral pathway activated by viral infection. Viral double-stranded (ds) RNA activates OAS isoforms that synthesize the second messenger 2-5A, which binds and activates the pseudokinase-endoribonuclease RNase L. In cells, OAS activation is tamped down by ADAR1, an adenosine deaminase that destabilizes dsRNA. Mutation of ADAR1 is one cause of Aicardi-Goutières syndrome (AGS), an interferonopathy in children. ADAR1 deficiency in human cells can lead to RNase L activation and subsequent cell death. To evaluate RNase L as a possible therapeutic target for AGS, we sought to identify small-molecule inhibitors of RNase L. A 500-compound library of protein kinase inhibitors was screened for modulators of RNase L activity in vitro. We identified ellagic acid (EA) as a hit with 10-fold higher selectivity against RNase L compared with its nearest paralog, IRE1. SAR analysis identified valoneic acid dilactone (VAL) as a superior inhibitor of RNase L, with 100-fold selectivity over IRE1. Mechanism-of-action analysis indicated that EA and VAL do not bind to the pseudokinase domain of RNase L despite acting as ATP competitive inhibitors of the protein kinase CK2. VAL is nontoxic and functional in cells, although with a 1,000-fold decrease in potency, as measured by RNA cleavage activity in response to treatment with dsRNA activator or by rescue of cell lethality resulting from self dsRNA induced by ADAR1 deficiency. These studies lay the foundation for understanding novel modes of regulating RNase L function using small-molecule inhibitors and avenues of therapeutic potential.

Polo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity. We localize the key determinants of phospho-Bora function to a 100 amino acid region encompassing two short Tpx2-like motifs and a phosphoSerine-Proline motif at Serine 112, through which Bora binds AURKA. The latter substitutes in trans for the Thr288 phospho-regulatory site of AURKA, which is essential for an active conformation of the kinase domain. We demonstrate the importance of these determinants for Bora function in mitotic entry both in Xenopus egg extracts and in human cells. Our findings unveil the activation mechanism of AURKA that is critical for mitotic entry., Tavernier et al. decipher the mechanism by which the intrinsically disordered protein Bora, phosphorylated by Cyclin-Cdk, potentiates AURKA activity towards Polo-like kinase 1. Furthermore, they demonstrate the importance of this mechanism for timely mitotic entry in Xenopus and human cells.

Pharmacological control of the ubiquitin-proteasome system (UPS) is of intense interest in drug discovery. Here, we report the development of chemical inhibitors of the ubiquitin-conjugating (E2) enzyme CDC34A (also known as UBE2R1), which donates activated ubiquitin to the cullin-RING ligase (CRL) family of ubiquitin ligase (E3) enzymes. A FRET-based interaction assay was used to screen for novel compounds that stabilize the noncovalent complex between CDC34A and ubiquitin, and thereby inhibit the CDC34A catalytic cycle. An isonipecotamide hit compound was elaborated into analogs with ~1000-fold increased potency in stabilizing the CDC34A-ubiquitin complex. These analogs specifically inhibited CDC34A-dependent ubiquitination in vitro and stabilized an E2~ubiquitin thioester reaction intermediate in cells. The x-ray crystal structure of a CDC34A-ubiquitin-inhibitor complex uncovered the basis for analog structure-activity relationships. The development of chemical stabilizers of the CDC34A-ubiquitin complex illustrates a general strategy for de novo discovery of molecular glue compounds that stabilize weak protein interactions.

PDZ domains are key players in signalling pathways. These modular domains generally recognize short linear C-terminal stretches of sequences in proteins that organize the formation of complex multi-component assemblies. The development of new methodologies for the characterization of the molecular principles governing these interactions is critical to fully understand the functional diversity of the family and to elucidate biological functions for family members. Here, we applied an in vitro evolution strategy to explore comprehensively the capacity of PDZ domains for specific recognition of different amino acids at a key position in C-terminal peptide ligands. We constructed a phage-displayed library of the Erbin PDZ domain by randomizing the binding site

We previously described structural and functional characterization of the first ubiquitin variant (UbV), UbV.v27.1, engineered by phage display to bind with high affinity to a specific ubiquitin interacting motif (UIM). We identified two substitutions relative to ubiquitin (Gly10Val/His68Tyr) that were critical for enhancing binding affinity but could only rationalize the mechanism of action of the Tyr68 substitution. Here, we extend our characterization and uncover the mechanism by which the Val10 substitution enhances binding affinity. We show that Val10 in UbV.v27.1 drives UbV dimerization through an intermolecular β‐strand exchange. Dimerization serves to increase the contact surface between the UIM and UbV and also affords direct contacts between two UIMs through an overall 2:2 binding stoichiometry. Our identification of the role of Val10 in UbV dimerization suggests a general means for the development of dimeric UbVs with improved affinity and specificity relative to their monomeric UbV counterparts. Statement: Previously, we used phage display to engineer a UbV that bound tightly and specifically to a UIM. Here, we discovered that tight binding is partly due to the dimerization of the UbV, which increases the contact surface between the UbV and UIM. We show that UbV dimerization is dependent on the Gly10Val substitution, and posit that dimerization may provide a general means for engineering UbVs with improved binding properties.

Significance Drugs that cause epigenetic modification of DNA, such as 5-azacytidine (AZA), are used clinically to treat myelodysplastic syndromes and acute myeloid leukemia. In addition, AZA is being investigated for use against a range of different types of solid tumors, including lung and colorectal cancers. Treatment with AZA causes demethylation of DNA, thus increasing RNA synthesis, including the synthesis of double-stranded RNA, which is otherwise produced in virus-infected cells. We determined that cell death in response to AZA requires the antiviral enzyme RNase L. The results identify a drug target for enhancing the anticancer activity and reducing the toxicity of AZA and related drugs., Drugs that reverse epigenetic silencing, such as the DNA methyltransferase inhibitor (DNMTi) 5-azacytidine (AZA), have profound effects on transcription and tumor cell survival. AZA is an approved drug for myelodysplastic syndromes and acute myeloid leukemia, and is under investigation for different solid malignant tumors. AZA treatment generates self, double-stranded RNA (dsRNA), transcribed from hypomethylated repetitive elements. Self dsRNA accumulation in DNMTi-treated cells leads to type I IFN production and IFN-stimulated gene expression. Here we report that cell death in response to AZA treatment occurs through the 2′,5′-oligoadenylate synthetase (OAS)-RNase L pathway. OASs are IFN-induced enzymes that synthesize the RNase L activator 2-5A in response to dsRNA. Cells deficient in RNase L or OAS1 to 3 are highly resistant to AZA, as are wild-type cells treated with a small-molecule inhibitor of RNase L. A small-molecule inhibitor of c-Jun NH2-terminal kinases (JNKs) also antagonizes RNase L-dependent cell death in response to AZA, consistent with a role for JNK in RNase L-induced apoptosis. In contrast, the rates of AZA-induced and RNase L-dependent cell death were increased by transfection of 2-5A, by deficiencies in ADAR1 (which edits and destabilizes dsRNA), PDE12 or AKAP7 (which degrade 2-5A), or by ionizing radiation (which induces IFN-dependent signaling). Finally, OAS1 expression correlates with AZA sensitivity in the NCI-60 set of tumor cell lines, suggesting that the level of OAS1 can be a biomarker for predicting AZA sensitivity of tumor cells. These studies may eventually lead to pharmacologic strategies for regulating the antitumor activity and toxicity of AZA and related drugs.

Summary Skp2 and cyclin A are cell-cycle regulators that control the activity of CDK2. Cyclin A acts as an activator and substrate recruitment factor of CDK2, while Skp2 mediates the ubiquitination and subsequent destruction of the CDK inhibitor protein p27. The N terminus of Skp2 can interact directly with cyclin A but is not required for p27 ubiquitination. To gain insight into this poorly understood interaction, we have solved the 3.2 A X-ray crystal structure of the N terminus of Skp2 bound to cyclin A. The structure reveals a bipartite mode of interaction with two motifs in Skp2 recognizing two discrete surfaces on cyclin A. The uncovered binding mechanism allows for a rationalization of the inhibitory effect of Skp2 on CDK2-cyclin A kinase activity toward the RxL motif containing substrates and raises the possibility that other intermolecular regulators and substrates may use similar non-canonical modes of interaction for cyclin targeting.

The KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway–Mowat syndrome, an autosomal-recessive disease causing childhood lethality. Kae1 catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine, but the precise roles of all other KEOPS subunits remain an enigma. Here we show using structure-guided studies that Cgi121 recruits tRNA to KEOPS by binding to its 3’ CCA tail. A composite model of KEOPS bound to tRNA reveals that all KEOPS subunits form an extended tRNA-binding surface that we have validated in vitro and in vivo to mediate the interaction with the tRNA substrate and its modification. These findings provide a framework for understanding the inner workings of KEOPS and delineate why all KEOPS subunits are essential., KEOPS is an evolutionary conserved complex with a core of four core subunits—Pcc1, Kae1, Bud32 and Cgi121—that catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine (t6A). Here the authors describe a Cgi121-tRNA crystal structure and new composite model of the KEOPS holo-enzyme-substrate complex that shed light on the t6A catalytic cycle and its regulation.

Saliva is an alternative biofluid to serum for detecting and monitoring IgG to SARS-CoV-2 spike and RBD antigens in COVID-19., While the antibody response to SARS-CoV-2 has been extensively studied in blood, relatively little is known about the antibody response in saliva and its relationship to systemic antibody levels. Here, we profiled by enzyme-linked immunosorbent assays (ELISAs) IgG, IgA and IgM responses to the SARS-CoV-2 spike protein (full length trimer) and its receptor-binding domain (RBD) in serum and saliva of acute and convalescent patients with laboratory-diagnosed COVID-19 ranging from 3–115 days post-symptom onset (PSO), compared to negative controls. Anti-SARS-CoV-2 antibody responses were readily detected in serum and saliva, with peak IgG levels attained by 16–30 days PSO. Longitudinal analysis revealed that anti-SARS-CoV-2 IgA and IgM antibodies rapidly decayed, while IgG antibodies remained relatively stable up to 105 days PSO in both biofluids. Lastly, IgG, IgM and to a lesser extent IgA responses to spike and RBD in the serum positively correlated with matched saliva samples. This study confirms that serum and saliva IgG antibodies to SARS-CoV-2 are maintained in the majority of COVID-19 patients for at least 3 months PSO. IgG responses in saliva may serve as a surrogate measure of systemic immunity to SARS-CoV-2 based on their correlation with serum IgG responses.

The RAF family kinases function in the RAS-ERK pathway to transmit signals from activated RAS to the downstream kinases MEK and ERK. This pathway regulates cell proliferation, differentiation and survival, enabling mutations in RAS and RAF to act as potent drivers of human cancers. Drugs targeting the prevalent oncogenic mutant BRAF(V600E) have shown great efficacy in the clinic, but long-term effectiveness is limited by resistance mechanisms that often exploit the dimerization-dependent process by which RAF kinases are activated. Here, we investigated a proteolysis-targeting chimera (PROTAC) approach to BRAF inhibition. The most effective PROTAC, termed P4B, displayed superior specificity and inhibitory properties relative to non-PROTAC controls in BRAF(V600E) cell lines. In addition, P4B displayed utility in cell lines harboring alternative BRAF mutations that impart resistance to conventional BRAF inhibitors. This work provides a proof of concept for a substitute to conventional chemical inhibition to therapeutically constrain oncogenic BRAF.

While the antibody response to SARS-CoV-2 has been extensively studied in blood, relatively little is known about the mucosal immune response and its relationship to systemic antibody levels. Since SARS-CoV-2 initially replicates in the upper airway, the antibody response in the oral cavity is likely an important parameter that influences the course of infection, but how it correlates to the antibody response in serum is not known. Here, we profile by enzyme linked immunosorbent assays (ELISAs) IgG, IgA and IgM responses to the SARS-CoV-2 spike protein (full length trimer) and its receptor binding domain (RBD) in serum (n=496) and saliva (n=90) of acute and convalescent patients with laboratory-diagnosed COVID-19 ranging from 3–115 days post-symptom onset (PSO), compared to negative controls. Anti-CoV-2 antibody responses were readily detected in serum and saliva, with peak IgG levels attained by 16–30 days PSO. Whereas anti-CoV-2 IgA and IgM antibodies rapidly decayed, IgG antibodies remained relatively stable up to 105 days PSO in both biofluids. In a surrogate neutralization ELISA (snELISA), neutralization activity peaks by 31–45 days PSO and slowly declines, though a clear drop is detected at the last blood draw (105–115 days PSO). Lastly, IgG, IgM and to a lesser extent IgA responses to spike and RBD in the serum positively correlated with matched saliva samples. This study confirms that systemic and mucosal humoral IgG antibodies are maintained in the majority of COVID-19 patients for at least 3 months PSO. Based on their correlation with each other, IgG responses in saliva may serve as a surrogate measure of systemic immunity.One Sentence SummaryIn this manuscript, we report evidence for sustained SARS-CoV-2-specific IgG and transient IgA and IgM responses both at the site of infection (mucosae) and systemically in COVID-19 patients over 3 months and suggest that saliva could be used as an alternative biofluid for monitoring IgG to SARS-CoV-2 spike and RBD antigens.

The small GTPases H, K, and NRAS are molecular switches indispensable for proper regulation of cellular proliferation and growth. Several mutations in the genes encoding members of this protein family are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered variants of the Ras-binding domain (RBD) of the C-Raf proto-oncogene, Ser/Thr kinase (CRAF). These variants bound with high affinity with the effector-binding site of Ras in an active conformation. Structural characterization disclosed how the newly identified RBD mutations cooperate and thereby enhance affinity with the effector-binding site in Ras compared with WT RBD. The engineered RBD variants closely mimicked the interaction mode of naturally occurring Ras effectors and acted as dominant-negative affinity reagents that block Ras signal transduction. Experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling, reducing cell growth and inducing apoptosis. Using these optimized RBD variants, we stratified patient-derived colorectal cancer organoids with known Ras mutational status according to their response to Ras inhibition. These results revealed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition, suggesting that not all of these tumors required Ras signaling for proliferation. In summary, by engineering the Ras/Raf interface of the CRAF-RBD, we identified potent and selective inhibitors of Ras in its active conformation that outcompete binding of Ras-signaling effectors.

Introduction Sub-cutaneous panniculitis T-cell lymphomas (SPTCL), a rare non-Hodgkin lymphoma, can be associated with hemophagocytic lymphohistiocytosis (HLH), a life-threatening activation of the immune system which adversely impacts survival. T-cell immunoglobulin mucin 3 (TIM-3) is a modulator of immune responses expressed on subgroups of T- and innate immune cells. In this work we describe the first germline variants associated with SPTCL, which are in the TIM-3 gene. Methods We sequenced 27 SPTCL cases to identify gene variants. We performed TIM-3 functional analysis on immune cells from patients and HEK293 cells engineered to overexpress wild-type or mutant TIM-3. Results We identified homozygous, germline, loss-of-function, missense variants in highly conserved residues of TIM-3, namely p.Y82C and p.I97M in about 60% (16/27) of SPTCL cases. These samples were drawn from cases series across 3 continents. Patients with bi-allelic TIM-3 mutations were younger at diagnosis, and several had life-threatening HLH and severe disease course. TIM-3 mutations show specific geographic distribution. Y82C TIM-3 mutations occur on a founder chromosome in patients with East-Asian and Polynesian ancestry, while I97M TIM-3 is observed in Caucasians. Both variants induce protein misfolding and cytoplasmic retention of TIM-3. Loss of TIM-3 membrane expression in TIM-3 mutants abrogates the PD-1/PDL-1 checkpoint and prevents the termination of a Th1-immune response. In HEK293 cells, mutant TIM-3 was not expressed on the cell surface. Defective TIM-3 expression leads to persistent immune activation with increased production of inflammatory cytokines including TNF-alpha and IL-1beta by innate immune cells. Conclusion Our findings highlight HLH/SPTCL as a new genetic entity where loss of the TIM-3 immune checkpoint is associated with T-cell infiltration of adipose tissue and inflammasome activation. This is the first causative germline defect identified in SPTCL. While our findings indicate that TIM-3-mutant HLH/SPTCL benefit from immunomodulation, therapeutic repression of the TIM-3 checkpoint could have serious adverse consequences. Disclosures Prince: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

In most solid tumors, the Hippo pathway is inactivated through poorly understood mechanisms that result in the activation of the transcriptional regulators, YAP and TAZ. Here, we identify NUAK2 as a YAP/TAZ activator that directly inhibits LATS-mediated phosphorylation of YAP/TAZ and show that NUAK2 induction by YAP/TAZ and AP-1 is required for robust YAP/TAZ signaling. Pharmacological inhibition or loss of NUAK2 reduces the growth of cultured cancer cells and mammary tumors in mice. Moreover, in human patient samples, we show that NUAK2 expression is elevated in aggressive, high-grade bladder cancer and strongly correlates with a YAP/TAZ gene signature. These findings identify a positive feed forward loop in the Hippo pathway that establishes a key role for NUAK2 in enforcing the tumor-promoting activities of YAP/TAZ. Our results thus introduce a new opportunity for cancer therapeutics by delineating NUAK2 as a potential target for re-engaging the Hippo pathway., The Hippo pathway is frequently dysregulated in cancer. Here, the authors identify NUAK2 as negative regulator of the Hippo pathway from a siRNA kinome screen and show that NUAK2 promotes YAP/TAZ nuclear localisation while NUAK2 is a transcriptional target of YAP/TAZ, thus providing a feed forward loop to promote tumorigenesis.

Metformin is a first-line drug for the treatment of individuals with type 2 diabetes, yet its precise mechanism of action remains unclear. Metformin exerts its anti-hyperglycemic action primarily through lowering of hepatic glucose production (HGP). This suppression is thought to be mediated through inhibition of mitochondrial respiratory complex I, and thus elevation of 5’-adenosine monophosphate (AMP) levels and the activation of AMP-activated protein kinase (AMPK), though this proposition has been challenged given results in mice lacking hepatic AMPK. Here, we report that the AMP-inhibited enzyme fructose-1,6-bisphosphatase-1 (FBP1, EC 3.1.3.11), a rate-controlling enzyme in gluconeogenesis, functions as a major contributor to the therapeutic action of metformin. We identified a point mutation in FBP1 that renders it insensitive to AMP while sparing regulation by fructose-2,6-bisphosphate (F-2,6-P2) and knockin (KI) of this mutant into mice significantly reduces their response to metformin treatment. We observe this during a metformin tolerance test and in a metformin-euglycemic clamp that we have developed. The anti-hyperglycemic effect of metformin in high fat diet-fed diabetic FBP1 KI mice was also significantly blunted compared to wild-type controls. Collectively, we show a new mechanism of action of metformin, while providing further evidence that molecular targeting of FBP1 can have anti-hyperglycemic effects.

RAF family kinases have prominent roles in cancer. Their activation is dependent on dimerization of their kinase domains, which has emerged as a hindrance for drug development. In mammals, RAF family kinases include three catalytically competent enzymes (ARAF, BRAF and CRAF) and two pseudokinases (KSR1 and KSR2) that have been described as scaffolds owing to their apparent ability to bridge RAF isoforms and their substrate, mitogen-activated protein kinase kinase (MEK). Kinase suppressor of Ras (KSR) pseudokinases were also shown to dimerize with kinase-competent RAFs to stimulate catalysis allosterically. Although GTP-bound RAS can modulate the dimerization of RAF isoforms by engaging their RAS-binding domains, KSR1 and KSR2 lack an RAS-binding domain and therefore the regulatory principles underlying their dimerization with other RAF family members remain unknown. Here we show that the selective heterodimerization of BRAF with KSR1 is specified by direct contacts between the amino-terminal regulatory regions of each protein, comprising in part a novel domain called BRS in BRAF and the coiled-coil-sterile α motif (CC-SAM) domain in KSR1. We also discovered that MEK binding to the kinase domain of KSR1 asymmetrically drives BRAF-KSR1 heterodimerization, resulting in the concomitant stimulation of BRAF catalytic activity towards free MEK molecules. These findings demonstrate that KSR-MEK complexes allosterically activate BRAF through the action of N-terminal regulatory region and kinase domain contacts and challenge the accepted role of KSR as a scaffold for MEK recruitment to RAF.

MOB1 is a multifunctional protein best characterized for its integrative role in regulating Hippo and NDR pathway signaling in metazoans and the Mitotic Exit Network in yeast. Human MOB1 binds both the upstream kinases MST1 and MST2 and the downstream AGC group kinases LATS1, LATS2, NDR1, and NDR2. Binding of MOB1 to MST1 and MST2 is mediated by its phosphopeptide-binding infrastructure, the specificity of which matches the phosphorylation consensus of MST1 and MST2. On the other hand, binding of MOB1 to the LATS and NDR kinases is mediated by a distinct interaction surface on MOB1. By assembling both upstream and downstream kinases into a single complex, MOB1 facilitates the activation of the latter by the former through a trans-phosphorylation event. Binding of MOB1 to its upstream partners also renders MOB1 a substrate, which serves to differentially regulate its two protein interaction activities (at least in vitro). Our previous interaction proteomics analysis revealed that beyond associating with MST1 (and MST2), MOB1A and MOB1B can associate in a phosphorylation-dependent manner with at least two other signaling complexes, one containing the Rho guanine exchange factors (DOCK6-8) and the other containing the serine/threonine phosphatase PP6. Whether these complexes are recruited through the same mode of interaction as MST1 and MST2 remains unknown. Here, through a comprehensive set of biochemical, biophysical, mutational and structural studies, we quantitatively assess how phosphorylation of MOB1A regulates its interaction with both MST kinases and LATS/NDR family kinases in vitro. Using interaction proteomics, we validate the significance of our in vitro studies and also discover that the phosphorylation-dependent recruitment of PP6 phosphatase and Rho guanine exchange factor protein complexes differ in key respects from that elucidated for MST1 and MST2. Together our studies confirm and extend previous work to delineate the intricate regulatory steps in key signaling pathways.

Ubiquitin interacting motifs (UIMs) are short α-helices found in a number of eukaryotic proteins. UIMs interact weakly but specifically with ubiquitin conjugated to other proteins, and in so doing, mediate specific cellular signals. Here we used phage display to generate ubiquitin variants (UbVs) targeting the N-terminal UIM of the yeast Vps27 protein. Selections yielded UbV.v27.1, which recognized the cognate UIM with high specificity relative to other yeast UIMs and bound with an affinity more than two orders of magnitude higher than that of ubiquitin. Structural and mutational studies of the UbV.v27.1-UIM complex revealed the molecular details for the enhanced affinity and specificity of UbV.v27.1, and underscored the importance of changes at the binding interface as well as at positions that do not contact the UIM. Our study highlights the power of the phage display approach for selecting UbVs with unprecedented affinity and high selectivity for particular α-helical UIM domains within proteomes, and it establishes a general approach for the development of inhibitors targeting interactions of this type.

The KEOPS/EKC complex is a tRNA modification complex involved in the biosynthesis of N6-threonylcarbamoyladenosine (t6A), a universally conserved tRNA modification found on ANN-codon recognizing tRNAs. In archaea and eukaryotes, KEOPS is composed of OSGEP/Kae1, PRPK/Bud32, TPRKB/Cgi121 and LAGE3/Pcc1. In fungi, KEOPS contains an additional subunit, Gon7, whose orthologs outside of fungi, if existent, remain unidentified. In addition to displaying defective t6A biosynthesis, Saccharomyces cerevisiae strains harboring KEOPS mutations are compromised for telomere homeostasis, growth and transcriptional co-activation. To identify a Gon7 ortholog in multicellular eukaryotes as well as to uncover KEOPS-interacting proteins that may link t6A biosynthesis to the diverse set of KEOPS mutant phenotypes, we conducted a proteomic analysis of human KEOPS. This work identified 152 protein interactors, one of which, C14ORF142, interacted strongly with all four KEOPS subunits, suggesting that it may be a core component of human KEOPS. Further characterization of C14ORF142 revealed that it shared a number of biophysical and biochemical features with fungal Gon7, suggesting that C14ORF142 is the human ortholog of Gon7. In addition, our proteomic analysis identified specific interactors for different KEOPS subcomplexes, hinting that individual KEOPS subunits may have additional functions outside of t6A biosynthesis.

To understand the molecular evolution of functional diversity in protein families, we comprehensively investigated the consequences of all possible mutation combinations separating two peptide-binding domains with highly divergent specificities. We analyzed the Erbin PDZ domain (Erbin-PDZ), which exhibits canonical type I specificity, and a synthetic Erbin-PDZ variant (E-14) that differs at six positions and exhibits an atypical specificity that closely resembles that of the natural Pdlim4 PDZ domain (Pdlim4-PDZ). We constructed a panel of 64 PDZ domains covering all possible transitions between Erbin-PDZ and E-14 (i.e., the panel contained variants with all possible combinations of either the Erbin-PDZ or E-14 sequence at the six differing positions). We assessed the specificity profiles of the 64 PDZ domains using a C-terminal phage-displayed peptide library containing all possible genetically encoded heptapeptides. The specificity profiles clustered into six distinct groups, showing that intermediate domains can be nodes for the evolution of divergent functions. Remarkably, three substitutions were sufficient to convert the specificity of Erbin-PDZ to that of Pdlim4-PDZ, whereas Pdlim4-PDZ contains 71 differences relative to Erbin-PDZ. X-ray crystallography revealed the structural basis for specificity transition: a single substitution in the center of the binding site, supported by contributions from auxiliary substitutions, altered the main chain conformation of the peptide ligand to resemble that of ligands bound to Pdlim4-PDZ. Our results show that a very small set of mutations can dramatically alter protein specificity, and these findings support the hypothesis whereby complex protein functions evolve by gene duplication followed by cumulative mutations.

Summary Pseudoenzymes have been identified across a diverse range of enzyme classes and fulfill important cellular functions. Examples of pseudoenzymes exist within ubiquitin conjugating and deubiquitinase (DUB) protein families. Here we characterize FAM105A/OTULINL, the only putative pseudodeubiquitinase of the ovarian tumor protease (OTU domain) family in humans. The crystal structure of FAM105A revealed that the OTU domain possesses structural deficiencies in both active site and substrate-binding infrastructure predicted to impair normal DUB function. We confirmed the absence of catalytic function against all ubiquitin linkages and an inability of FAM105A to bind ubiquitin compared with catalytically active FAM105B/OTULIN. FAM105A co-localized with KDEL markers and Lamin B1 at the endoplasmic reticulum (ER) and nuclear envelope, respectively. Accordingly, the FAM105A interactome exhibited significant enrichment in proteins localized to the ER/outer nuclear, Golgi and vesicular membranes. In light of undetectable deubiquitinase activity, we posit that FAM105A/OTULINL functions through its ability to mediate protein-protein interactions.

[Image: see text] One effective means to achieve inhibitor specificity for RAF kinases, an important family of cancer drug targets, has been to target the monomeric inactive state conformation of the kinase domain, which, unlike most other kinases, can accommodate sulfonamide-containing drugs such as vemurafenib and dabrafenib because of the presence of a unique pocket specific to inactive RAF kinases. We previously reported an alternate strategy whereby rigidification of a nonselective pyrazolo[3,4-d]pyrimidine-based inhibitor through ring closure afforded moderate but appreciable increases in selectivity for RAF kinases. Here, we show that a further application of the rigidification strategy to a different pyrazolopyrimidine-based scaffold dramatically improved selectivity for RAF kinases. Crystal structure analysis confirmed our inhibitor design hypothesis revealing that 2l engages an active-like state conformation of BRAF normally associated with poorly discriminating inhibitors. When screened against a panel of distinct cancer cell lines, the optimized inhibitor 2l primarily inhibited the proliferation of the expected BRAF(V600E)-harboring cell lines consistent with its kinome selectivity profile. These results suggest that rigidification could be a general and powerful strategy for enhancing inhibitor selectivity against protein kinases, which may open up therapeutic opportunities not afforded by other approaches.

The small GTPases H, K, and NRAS are molecular switches that are indispensable for proper regulation of cellular proliferation and growth. Mutations in this family of proteins are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered novel variants of the Ras-binding domain (RBD) of the kinase CRAF. These variants bound with high affinity to the effector binding site of active Ras. Structural characterization showed how the newly identified mutations cooperate to enhance affinity to the effector binding site compared to RBDwt. The engineered RBD variants closely mimic the interaction mode of naturally occurring Ras effectors and as dominant negative affinity reagent block their activation. Experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling leading to a reduced growth and inductions of apoptosis. Using the optimized RBD variants, we stratified patient-derived colorectal cancer organoids according to Ras dependency, which showed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition.

Subcutaneous panniculitis-like T cell lymphoma (SPTCL), a non-Hodgkin lymphoma, can be associated with hemophagocytic lymphohistiocytosis (HLH), a life-threatening immune activation that adversely affects survival1,2. T cell immunoglobulin mucin 3 (TIM-3) is a modulator of immune responses expressed on subgroups of T and innate immune cells. We identify in ~60% of SPTCL cases germline, loss-of-function, missense variants altering highly conserved residues of TIM-3, c.245A>G (p.Tyr82Cys) and c.291A>G (p.Ile97Met), each with specific geographic distribution. The variant encoding p.Tyr82Cys TIM-3 occurs on a potential founder chromosome in patients with East Asian and Polynesian ancestry, while p.Ile97Met TIM-3 occurs in patients with European ancestry. Both variants induce protein misfolding and abrogate TIM-3’s plasma membrane expression, leading to persistent immune activation and increased production of inflammatory cytokines, including tumor necrosis factor-α and interleukin-1β, promoting HLH and SPTCL. Our findings highlight HLH–SPTCL as a new genetic entity and identify mutations causing TIM-3 alterations as a causative genetic defect in SPTCL. While HLH–SPTCL patients with mutant TIM-3 benefit from immunomodulation, therapeutic repression of the TIM-3 checkpoint may have adverse consequences.

The tumor suppressor and deubiquitinase (DUB) BAP1 and its Drosophila ortholog Calypso assemble DUB complexes with the transcription regulators Additional sex combs-like (ASXL1, ASXL2, ASXL3) and Asx respectively. ASXLs and Asx use their DEUBiquitinase ADaptor (DEUBAD) domain to stimulate BAP1/Calypso DUB activity. Here we report that monoubiquitination of the DEUBAD is a general feature of ASXLs and Asx. BAP1 promotes DEUBAD monoubiquitination resulting in an increased stability of ASXL2, which in turn stimulates BAP1 DUB activity. ASXL2 monoubiquitination is directly catalyzed by UBE2E family of Ubiquitin-conjugating enzymes and regulates mammalian cell proliferation. Remarkably, Calypso also regulates Asx monoubiquitination and transgenic flies expressing monoubiquitination-defective Asx mutant exhibit developmental defects. Finally, the protein levels of ASXL2, BAP1 and UBE2E enzymes are highly correlated in mesothelioma tumors suggesting the importance of this signaling axis for tumor suppression. We propose that monoubiquitination orchestrates a molecular symbiosis relationship between ASXLs and BAP1., Additional sex combs-like (ASXLs) stimulate BAP1 deubiquitinase activity to induce tumor suppression, but how these complexes work in coordination in vivo is unclear. Here, the authors show the mutually reinforcing roles of BAP1 and ASXLs such that BAP1 promotes DEUBAD monoubiquitination of ASXL2, which in turn stimulates BAP1 DUB activity.

A cryo-electron microscopy structure of the DNA damage repair protein 53BP1 bound to a nucleosome illuminates the way 53BP1 recognizes two types of histone modifications (a methyl group and a ubiquitin moiety), and provides insight into the highly specified recognition and recruitment of 53BP1 to modified chromatin. Tudor domain proteins — containing a characteristic repeated structure that recognizes methylated arginine residues — mediate various protein–protein interactions, and Tudor domains are also employed as motifs to recognize different types of histone methylation marks. 53BP1, a protein involved early in the DNA damage response that determines the pathway of repair, has tandem Tudor domains. Daniel Durocher and colleagues have determined the cryo-electron microscopy structure of a 53BP1 dimer bound to a nucleosome core particle containing two types of histone modifications: a methyl group and a ubiquitin moiety. The structure illustrates how dual recognition of both marks as well as elements on the histones themselves mediates highly specified recognition and recruitment of 53BP1. DNA double-strand breaks (DSBs) elicit a histone modification cascade that controls DNA repair1,2,3. This pathway involves the sequential ubiquitination of histones H1 and H2A by the E3 ubiquitin ligases RNF8 and RNF168, respectively4,5,6,7,8. RNF168 ubiquitinates H2A on lysine 13 and lysine 15 (refs 7, 8) (yielding H2AK13ub and H2AK15ub, respectively), an event that triggers the recruitment of 53BP1 (also known as TP53BP1) to chromatin flanking DSBs9,10. 53BP1 binds specifically to H2AK15ub-containing nucleosomes through a peptide segment termed the ubiquitination-dependent recruitment motif (UDR), which requires the simultaneous engagement of histone H4 lysine 20 dimethylation (H4K20me2) by its tandem Tudor domain10,11. How 53BP1 interacts with these two histone marks in the nucleosomal context, how it recognizes ubiquitin, and how it discriminates between H2AK13ub and H2AK15ub is unknown. Here we present the electron cryomicroscopy (cryo-EM) structure of a dimerized human 53BP1 fragment bound to a H4K20me2-containing and H2AK15ub-containing nucleosome core particle (NCP-ubme) at 4.5 A resolution. The structure reveals that H4K20me2 and H2AK15ub recognition involves intimate contacts with multiple nucleosomal elements including the acidic patch. Ubiquitin recognition by 53BP1 is unusual and involves the sandwiching of the UDR segment between ubiquitin and the NCP surface. The selectivity for H2AK15ub is imparted by two arginine fingers in the H2A amino-terminal tail, which straddle the nucleosomal DNA and serve to position ubiquitin over the NCP-bound UDR segment. The structure of the complex between NCP-ubme and 53BP1 reveals the basis of 53BP1 recruitment to DSB sites and illuminates how combinations of histone marks and nucleosomal elements cooperate to produce highly specific chromatin responses, such as those elicited following chromosome breaks.

KEOPS is an ancient protein complex required for the biosynthesis of N6-threonylcarbamoyladenosine (t(6)A), a universally conserved tRNA modification found on all ANN-codon recognizing tRNAs. KEOPS consist minimally of four essential subunits, namely the proteins Kae1, Bud32, Cgi121 and Pcc1, with yeast possessing the fifth essential subunit Gon7. Bud32, Cgi121, Pcc1 and Gon7 appear to have evolved to regulate the central t(6)A biosynthesis function of Kae1, but their precise function and mechanism of action remains unclear. Pcc1, in particular, binds directly to Kae1 and by virtue of its ability to form dimers in solution and in crystals, Pcc1 was inferred to function as a dimerization module for Kae1 and therefore KEOPS. We now present a 3.4 Å crystal structure of a dimeric Kae1-Pcc1 complex providing direct evidence that Pcc1 can bind and dimerize Kae1. Further biophysical analysis of a complete archaeal KEOPS complex reveals that Pcc1 facilitates KEOPS dimerization in vitro Interestingly, while Pcc1-mediated dimerization of KEOPS is required to support the growth of yeast, it is dispensable for t(6)A biosynthesis by archaeal KEOPS in vitro, raising the question of how precisely Pcc1-mediated dimerization impacts cellular biology.

Skp1-Cul1-F-box (SCF) E3 ligases play key roles in multiple cellular processes through ubiquitination and subsequent degradation of substrate proteins. Although Skp1 and Cul1 are invariant components of all SCF complexes, the 69 different human F-box proteins are variable substrate binding modules that determine specificity. SCF E3 ligases are activated in many cancers and inhibitors could have therapeutic potential. Here, we used phage display to develop specific ubiquitin-based inhibitors against two F-box proteins, Fbw7 and Fbw11. Unexpectedly, the ubiquitin variants bind at the interface of Skp1 and F-box proteins and inhibit ligase activity by preventing Cul1 binding to the same surface. Using structure-based design and phage display, we modified the initial inhibitors to generate broad-spectrum inhibitors that targeted many SCF ligases, or conversely, a highly specific inhibitor that discriminated between even the close homologs Fbw11 and Fbw1. We propose that most F-box proteins can be targeted by this approach for basic research and for potential cancer therapies.