Poly ADP ribosyltransferases play a critical role in DNA repair and cell death, and PARP1 is a particularly important therapeutic target for the treatment of breast cancer due to its synthetic lethal relationship with BRCA1/2. Numerous PARP1 inhibitors have been developed, and their efficacy in cancer treatment is attributed to both the inhibition of enzymatic activity and their ability to trap PARP1 on to the damaged DNA, which is cytotoxic. Of the clinical PARP inhibitors, talazoparib is the most effective at trapping PARP1 on damaged DNA. Biochemically, talazoparib is also suspected to be a potent inhibitor of PARP5a/b (tankyrase1/2), which is an important regulator of Wnt/β-catenin pathway. Here we show using competition experiments in cell lysate that, at a clinically relevant concentration, talazoparib can potentially bind and engage tankyrase1. Using surface plasmon resonance, we measured the dissociation constants of talazoparib, olaparib, niraparib and veliparib for their interaction with PARP1 and tankyrase1. The results show that talazoparib has strong affinity for PARP1 as well as uniquely strong affinity for tankyrase1. Finally, we used crystallography and hydrogen deuterium exchange mass spectroscopy to dissect the molecular mechanism of differential selectivity of these PARP1 inhibitors. From these data, we conclude that subtle differences between the ligand binding sites of PARP1 and tankyrase1, differences in the electrostatic nature of the ligands, protein dynamics, and ligand conformational energetics contribute to the different pharmacology of these PARP1 inhibitors. These results will help in the design of drugs to treat Wnt-β-catenin pathway-related cancers, such as colorectal cancers.

The phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling pathway is a frequently dysregulated pathway in human cancer, and PI3Kα is one of the most frequently mutated kinases in human cancer. A PI3Kα-selective inhibitor may provide the opportunity to spare patients the side effects associated with broader inhibition of the class I PI3K family. Here, we describe our efforts to discover a PI3Kα-selective inhibitor by applying structure-based drug design (SBDD) and computational analysis. A novel series of compounds, exemplified by 2,2-difluoroethyl (3S)-3-{[2'-amino-5-fluoro-2-(morpholin-4-yl)-4,5'-bipyrimidin-6-yl]amino}-3-(hydroxymethyl)pyrrolidine-1-carboxylate (1) (PF-06843195), with high PI3Kα potency and unique PI3K isoform and mTOR selectivity were discovered. We describe here the details of the design and synthesis program that lead to the discovery of 1.

S-Adenosyl-L-methionine (SAM) is an enzyme cofactor used in methyl transfer reactions and polyamine biosynthesis. The biosynthesis of SAM from ATP and L-methionine is performed by the methionine adenosyltransferase enzyme family (Mat; EC 2.5.1.6). Human methionine adenosyltransferase 2A (Mat2A), the extrahepatic isoform, is often deregulated in cancer. We identified a Mat2A inhibitor, PF-9366, that binds an allosteric site on Mat2A that overlaps with the binding site for the Mat2A regulator, Mat2B. Studies exploiting PF-9366 suggested a general mode of Mat2A allosteric regulation. Allosteric binding of PF-9366 or Mat2B altered the Mat2A active site, resulting in increased substrate affinity and decreased enzyme turnover. These data support a model whereby Mat2B functions as an inhibitor of Mat2A activity when methionine or SAM levels are high, yet functions as an activator of Mat2A when methionine or SAM levels are low. The ramification of Mat2A activity modulation in cancer cells is also described.

Tumors use tryptophan-catabolizing enzymes such as indoleamine 2,3-dioxygenase (IDO-1) to induce an immunosuppressive environment. IDO-1 is induced in response to inflammatory stimuli and promotes immune tolerance through effector T-cell anergy and enhanced Treg function. As such, IDO-1 is a nexus for the induction of a key immunosuppressive mechanism and represents an important immunotherapeutic target in oncology. Starting from HTS hit 5, IDO-1 inhibitor 6 (EOS200271/PF-06840003) has been developed. The structure activity relationship around 6 is described and rationalized using the X-ray crystal structure of 6 bound to human IDO-1, which shows that 6, differently from most of the IDO-1 inhibitors described so far, does not bind to the heme iron atom and has a novel binding mode. Clinical candidate 6 shows good potency in an IDO-1 human whole blood assay and also shows a very favorable ADME profile leading to favorable predicted human pharmacokinetic properties, including a predicted half-life of 16-19 h.

Autophagosome formation and specific substrate recruitment during autophagy require ligation of the ubiquitin-like protein (UBL) Atg8 to the head group of the lipid phosphatidylethanolamine. Atg8 lipidation is mediated by distinctive UBL cascades involving autophagy-specific E1, E2, and E3 enzymes that differ substantially in sequence from components of other UBL conjugation cascades. Structural studies are important for elucidating the roles of Atg proteins that regulate multiple steps involved in autophagy. This chapter describes methods to prepare and crystallize selected proteins and complexes involved in autophagy UBL conjugation pathways, as a guide for strategies for structural and biochemical characterization of Atg proteins.

Sulfonyl fluoride (SF)-based activity probes have become important tools in chemical biology. Herein, exploiting the relative chemical stability of SF to carry out a number of unprecedented SF-sparing functional group manipulations, we report the chemoselective synthesis of a toolbox of highly functionalized aryl SF monomers that we used to quickly prepare SF chemical biology probes. In addition to SF, the monomers bear an embedded click handle (a terminal alkyne that can perform copper(I)-mediated azide-alkyne cycloaddition). The monomers can be used either as fragments to prepare clickable SF analogues of drugs (biologically active compounds) bearing an aryl ring or, alternatively, attached to drugs as minimalist clickable aryl SF substituents.

Insults to ER homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2α phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as an important facet of eukaryotic translational control.

Central to most forms of autophagy are two ubiquitin-like proteins (UBLs), Atg8 and Atg12, which play important roles in autophagosome biogenesis, substrate recruitment to autophagosomes, and other aspects of autophagy. Typically, UBLs are activated by an E1 enzyme that (1) catalyzes adenylation of the UBL C terminus, (2) transiently covalently captures the UBL through a reactive thioester bond between the E1 active site cysteine and the UBL C terminus, and (3) promotes transfer of the UBL C terminus to the catalytic cysteine of an E2 conjugating enzyme. The E2, and often an E3 ligase enzyme, catalyzes attachment of the UBL C terminus to a primary amine group on a substrate. Here, we summarize our recent work reporting the structural and mechanistic basis for E1-E2 protein interactions in autophagy.

Ubiquitin-like proteins (UBLs) are activated, transferred and conjugated by E1-E2-E3 enzyme cascades. E2 enzymes for canonical UBLs such as ubiquitin, SUMO, and NEDD8 typically use common surfaces to bind to E1 and E3 enzymes. Thus, canonical E2s are required to disengage from E1 prior to E3-mediated UBL ligation. However, E1, E2, and E3 enzymes in the autophagy pathway are structurally and functionally distinct from canonical enzymes, and it has not been possible to predict whether autophagy UBL cascades are organized according to the same principles. Here, we address this question for the pathway mediating lipidation of the human autophagy UBL, LC3. We utilized bioinformatic and experimental approaches to identify a distinctive region in the autophagy E2, Atg3, that binds to the autophagy E3, Atg12∼Atg5-Atg16. Short peptides corresponding to this Atg3 sequence inhibit LC3 lipidation in vitro. Notably, the E3-binding site on Atg3 overlaps with the binding site for the E1, Atg7. Accordingly, the E3 competes with Atg7 for binding to Atg3, implying that Atg3 likely cycles back and forth between binding to Atg7 for loading with the UBL LC3 and binding to E3 to promote LC3 lipidation. The results show that common organizational principles underlie canonical and noncanonical UBL transfer cascades, but are established through distinct structural features.

Comment on: Taherbhoy AM, et al. Mol Cell 2011; 44:451–61

The diversity of ubiquitin (Ub)-dependent signaling is attributed to the ability of this small protein to form different types of covalently linked polyUb chains and to the existence of Ub binding proteins that interpret this molecular syntax. We used affinity capture/mass spectrometry to identify ALIX, a component of the ESCRT pathway, as a Ub binding protein. We report that the V domain of ALIX binds directly and selectively to K63-linked polyUb chains, exhibiting a strong preference for chains composed of more than three Ub. Sequence analysis identified two potential Ub binding sites on a single α-helical surface within the coiled-coil region of the V domain. Mutation of these putative Ub binding sites inhibited polyUb binding to the isolated V domain in vitro and impaired budding of lentiviruses. These data reveal an important role for K63 polyUb binding by ALIX in retroviral release.

Pathognomonic accumulation of ubiquitin (Ub) conjugates in human neurodegenerative diseases, such as Huntington’s disease, suggests that highly aggregated proteins interfere with 26S proteasome activity. In this paper, we examine possible mechanisms by which an N-terminal fragment of mutant huntingtin (htt; N-htt) inhibits 26S function. We show that ubiquitinated N-htt—whether aggregated or not—did not choke or clog the proteasome. Both Ub-dependent and Ub-independent proteasome reporters accumulated when the concentration of mutant N-htt exceeded a solubility threshold, indicating that stabilization of 26S substrates is not linked to impaired Ub conjugation. Above this solubility threshold, mutant N-htt was rapidly recruited to cytoplasmic inclusions that were initially devoid of Ub. Although synthetically polyubiquitinated N-htt competed with other Ub conjugates for access to the proteasome, the vast majority of mutant N-htt in cells was not Ub conjugated. Our data confirm that proteasomes are not directly impaired by aggregated N-terminal fragments of htt; instead, our data suggest that Ub accumulation is linked to impaired function of the cellular proteostasis network.

UBLs (ubiquitin-like proteins) are a major class of eukaryotic post-translational modifiers. UBLs are attached to numerous cellular proteins and to other macromolecules, thereby regulating a wide array of cellular processes. In this chapter we highlight a subset of UBLs and describe their regulatory roles in the cell.

The protein ubiquitin is an important post-translational modifier that regulates a wide variety of biological processes. In cells, ubiquitin is apportioned among distinct pools, which include a variety of free and conjugated species. Although maintenance of a dynamic and complex equilibrium among ubiquitin pools is crucial for cell survival, the tools necessary to quantify each cellular ubiquitin pool have been limited. We have developed a quantitative mass spectrometry approach to measure cellular concentrations of ubiquitin species using isotope-labeled protein standards and applied it to characterize ubiquitin pools in cells and tissues. Our method is convenient, adaptable and should be a valuable tool to facilitate our understanding of this important signaling molecule.

Genetic inactivation of autophagy in liver or brain leads to the appearance of ubiquitin- and p62-positive inclusions coincident with liver dysfunction and neurodegeneration, respectively. In our recent study we measured the abundance of polyubiquitin species in autophagy-deficient tissues and demonstrated that a specific polyubiquitin chain linkage is not the decisive autophagic substrate-targeting signal. Instead our data suggest that aggregation or oligomerization of a misfolded protein, in the absence of detectable polyubiquitin modification, is an important signal for autophagic degradation. We determined that the ubiquitin accumulation observed upon autophagy inhibition is caused by p62-mediated activation of Nrf2 resulting in global transcriptional changes to ubiquitin-associated genes. Thus, substrate polyubiquitination does not appear to be the major autophagy substrate-targeting signal and the primary role of p62 appears to be Nrf2 activation, not ubiquitin-dependent substrate degradation. Selective targeting of proteins to distinct subcellular machineries is fundamental to the regulation of cellular decisions between catabolism and anabolism. In particular, ubiquitin (Ub)-mediated targeting of proteins is essential in maintaining cellular homeostasis, and has been predominantly associated with nonlysosomal-mediated degradation. However, liver- and brain-specific autophagy knockout mice exhibit accumulation of Ub- and p62-positive inclusions suggesting that Ub-modification targets cargo for selective autophagic degradation. Subsequent reports have established the requirement of p62 oligomerization for the appearance of Ub-positive inclusions and suggest that p62 is a selective ‘autophagy adaptor’ for recognition and delivery of Ub-modified cargo to autophagosomes. Autophagy contributes to the detoxification of misfolded, aggregated proteins commonly associated with neurodegenerative disorders (Alzheimer disease, Parkinson disease, Huntington disease, and Lou Gehrig's disease [amyotrophic lateral sclerosis] and prion encephalophathies) and the presence of these misfolded proteins also correlates with the appearance of Ub- and p62-positive inclusions. Revealing how proteins are ‘marked’ for selective recognition by the autophagy machinery is essential. To determine whether specific polyubiquitin linkages target substrates for selective autophagic degradation we used Ub absolute quantification (AQUA) mass spectrometry to measure the amount of Ub that accumulates in both liver- and brain-specific autophagy knockout mouse models. We observed a global increase in all Ub isopeptide and non-isopeptide species and the results were similar between two different autophagy-deficient models in two separate tissues. Moreover the increased levels of Ub conjugates observed in samples of Atg7-null tissues were suppressed when this mutation was combined with deletions of p62 or Nrf2. These results are significant for two reasons. First, Nrf2 controls expression levels of detoxification enzymes by regulating genes that contain an antioxidant response element (ARE). If substrate polyubiquitination was the major autophagy targeting signal then we would not expect Ub accumulation in autophagy-deficient tissue to depend upon the presence of this transcription factor. Second, whereas p62 has been hypothesized to be an adaptor facilitating the autophagic degradation of ubiquitinated substrates, it has been previously shown that loss of p62 protects tissues from autophagy deficiency by preventing Nrf2 activation. Our observations are consistent with a role for p62 in controlling Nrf2—and not with a role as a simple Ub-dependent autophagy adaptor. Our findings suggest that accumulation of Ub during autophagy deficiency is a result of Nrf2 stress signaling downstream of the multifunctional scaffolding protein p62 rather than polyubiquitin functioning as the substrate targeting signal for selective autophagy. To identify Ub-related Nrf2 target genes that might explain the Ub dysregulation observed in autophagy-deficient tissues, we used bioinformatics, functional genomics and RT-PCR. Nrf2 (and its heterodimeric binding partner Maf) transcription factor-binding sites were found to be enriched among Ub-associated genes and the autophagy network. Within the autophagy network, there are 35 Ub-associated Nrf2 targets, and 22 are differentially affected in Atg7-/- mice compared to wild-type mice. Importantly, these changes are reversed in the Atg7-/Nrf2-double knockout mice confirming Nrf2-dependent regulation. Our overall interpretation of these data is that autophagy deficiency results in a stress response that activates Nrf2, globally affecting numerous Ub-related proteins. It is important to note that upregulation of ARE-containing genes has been reported for numerous neurodegenerative disorders. Understanding the mechanistic details of how Nrf2 binds ARE elements within Ub-associated genes, with what binding partner it binds, and Nrf2 nuclear/cytoplasmic trafficking in response to autophagy inhibition/p62 accumulation will reveal the physiological relevance of Nrf2-p62 Ub-associated signaling in neurodegenerative disorders. Since our results from mouse indicate that polyubiquitination is not the major autophagy substrate targeting signal, we used quantitative mass spectrometry and flow cytometry to measure Ub-modification of a selective autophagy substrate in an autophagy-regulatable stable cell line. Ub has been implicated to function as a signal in several forms of selective autophagy such as pexophagy, mitophagy and xenophagy. However, in these studies, the dependence of p62 and Ub on autophagic clearance was shown by immunofluorescence studies in which the absence of p62 results in the loss of substrate puncta formation or Ub colocalization with the substrate. Colocalization, though indicative of a signaling role, does not demonstrate covalent Ub modification of substrates. Our work addresses directly whether substrate modification by polyubiquitin chains targets proteins for selective autophagy and enables us to identify additional features of selective substrates. We developed a flow cytometry cell-based assay to measure selective autophagy using an autophagy-regulatable cell line stably expressing a bicistronic reporter construct containing both the misfolded, aggregation-prone protein huntingtin with an expanded polyglutamine tract (htt(Q47)) fused to GFP (green fluorescent protein), and the non-aggregation-prone protein cherry chFP (cherry fluorescent protein). Following autophagy shut-off, the reporter cell lines provide the ability to quantify and compare the relative accumulation of the two different fluorescent reporters, where autophagic selectivity is indicated by a ratio of greater than one. Using our flow cytometry assay we measured the selective accumulation of the aggregation-prone protein htt(Q47) compared to the non-aggregation prone protein chFP and although there is global accumulation of polyubiquitin chains following autophagy shut-off there is no increase in polyubiquitin-modified htt(Q47). Overall, these data demonstrate that aggregation or oligomerization of a misfolded protein, in the absence of detectable Ub-modification, results in selective accumulation following genetic ablation of autophagy. Based on these observations the major conclusion of our manuscript is that oligomerization targets proteins for selective autophagy in mammalian cells, and that polyubiquitination does not appear to be the major autophagy targeting signal; the primary role of p62 in autophagy deficiency appears to be Nrf2 activation, not Ub-dependent substrate degradation. Moreover, our data also demonstrate the broad importance of Nrf2-driven Ub signaling as an important cellular detoxification mechanism acting in addition to the arsenal of Nrf2-oxidative stress genes, and suggest that sustained activation could be detrimental to the cell (Fig. 1). Figure 1 Schematic of Ub-associated signaling regulated by the Nrf2-p62 axis. The Keap1-Cul3-Rbx1 E3 Ub ligase maintains low levels of Nrf2 in the cell. In response to oxidative insult, accumulation of misfolded, aggregation-prone proteins or additional unknown ...

Atg7 is a noncanonical, homodimeric E1 enzyme that interacts with the noncanonical E2 enzyme, Atg3, to mediate conjugation of the ubiquitin-like protein (UBL) Atg8 during autophagy. Here we report that the unique N-terminal domain of Atg7 (Atg7(NTD)) recruits a unique "flexible region" from Atg3 (Atg3(FR)). The structure of an Atg7 (NTD)-Atg3(FR) complex reveals hydrophobic residues from Atg3 engaging a conserved groove in Atg7, important for Atg8 conjugation. We also report the structure of the homodimeric Atg7 C-terminal domain, which is homologous to canonical E1s and bacterial antecedents. The structures, SAXS, and crosslinking data allow modeling of a full-length, dimeric (Atg7 similar to Atg8-Atg3)(2) complex. The model and biochemical data provide a rationale for Atg7 dimerization: Atg8 is transferred in trans from the catalytic cysteine of one Atg7 protomer to Atg3 bound to the N-terminal domain of the opposite Atg7 protomer within the homodimer. The studies reveal a distinctive E1 similar to UBL-E2 architecture for enzymes mediating autophagy.