Control of nutrient stress-induced metabolic reprogramming by PKCζ in tumorigenesis.

Tumor cells have high-energetic and anabolic needs and are known to adapt their metabolism to be able to survive and keep proliferating under conditions of nutrient stress. We show that PKCζ deficiency promotes the plasticity necessary for cancer cells to reprogram their metabolism to utilize glutamine through the serine biosynthetic pathway in the absence of glucose. PKCζ represses the expression of two key enzymes of the pathway, PHGDH and PSAT1, and phosphorylates PHGDH at key residues to inhibit its enzymatic activity. Interestingly, the loss of PKCζ in mice results in enhanced intestinal tumorigenesis and increased levels of these two metabolic enzymes, whereas patients with low levels of PKCζ have a poor prognosis. Furthermore, PKCζ and caspase-3 activities are correlated with PHGDH levels in human intestinal tumors. Taken together, this demonstrates that PKCζ is a critical metabolic tumor suppressor in mouse and human cancer.

Structure of a HOIP/E2~ubiquitin complex reveals RBR E3 ligase mechanism and regulation.

Ubiquitination is a central process affecting all facets of cellular signalling and function. A critical step in ubiquitination is the transfer of ubiquitin from an E2 ubiquitin-conjugating enzyme to a substrate or a growing ubiquitin chain, which is mediated by E3 ubiquitin ligases. RING-type E3 ligases typically facilitate the transfer of ubiquitin from the E2 directly to the substrate. The RING-between-RING (RBR) family of RING-type E3 ligases, however, breaks this paradigm by forming a covalent intermediate with ubiquitin similarly to HECT-type E3 ligases. The RBR family includes Parkin and HOIP, the central catalytic factor of the LUBAC (linear ubiquitin chain assembly complex). While structural insights into the RBR E3 ligases Parkin and HHARI in their overall auto-inhibited forms are available, no structures exist of intact fully active RBR E3 ligases or any of their complexes. Thus, the RBR mechanism of action has remained largely unknown. Here we present the first structure, to our knowledge, of the fully active human HOIP RBR in its transfer complex with an E2~ubiquitin conjugate, which elucidates the intricate nature of RBR E3 ligases. The active HOIP RBR adopts a conformation markedly different from that of auto-inhibited RBRs. HOIP RBR binds the E2~ubiquitin conjugate in an elongated fashion, with the E2 and E3 catalytic centres ideally aligned for ubiquitin transfer, which structurally both requires and enables a HECT-like mechanism. In addition, three distinct helix-IBR-fold motifs inherent to RBRs form ubiquitin-binding regions that engage the activated ubiquitin of the E2~ubiquitin conjugate and, surprisingly, an additional regulatory ubiquitin molecule. The features uncovered reveal critical states of the HOIP RBR E3 ligase cycle, and comparison with Parkin and HHARI suggests a general mechanism for RBR E3 ligases.

Rosetta Stone of NLR Innate Immunity.

The formation of NLR inflammasomes is a central step in the initiation of the innate immune response. Two recent publications describe the structure of the NAIP2-NLRC4 inflammasome and derive an elegant model of NLR inflammasome formation, whereby binding of the pathogen-molecule-bound NLR NAIP2 to NLRC4 leads to the activation of NLRC4 and initiation of self-propagating NLRC4 inflammasome formation.

Association of the breast cancer antiestrogen resistance protein 1 (BCAR1) and BCAR3 scaffolding proteins in cell signaling and antiestrogen resistance.

Most breast cancers are estrogen receptor-positive and treated with antiestrogens, but aberrant signaling networks can induce drug resistance. One of these networks involves the scaffolding protein BCAR1/p130CAS, which regulates cell growth and migration/invasion. A less investigated scaffolding protein that also confers antiestrogen resistance is the SH2 domain-containing protein BCAR3. BCAR1 and BCAR3 bind tightly to each other through their C-terminal domains, thus potentially connecting their associated signaling networks. However, recent studies using BCAR1 and BCAR3 interaction mutants concluded that association between the two proteins is not critical for many of their interrelated activities regulating breast cancer malignancy. We report that these previously used BCAR mutations fail to cause adequate loss-of-function of the complex. By using structure-based BCAR1 and BCAR3 mutants that lack the ability to interact, we show that BCAR3-induced antiestrogen resistance in MCF7 breast cancer cells critically depends on its ability to bind BCAR1. Interaction with BCAR3 increases the levels of phosphorylated BCAR1, ultimately potentiating BCAR1-dependent antiestrogen resistance. Furthermore, antiestrogen resistance in cells overexpressing BCAR1/BCAR3 correlates with increased ERK1/2 activity. Inhibiting ERK1/2 through overexpression of the regulatory protein PEA15 negates the resistance, revealing a key role for ERK1/2 in BCAR1/BCAR3-induced antiestrogen resistance. Reverse-phase protein array data show that PEA15 levels in invasive breast cancers correlate with patient survival, suggesting that PEA15 can override ERK1/2 activation by BCAR1/BCAR3 and other upstream regulators. We further uncovered that the BCAR3-related NSP3 can also promote antiestrogen resistance. Thus, strategies to disrupt BCAR1-BCAR3/NSP3 complexes and associated signaling networks could ultimately lead to new breast cancer therapies.

The Fas-FADD death domain complex structure unravels signalling by receptor clustering.

The death inducing signalling complex (DISC) formed by Fas receptor, FADD (Fas-associated death domain protein) and caspase 8 is a pivotal trigger of apoptosis. The Fas-FADD DISC represents a receptor platform, which once assembled initiates the induction of programmed cell death. A highly oligomeric network of homotypic protein interactions comprised of the death domains of Fas and FADD is at the centre of DISC formation. Thus, characterizing the mechanistic basis for the Fas-FADD interaction is crucial for understanding DISC signalling but has remained unclear largely because of a lack of structural data. We have successfully formed and isolated the human Fas-FADD death domain complex and report the 2.7 A crystal structure. The complex shows a tetrameric arrangement of four FADD death domains bound to four Fas death domains. We show that an opening of the Fas death domain exposes the FADD binding site and simultaneously generates a Fas-Fas bridge. The result is a regulatory Fas-FADD complex bridge governed by weak protein-protein interactions revealing a model where the complex itself functions as a mechanistic switch. This switch prevents accidental DISC assembly, yet allows for highly processive DISC formation and clustering upon a sufficient stimulus. In addition to depicting a previously unknown mode of death domain interactions, these results further uncover a mechanism for receptor signalling solely by oligomerization and clustering events.

The CARD plays a critical role in ASC foci formation and inflammasome signalling.

The ASC (apoptosis speck-like protein) is a key component of multimeric protein complexes that mediate inflammation and host defence. Comprising a PYD (Pyrin) domain and a CARD (caspase activation and recruitment domain), ASC functions downstream of NLRs (nucleotide-binding domain, leucine-rich repeat-containing receptors) and AIM2 (absent in melanoma 2) through the formation of supramolecular structures termed inflammasomes. However, the mechanism underlying ASC signalling and its dependency on oligomeric arrangements in inflammasome formation remain poorly understood. When expressed in cells, ASC forms discrete foci (called 'specks') typically with one speck per cell. We employed a BiFC (bimolecular fluorescence complementation) system to investigate and visualize ASC foci formation in living cells. We demonstrated that the CARD of ASC plays a central role in ASC inflammasome assembly, representing the minimal unit capable of forming foci in conjunction with the caspase 1 CARD. Mutational studies point to multiple surfaces on the ASC CARD and two predominant areas on the caspase 1 CARD mediating the formation of ASC/caspase 1 foci. The lack of foci formation for ASC CARD mutants correlates with a loss of IL-1ß (interleukin 1ß) processing in response to NLRP (NLR family, PYD domain-containing) 3 or AIM2 agonists in RAW264.7 cell reconstitution assays. Analogously, we show that productive formation of the Salmonella typhimurium-induced NLRC4 (NLR family CARD domain-containing protein 4) inflammasome is dependent on ASC-CARD-mediated platform formation. Thus the results of the present study depict a central role of CARDs in the formation of ASC signalling platforms and provide an important tool for investigation of CARD-dependent networks.

Whole-exome-sequencing-based discovery of human FADD deficiency.

Germline mutations in FASL and FAS impair Fas-dependent apoptosis and cause recessively or dominantly inherited autoimmune lymphoproliferative syndrome (ALPS). Patients with ALPS typically present with no other clinical phenotype. We investigated a large, consanguineous, multiplex kindred in which biological features of ALPS were found in the context of severe bacterial and viral disease, recurrent hepatopathy and encephalopathy, and cardiac malformations. By a combination of genome-wide linkage and whole-exome sequencing, we identified a homozygous missense mutation in FADD, encoding the Fas-associated death domain protein (FADD), in the patients. This FADD mutation decreases steady-state protein levels and impairs Fas-dependent apoptosis in vitro, accounting for biological ALPS phenotypes in vivo. It also impairs Fas-independent signaling pathways. The observed bacterial infections result partly from functional hyposplenism, and viral infections result from impaired interferon immunity. We describe here a complex clinical disorder, its genetic basis, and some of the key mechanisms underlying its pathogenesis. Our findings highlight the key role of FADD in Fas-dependent and Fas-independent signaling pathways in humans.

Design, synthesis and evaluation of inhibitor of apoptosis protein (IAP) antagonists that are highly selective for the BIR2 domain of XIAP.

We recently reported the systematic ligand-based rational design and synthesis of monovalent Smac mimetics that bind preferentially to the BIR2 domain of the anti-apoptotic protein XIAP. Expanded structure-activity relationship (SAR) studies around these peptidomimetics led to compounds with significantly improved selectivity (>60-fold) for the BIR2 domain versus the BIR3 domain of XIAP. The potent and highly selective IAP antagonist 8q (ML183) sensitized TRAIL-resistant prostate cancer cells to apoptotic cell death, highlighting the merit of this probe compound as a valuable tool to investigate the biology of XIAP.

Distinctive binding of three antagonistic peptides to the ephrin-binding pocket of the EphA4 receptor.

The EphA4 receptor tyrosine kinase interacts with ephrin ligands to regulate many processes, ranging from axon guidance and nerve regeneration to cancer malignancy. Thus antagonists that inhibit ephrin binding to EphA4 could be useful for a variety of research and therapeutic applications. In the present study we characterize the binding features of three antagonistic peptides (KYL, APY and VTM) that selectively target EphA4 among the Eph receptors. Isothermal titration calorimetry analysis demonstrated that all three peptides bind to the ephrin-binding domain of EphA4 with low micromolar affinity. Furthermore, the effects of a series of EphA4 mutations suggest that the peptides interact in different ways with the ephrin-binding pocket of EphA4. Chemical-shift changes observed by NMR spectroscopy upon binding of the KYL peptide involve many EphA4 residues, consistent with extensive interactions and possibly receptor conformational changes. Additionally, systematic replacement of each of the 12 amino acids of KYL and VTM identify the residues critical for EphA4, binding. The peptides exhibit a long half-life in cell culture medium which, with their substantial binding affinity and selectivity for EphA4, makes them excellent research tools to modulate EphA4 function.

Lipidation and PEGylation Strategies to Prolong the in Vivo Half-Life of a Nanomolar EphA4 Receptor Antagonist.

The EphA4 receptor tyrosine kinase plays a role in neurodegenerative diseases, inhibition of nerve regeneration, cancer progression and other diseases. Therefore, EphA4 inhibition has potential therapeutic value. Selective EphA4 kinase inhibitors are not available, but we identified peptide antagonists that inhibit ephrin ligand binding to EphA4 with high specificity. One of these peptides is the cyclic APY-d3 (ßAPYCVYRßASWSC-NH2), which inhibits ephrin-A5 ligand binding to EphA4 with low nanomolar binding affinity and is highly protease resistant. Here we describe modifications of APY-d3 that yield two different key derivatives with greatly increased half-lives in the mouse circulation, the lipidated APY-d3-laur8 and the PEGylated APY-d3-PEG4. These two derivatives inhibit ligand induced EphA4 activation in cells with sub-micromolar potency. Since they retain high potency and specificity for EphA4, lipidated and PEGylated APY-d3 derivatives represent new tools for discriminating EphA4 activities in vivo and for preclinical testing of EphA4 inhibition in animal disease models.

FLIP(L) induces caspase 8 activity in the absence of interdomain caspase 8 cleavage and alters substrate specificity.

Caspase 8 is an initiator caspase that is activated by death receptors to initiate the extrinsic pathway of apoptosis. Caspase 8 activation involves dimerization and subsequent interdomain autoprocessing of caspase 8 zymogens, and recently published work has established that elimination of the autoprocessing site of caspase 8 abrogates its pro-apoptotic function while leaving its proliferative function intact. The observation that the developmental abnormalities of caspase 8-deficient mice are shared by mice lacking the dimerization adapter FADD (Fas-associated death domain) or the caspase paralogue FLIP(L) [FLICE (FADD-like interleukin 1ß-converting enzyme)-inhibitory protein, long form] has led to the hypothesis that FADD-dependent formation of heterodimers between caspase 8 and FLIP(L) could mediate the developmental role of caspase 8. In the present study, using an inducible dimerization system we demonstrate that cleavage of the catalytic domain of caspase 8 is crucial for its activity in the context of activation by homodimerization. However, we find that use of FLIP(L) as a partner for caspase 8 in dimerization-induced activation rescues the requirement for intersubunit linker proteolysis in both protomers. Moreover, before processing, caspase 8 in complex with FLIP(L) does not generate a fully active enzyme, but an attenuated species able to process only selected natural substrates. Based on these results we propose a mechanism of caspase 8 activation by dimerization in the presence of FLIP(L), as well as a mechanism of caspase 8 functional divergence in apoptotic and non-apoptotic pathways.

Identification of an artificial peptide motif that binds and stabilizes reduced human DJ-1.

Although the precise biochemical function of DJ-1 remains unclear, it has been found to exert cytoprotective activity against oxidative stress. Cys106 is central to this function since it has a distinctly low pK(a) rendering it extremely susceptible for oxidation. This characteristic, however, also poses a severe hindrance to obtain reduced DJ-1 for in vitro investigation. We have developed an approach to produce recombinant human DJ-1 in its reduced form as a bona fide basis for exploring the redox capacities of the protein. We solved the crystal structure of this DJ-1 at 1.56Å resolution, allowing us to capture Cys106 in the reduced state for the first time. The dimeric structure reveals one molecule of DJ-1 in its reduced state while the other exhibits the characteristics of a mono-oxygenated cysteine. Comparison with previous structures indicates the absence of redox dependent global conformational changes in DJ-1. The capture of reduced Cys106 is facilitated by stabilization within the putative active site achieved through a glutamate side chain. This side chain is provided by a crystallographic neighbor as part of a 'Leu-Glu' motif, which was added to the C-terminus of DJ-1. In the structure this motif binds DJ-1 in close proximity to Cys106 through extended hydrophilic and hydrophobic interactions depicting a distinct binding pocket, which can serve as a basis for compound development targeting DJ-1.

Design, synthesis and evaluation of monovalent Smac mimetics that bind to the BIR2 domain of the anti-apoptotic protein XIAP.

We report the systematic rational design and synthesis of new monovalent Smac mimetics that bind preferentially to the BIR2 domain of the anti-apoptotic protein XIAP. Characterization of compounds in vitro (including 9i; ML101) led to the determination of key structural requirements for BIR2 binding affinity. Compounds 9h and 9j sensitized TRAIL-resistant breast cancer cells to apoptotic cell death, highlighting the value of these probe compounds as tools to investigate the biology of XIAP.

Structure of the apoptotic protease-activating factor 1 bound to ADP.

Apoptosis is executed by caspases, which undergo proteolytic activation in response to cell death stimuli. The apoptotic protease-activating factor 1 (Apaf-1) controls caspase activation downstream of mitochondria. During apoptosis, Apaf-1 binds to cytochrome c and in the presence of ATP/dATP forms an apoptosome, leading to the recruitment and activation of the initiator caspase, caspase-9 (ref. 2). The mechanisms underlying Apaf-1 function are largely unknown. Here we report the 2.2-A crystal structure of an ADP-bound, WD40-deleted Apaf-1, which reveals the molecular mechanism by which Apaf-1 exists in an inactive state before ATP binding. The amino-terminal caspase recruitment domain packs against a three-layered alpha/beta fold, a short helical motif and a winged-helix domain, resulting in the burial of the caspase-9-binding interface. The deeply buried ADP molecule serves as an organizing centre to strengthen interactions between these four adjoining domains, thus locking Apaf-1 in an inactive conformation. Apaf-1 binds to and hydrolyses ATP/dATP and their analogues. The binding and hydrolysis of nucleotides seem to drive conformational changes that are essential for the formation of the apoptosome and the activation of caspase-9.

Activation and specificity of human caspase-10.

Two apical caspases, caspase-8 and -10, are involved in the extrinsic death receptor pathway in humans, but it is mainly caspase-8 in its apoptotic and nonapoptotic functions that has been an intense research focus. In this study we concentrate on caspase-10, its mechanism of activation, and the role of the intersubunit cleavage. Our data obtained through in vitro dimerization assays strongly suggest that caspase-10 follows the proximity-induced dimerization model for apical caspases. Furthermore, we compare the specificity and activity of the wild-type protease with a mutant incapable of autoprocessing by using positional scanning substrate analysis and cleavage of natural protein substrates. These experiments reveal a striking difference between the wild type and the mutant, leading us to hypothesize that the single chain enzyme has restricted activity on most proteins but high activity on the proapoptotic protein Bid, potentially supporting a prodeath role for both cleaved and uncleaved caspase-10.

Caspase mechanisms.

The main effectors of apoptosis encompass proteases from the caspase family, which reside as latent precursors in most nucleated animal cells. The apoptotic caspases constitute a minimal two-step signaling pathway. The apical (initiator) caspases are activated within oligomeric signaling complexes in response to apoptotic stimuli. Their mechanism of activation probably results from proximity-induced clustering to the dimeric active forms. Once activated, the apical caspases directly activate the executioner (effector) caspases by limited proteolytic cleavage. The distinct activation mechanisms explain how an apoptotic stimulus is converted to proteolytic activity, and how this activity is amplified to allow for limited proteolysis of the dozens of protein substrates whose cleavage is required for efficient apoptosis.

A structure of the human apoptosome at 12.8 A resolution provides insights into this cell death platform.

Apaf-1 and cytochrome c coassemble in the presence of dATP to form the apoptosome. We have determined a structure of the apoptosome at 12.8 A resolution by using electron cryomicroscopy and single-particle methods. We then docked appropriate crystal structures into the map to create an accurate domain model. Thus, we found that seven caspase recruitment domains (CARDs) form a central ring within the apoptosome. At a larger radius, seven copies of the nucleotide binding and oligomerization domain (NOD) associate laterally to form the hub, which encircles the CARD ring. Finally, an arm-like helical domain (HD2) links each NOD to a pair of beta propellers, which bind a single cytochrome c. This model provides insights into the roles of dATP and cytochrome c in assembly. Our structure also reveals how a CARD ring and the central hub combine to create a platform for procaspase-9 activation.

Structural Studies of ERK2 Protein Complexes.

ERK1 and ERK2 (ERK1/2) are the primary effector kinases of the RAS-RAF-MEK-ERK signaling pathway. A variety of substrates and regulatory partners associate with ERK1/2 through distinct D-peptide- and DEF-docking sites on their kinase domains. While understanding of D-peptides that bind to ERK1/2 has become increasingly clear over the last decade, only more recently have structures of proteins interacting with other binding sites on ERK1/2 become available. PEA-15 is a 130-residue ERK1/2 regulator that engages both the D-peptide- and DEF-docking sites of ERK kinases, and directly sequesters the ERK2 activation loop in various different phosphorylation states. Here we describe the methods used to derive crystallization-grade complexes of ERK2-PEA-15, which may also be adapted for other regulators that associate with the activation loop of ERK1/2.

Structure-Guided Strategy for the Development of Potent Bivalent ERK Inhibitors.

ERK is the effector kinase of the RAS-RAF-MEK-ERK signaling cascade, which promotes cell transformation and malignancy in many cancers and is thus a major drug target in oncology. Kinase inhibitors targeting RAF or MEK are already used for the treatment of certain cancers, such as melanoma. Although the initial response to these drugs can be dramatic, development of drug resistance is a major challenge, even with combination therapies targeting both RAF and MEK. Importantly, most resistance mechanisms still rely on activation of the downstream effector kinase ERK, making it a promising target for drug development efforts. Here, we report the design and structural/functional characterization of a set of bivalent ERK inhibitors that combine a small molecule inhibitor that binds to the ATP-binding pocket with a peptide that selectively binds to an ERK protein interaction surface, the D-site recruitment site (DRS). Our studies show that the lead bivalent inhibitor, SBP3, has markedly improved potency compared to the small molecule inhibitor alone. Unexpectedly, we found that SBP3 also binds to several ERK-related kinases that contain a DRS, highlighting the importance of experimentally verifying the predicted specificity of bivalent inhibitors. However, SBP3 does not target any other kinases belonging to the same CMGC branch of the kinome. Additionally, our modular click chemistry inhibitor design facilitates the generation of different combinations of small molecule inhibitors with ERK-targeting peptides.

Modifications of a Nanomolar Cyclic Peptide Antagonist for the EphA4 Receptor To Achieve High Plasma Stability.

EphA4 is a receptor tyrosine kinase with a critical role in repulsive axon guidance and synaptic function. However, aberrant EphA4 activity can inhibit neural repair after injury and exacerbate neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer's. We previously identified the cyclic peptide APY-d2 (APYCVYRßASWSC-nh2, containing a disulfide bond) as a potent and selective EphA4 antagonist. However, APY-d2 lacks sufficient plasma stability to be useful for EphA4 inhibition in vivo through peripheral administration. Using structure-activity relationship studies, we show that protecting the peptide N-terminus from proteolytic degradation dramatically increases the persistence of the active peptide in plasma and that a positively charged peptide N-terminus is essential for high EphA4 binding affinity. Among several improved APY-d2 derivatives, the cyclic peptides APY-d3 (ßAPYCVYRßASWSC-nh2) and APY-d4 (ßAPYCVYRßAEWEC-nh2) combine high stability in plasma and cerebrospinal fluid with slightly enhanced potency. These properties make them valuable research tools and leads toward development of therapeutics for neurological diseases.