Disruption of XIAP-RIP2 Association Blocks NOD2-Mediated Inflammatory Signaling.

Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling. Rather, the conformation of the RIP2 kinase domain functions to regulate binding to the XIAP-BIR2 domain. Effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction. Finally, we identify NOD2 signaling and XIAP-dependent ubiquitination sites on RIP2 and show that mutating these lysine residues adversely affects NOD2 pathway signaling. Overall, these results reveal a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signaling and provide a molecular basis for the design of innovative therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors.

AzidoTMT Enables Direct Enrichment and Highly Multiplexed Quantitation of Proteome-Wide Functional Residues.

Recent advances in targeted covalent inhibitors have aroused significant interest for their potential in drug development for difficult therapeutic targets. Proteome-wide profiling of functional residues is an integral step of covalent drug discovery aimed at defining actionable sites and evaluating compound selectivity in cells. A classical workflow for this purpose is called IsoTOP-ABPP, which employs an activity-based probe and two isotopically labeled azide-TEV-biotin tags to mark, enrich, and quantify proteome from two samples. Here we report a novel isobaric 11plex-AzidoTMT reagent and a new workflow, named AT-MAPP, that significantly expands multiplexing power as compared to the original isoTOP-ABPP. We demonstrate its application in identifying cysteine on- and off-targets using a KRAS G12C covalent inhibitor ARS-1620. However, changes in some of these hits can be explained by modulation at the protein and post-translational levels. Thus, it would be crucial to interrogate site-level bona fide changes in concurrence to proteome-level changes for corroboration. In addition, we perform a multiplexed covalent fragment screening using four acrylamide-based compounds as a proof-of-concept. This study identifies a diverse set of liganded cysteine residues in a compound-dependent manner with an average hit rate of 0.07% in intact cell. Lastly, we screened 20 sulfonyl fluoride-based compounds to demonstrate that the AT-MAPP assay is flexible for noncysteine functional residues such as tyrosine and lysine. Overall, we envision that 11plex-AzidoTMT will be a useful addition to the current toolbox for activity-based protein profiling and covalent drug development.

Primary Amine Tethered Small Molecules Promote the Degradation of X-Linked Inhibitor of Apoptosis Protein.

We hypothesized that the proximity-driven ubiquitylation of E3-interacting small molecules could affect the degradation of E3 ubiquitin ligases. A series of XIAP BIR2 domain-binding small molecules was modified to append a nucleophilic primary amine. This modification transforms XIAP binders into inducers of XIAP degradation. The degradation of XIAP is E1- and proteasome-dependent, dependent on the ligase function of XIAP, and is rescued by subtle modifications of the small molecule that would obviate ubiquitylation. We demonstrate in vitro ubiquitylation of the small molecule that is dependent on its interaction with XIAP. Taken together, these results demonstrate the designed ubiquitylation of an engineered small molecule and a novel approach for the degradation of E3 ubiquitin ligases.

Phosphorylation of NLRC4 is critical for inflammasome activation.

NLRC4 is a cytosolic member of the NOD-like receptor family that is expressed in innate immune cells. It senses indirectly bacterial flagellin and type III secretion systems, and responds by assembling an inflammasome complex that promotes caspase-1 activation and pyroptosis. Here we use knock-in mice expressing NLRC4 with a carboxy-terminal 3×Flag tag to identify phosphorylation of NLRC4 on a single, evolutionarily conserved residue, Ser 533, following infection of macrophages with Salmonella enterica serovar Typhimurium (also known as Salmonella typhimurium). Western blotting with a NLRC4 phospho-Ser 533 antibody confirmed that this post-translational modification occurs only in the presence of stimuli known to engage NLRC4 and not the related protein NLRP3 or AIM2. Nlrc4(-/-) macrophages reconstituted with NLRC4 mutant S533A, unlike those reconstituted with wild-type NLRC4, did not activate caspase-1 and pyroptosis in response to S. typhimurium, indicating that S533 phosphorylation is critical for NLRC4 inflammasome function. Conversely, phosphomimetic NLRC4 S533D caused rapid macrophage pyroptosis without infection. Biochemical purification of the NLRC4-phosphorylating activity and a screen of kinase inhibitors identified PRKCD (PKCδ) as a candidate NLRC4 kinase. Recombinant PKCδ phosphorylated NLRC4 S533 in vitro, immunodepletion of PKCδ from macrophage lysates blocked NLRC4 S533 phosphorylation in vitro, and Prkcd(-/-) macrophages exhibited greatly attenuated caspase-1 activation and IL-1ß secretion specifically in response to S. typhimurium. Phosphorylation-defective NLRC4 S533A failed to recruit procaspase-1 and did not assemble inflammasome specks during S. typhimurium infection, so phosphorylation of NLRC4 S533 probably drives conformational changes necessary for NLRC4 inflammasome activity and host innate immunity.

OTUB1 modulates c-IAP1 stability to regulate signalling pathways.

The cellular inhibitor of apoptosis (c-IAP) proteins are E3 ubiquitin ligases that are critical regulators of tumour necrosis factor (TNF) receptor (TNFR)-mediated signalling. Through their E3 ligase activity c-IAP proteins promote ubiquitination of receptor-interaction protein 1 (RIP1), NF-κB-inducing kinase (NIK) and themselves, and regulate the assembly of TNFR signalling complexes. Consequently, in the absence of c-IAP proteins, TNFR-mediated activation of NF-κB and MAPK pathways and the induction of gene expression are severely reduced. Here, we describe the identification of OTUB1 as a c-IAP-associated deubiquitinating enzyme that regulates c-IAP1 stability. OTUB1 disassembles K48-linked polyubiquitin chains from c-IAP1 in vitro and in vivo within the TWEAK receptor-signalling complex. Downregulation of OTUB1 promotes TWEAK- and IAP antagonist-stimulated caspase activation and cell death, and enhances c-IAP1 degradation. Furthermore, knockdown of OTUB1 reduces TWEAK-induced activation of canonical NF-κB and MAPK signalling pathways and modulates TWEAK-induced gene expression. Finally, suppression of OTUB1 expression in zebrafish destabilizes c-IAP (Birc2) protein levels and disrupts fish vasculature. These results suggest that OTUB1 regulates NF-κB and MAPK signalling pathways and TNF-dependent cell death by modulating c-IAP1 stability.

Mass spectrometric quantification of histone post-translational modifications by a hybrid chemical labeling method.

Mass spectrometry is a powerful alternative to antibody-based methods for the analysis of histone post-translational modifications (marks). A key development in this approach was the deliberate propionylation of histones to improve sequence coverage across the lysine-rich and hydrophilic tails that bear most modifications. Several marks continue to be problematic however, particularly di- and tri-methylated lysine 4 of histone H3 which we found to be subject to substantial and selective losses during sample preparation and liquid chromatography-mass spectrometry. We developed a new method employing a "one-pot" hybrid chemical derivatization of histones, whereby an initial conversion of free lysines to their propionylated forms under mild aqueous conditions is followed by trypsin digestion and labeling of new peptide N termini with phenyl isocyanate. High resolution mass spectrometry was used to collect qualitative and quantitative data, and a novel web-based software application (Fishtones) was developed for viewing and quantifying histone marks in the resulting data sets. Recoveries of 53 methyl, acetyl, and phosphoryl marks on histone H3.1 were improved by an average of threefold overall, and over 50-fold for H3K4 di- and tri-methyl marks. The power of this workflow for epigenetic research and drug discovery was demonstrated by measuring quantitative changes in H3K4 trimethylation induced by small molecule inhibitors of lysine demethylases and siRNA knockdown of epigenetic modifiers ASH2L and WDR5.

Ubiquitination profiling identifies sensitivity factors for IAP antagonist treatment.

Evasion of cell death is one crucial capability acquired by tumour cells to ward-off anti-tumour therapies and represents a fundamental challenge to sustaining clinical efficacy for currently available agents. Inhibitor of apoptosis (IAP) proteins use their ubiquitin E3 ligase activity to promote cancer cell survival by mediating proliferative signalling and blocking cell death in response to diverse stimuli. Using immunoaffinity enrichment and MS, ubiquitination sites on thousands of proteins were profiled upon initiation of cell death by IAP antagonists in IAP antagonist-sensitive and -resistant breast cancer cell lines. Our analyses identified hundreds of proteins with elevated levels of ubiquitin-remnant [K-GG (Lys-Gly-Gly)] peptides upon activation of cell death by the IAP antagonist BV6. The majority of these were observed in BV6-sensitive, but not-resistant, cells. Among these were known pro-apoptotic regulators, including CYC (cytochrome c), RIP1 (receptor-interacting protein 1) and a selection of proteins known to reside in the mitochondria or regulate NF-κB (nuclear factor κB) signalling. Analysis of early time-points revealed that IAP antagonist treatment stimulated rapid ubiquitination of NF-κB signalling proteins, including TRAF2 [TNF (tumour necrosis factor) receptor-associated factor 2], HOIL-1 (haem-oxidized iron-regulatory protein 2 ubiquitin ligase-1), NEMO (NF-κB essential modifier), as well as c-IAP1 (cellular IAP1) auto-ubiquitination. Knockdown of several NF-κB pathway members reduced BV6-induced cell death and TNF production in sensitive cell lines. Importantly, RIP1 was found to be constitutively ubiquitinated in sensitive breast-cancer cell lines at higher basal level than in resistant cell lines. Together, these data show the diverse and temporally defined roles of protein ubiquitination following IAP-antagonist treatment and provide critical insights into predictive diagnostics that may enhance clinical efficacy.

c-IAP1 and UbcH5 promote K11-linked polyubiquitination of RIP1 in TNF signalling.

Ubiquitin ligases are critical components of the ubiquitination process that determine substrate specificity and, in collaboration with E2 ubiquitin-conjugating enzymes, regulate the nature of polyubiquitin chains assembled on their substrates. Cellular inhibitor of apoptosis (c-IAP1 and c-IAP2) proteins are recruited to TNFR1-associated signalling complexes where they regulate receptor-stimulated NF-κB activation through their RING domain ubiquitin ligase activity. Using a directed yeast two-hybrid screen, we found several novel and previously identified E2 partners of IAP RING domains. Among these, the UbcH5 family of E2 enzymes are critical regulators of the stability of c-IAP1 protein following destabilizing stimuli such as TWEAK or CD40 signalling or IAP antagonists. We demonstrate that c-IAP1 and UbcH5 family promote K11-linked polyubiquitination of receptor-interacting protein 1 (RIP1) in vitro and in vivo. We further show that TNFα-stimulated NF-κB activation involves endogenous K11-linked ubiquitination of RIP1 within the TNFR1 signalling complex that is c-IAP1 and UbcH5 dependent. Lastly, NF-κB essential modifier efficiently binds K11-linked ubiquitin chains, suggesting that this ubiquitin linkage may have a signalling role in the activation of proliferative cellular pathways.

Simultaneous substrate and ubiquitin modification recognition by bispecific antibodies enables detection of ubiquitinated RIP1 and RIP2.

Ubiquitination is a posttranslational modification that is crucial for the dynamic regulation of diverse signaling pathways. To enhance our understanding of ubiquitination-mediated signaling, we generated a new class of bispecific antibodies that combine recognition of ubiquitination substrates and specific polyubiquitin linkages. RIP1-K63 and RIP1-linear (Lin) linkage polyubiquitin bispecific antibodies detected linkage-specific ubiquitination of the proinflammatory kinase RIP1 in cells and in tissues and revealed RIP1 ubiquitination by immunofluorescence. Similarly, ubiquitination of the RIP1-related kinase RIP2 with K63 or linear linkages was specifically detected with the RIP2-K63 and RIP2-Lin bispecific antibodies, respectively. Furthermore, using the RIP2-K63 and RIP2-Lin bispecific antibodies, we found prominent K63-linked and linear RIP2 ubiquitination in samples from patients with ulcerative colitis and Crohn's disease. We also developed a bispecific antibody (K63-Lin) that simultaneously recognizes K63-linked and linear ubiquitination of components of various signaling pathways. Together, these bispecific antibodies represent a new class of reagents with the potential to be developed for the detection of inflammatory biomarkers.

Furin-cleaved proprotein convertase subtilisin/kexin type 9 (PCSK9) is active and modulates low density lipoprotein receptor and serum cholesterol levels.

Proprotein convertase subtilisin/kexin 9 (PCSK9) regulates plasma LDL cholesterol levels by regulating the degradation of LDL receptors. Another proprotein convertase, furin, cleaves PCSK9 at Arg(218)-Gln(219) in the surface-exposed "218 loop." This cleaved form circulates in blood along with the intact form, albeit at lower concentrations. To gain a better understanding of how cleavage affects PCSK9 function, we produced recombinant furin-cleaved PCSK9 using antibody Ab-3D5, which binds the intact but not the cleaved 218 loop. Using Ab-3D5, we also produced highly purified hepsin-cleaved PCSK9. Hepsin cleaves PCSK9 at Arg(218)-Gln(219) more efficiently than furin but also cleaves at Arg(215)-Phe(216). Further analysis by size exclusion chromatography and mass spectrometry indicated that furin and hepsin produced an internal cleavage in the 218 loop without the loss of the N-terminal segment (Ser(153)-Arg(218)), which remained attached to the catalytic domain. Both furin- and hepsin-cleaved PCSK9 bound to LDL receptor with only 2-fold reduced affinity compared with intact PCSK9. Moreover, they reduced LDL receptor levels in HepG2 cells and in mouse liver with only moderately lower activity than intact PCSK9, consistent with the binding data. Single injection into mice of furin-cleaved PCSK9 resulted in significantly increased serum cholesterol levels, approaching the increase by intact PCSK9. These findings indicate that circulating furin-cleaved PCSK9 is able to regulate LDL receptor and serum cholesterol levels, although somewhat less efficiently than intact PCSK9. Therapeutic anti-PCSK9 approaches that neutralize both forms should be the most effective in preserving LDL receptors and in lowering plasma LDL cholesterol.

Improved quantitative mass spectrometry methods for characterizing complex ubiquitin signals.

Ubiquitinated substrates can be recruited to macromolecular complexes through interactions between their covalently bound ubiquitin (Ub) signals and Ub receptor proteins. To develop a functional understanding of the Ub system in vivo, methods are needed to determine the composition of Ub signals on individual substrates and in protein mixtures. Mass spectrometry has emerged as an important tool for characterizing the various forms of Ub. In the Ubiquitin-AQUA approach, synthetic isotopically labeled internal standard peptides are used to quantify unbranched peptides and the branched -GG signature peptides generated by trypsin digestion of Ub signals. Here we have built upon existing methods and established a comprehensive platform for the characterization of Ub signals. Digested peptides and isotopically labeled standards are analyzed either by selected reaction monitoring on a QTRAP mass spectrometer or by narrow window extracted ion chromatograms on a high resolution LTQ-Orbitrap. Additional peptides are now monitored to account for the N terminus of ubiquitin, linear polyUb chains, the peptides surrounding K33 and K48, and incomplete digestion products. Using this expanded battery of peptides, the total amount of Ub in a sample can be determined from multiple loci within the protein, minimizing possible confounding effects of complex Ub signals, digestion abnormalities, or use of mutant Ub in experiments. These methods have been useful for the characterization of in vitro, multistage ubiquitination and have now been extended to reactions catalyzed by multiple E2 enzymes. One question arising from in vitro studies is whether individual protein substrates in cells may be modified by multiple forms of polyUb. Here we have taken advantage of recently developed polyubiquitin linkage-specific antibodies recognizing K48- and K63-linked polyUb chains, coupled with these mass spectrometry methods, to further evaluate the abundance of mixed linkage Ub substrates in cultured mammalian cells. By combining these two powerful tools, we show that polyubiquitinated substrates purified from cells can be modified by mixtures of K48, K63, and K11 linkages.

An RNA Damage Response Network Mediates the Lethality of 5-FU in Clinically Relevant Tumor Types.

5-fluorouracil (5-FU) is a successful and broadly used anti-cancer therapeutic. A major mechanism of action of 5-FU is thought to be through thymidylate synthase (TYMS) inhibition resulting in dTTP depletion and activation of the DNA damage response. This suggests that 5-FU should synergize with other DNA damaging agents. However, we found that combinations of 5-FU and oxaliplatin or irinotecan failed to display any evidence of synergy in clinical trials, and resulted in sub-additive killing in a panel of colorectal cancer (CRC) cell lines. In seeking to understand this antagonism, we unexpectedly found that an RNA damage response during ribosome biogenesis dominates the drug's efficacy in tumor types for which 5-FU shows clinical benefit. 5-FU has an inherent bias for RNA incorporation, and blocking this greatly reduced drug-induced lethality, indicating that accumulation of damaged RNA is more deleterious than the lack of new RNA synthesis. Using 5-FU metabolites that specifically incorporate into either RNA or DNA revealed that CRC cell lines and patient-derived colorectal cancer organoids are inherently more sensitive to RNA damage. This difference held true in cell lines from other tissues in which 5-FU has shown clinical utility, whereas cell lines from tumor tissues that lack clinical 5-FU responsiveness typically showed greater sensitivity to the drug's DNA damage effects. Analysis of changes in the phosphoproteome and ubiquitinome shows RNA damage triggers the selective ubiquitination of multiple ribosomal proteins leading to autophagy-dependent rRNA catabolism and proteasome-dependent degradation of ubiquitinated ribosome proteins. Further, RNA damage response to 5-FU is selectively enhanced by compounds that promote ribosome biogenesis, such as KDM2A inhibitors. These results demonstrate the presence of a strong RNA damage response linked to apoptotic cell death, with clear utility of combinatorially targeting this response in cancer therapy.

B and T lymphocyte attenuator regulates B cell receptor signaling by targeting Syk and BLNK.

B and T lymphocyte attenuator (BTLA) functions as a negative regulator of T cell activation and proliferation. Although the role of BTLA in regulating T cell responses has been characterized, a thorough investigation into the precise molecular mechanisms involved in BTLA-mediated lymphocyte attenuation and, more specifically, its role in regulating B cell activation has not been presented. In this study, we have begun to elucidate the biochemical mechanisms by which BTLA functions to inhibit B cell activation. We describe the cell surface expression of BTLA on various human B cell subsets and confirm its ability to attenuate B cell proliferation upon associating with its known ligand, herpesvirus entry mediator (HVEM). BTLA associates with the BCR and, upon binding to HVEM, recruits the tyrosine phosphatase Src homology 2 domain-containing phosphatase 1 and reduces activation of signaling molecules downstream of the BCR. This is exemplified by a quantifiable decrease in tyrosine phosphorylation of the protein tyrosine kinase Syk, as measured by absolute quantification mass spectrometry. Furthermore, effector molecules downstream of BCR signaling, including the B cell linker protein, phospholipase Cgamma2, and NF-kappaB, display decreased activation and nuclear translocation, respectively, after BTLA activation by HVEM. These results begin to provide insight into the mechanism by which BTLA negatively regulates B cell activation and indicates that BTLA is an inhibitory coreceptor of the BCR signaling pathway and attenuates B cell activation by targeting the downstream signaling molecules Syk and B cell linker protein.

Ubiquitin binding modulates IAP antagonist-stimulated proteasomal degradation of c-IAP1 and c-IAP2(1).

A family of anti-apoptotic regulators known as IAP (inhibitor of apoptosis) proteins interact with multiple cellular partners and inhibit apoptosis induced by a variety of stimuli. c-IAP (cellular IAP) 1 and 2 are recruited to TNFR1 (tumour necrosis factor receptor 1)-associated signalling complexes, where they mediate receptor-induced NF-kappaB (nuclear factor kappaB) activation. Additionally, through their E3 ubiquitin ligase activities, c-IAP1 and c-IAP2 promote proteasomal degradation of NIK (NF-kappaB-inducing kinase) and regulate the non-canonical NF-kappaB pathway. In the present paper, we describe a novel ubiquitin-binding domain of IAPs. The UBA (ubiquitin-associated) domain of IAPs is located between the BIR (baculovirus IAP repeat) domains and the CARD (caspase activation and recruitment domain) or the RING (really interesting new gene) domain of c-IAP1 and c-IAP2 or XIAP (X-linked IAP) respectively. The c-IAP1 UBA domain binds mono-ubiquitin and Lys(48)- and Lys(63)-linked polyubiquitin chains with low-micromolar affinities as determined by surface plasmon resonance or isothermal titration calorimetry. NMR analysis of the c-IAP1 UBA domain-ubiquitin interaction reveals that this UBA domain binds the classical hydrophobic patch surrounding Ile(44) of ubiquitin. Mutations of critical amino acid residues in the highly conserved MGF (Met-Gly-Phe) binding loop of the UBA domain completely abrogate ubiquitin binding. These mutations in the UBA domain do not overtly affect the ubiquitin ligase activity of c-IAP1 or the participation of c-IAP1 and c-IAP2 in the TNFR1 signalling complex. Treatment of cells with IAP antagonists leads to proteasomal degradation of c-IAP1 and c-IAP2. Deletion or mutation of the UBA domain decreases this degradation, probably by diminishing the interaction of the c-IAPs with the proteasome. These results suggest that ubiquitin binding may be an important mechanism for rapid turnover of auto-ubiquitinated c-IAP1 and c-IAP2.

Coordinated ubiquitination and phosphorylation of RIP1 regulates necroptotic cell death.

Proper regulation of cell death signaling is crucial for the maintenance of homeostasis and prevention of disease. A caspase-independent regulated form of cell death called necroptosis is rapidly emerging as an important mediator of a number of human pathologies including inflammatory bowel disease and ischemia-reperfusion organ injury. Activation of necroptotic signaling through TNF signaling or organ injury leads to the activation of kinases receptor-interacting protein kinases 1 and 3 (RIP1 and RIP3) and culminates in inflammatory cell death. We found that, in addition to phosphorylation, necroptotic cell death is regulated by ubiquitination of RIP1 in the necrosome. Necroptotic RIP1 ubiquitination requires RIP1 kinase activity, but not necroptotic mediators RIP3 and MLKL (mixed lineage kinase-like). Using immunoaffinity enrichment and mass spectrometry, we profiled numerous ubiquitination events on RIP1 that are triggered during necroptotic signaling. Mutation of a necroptosis-related ubiquitination site on RIP1 reduced necroptotic cell death and RIP1 ubiquitination and phosphorylation, and disrupted the assembly of RIP1 and RIP3 in the necrosome, suggesting that necroptotic RIP1 ubiquitination is important for maintaining RIP1 kinase activity in the necrosome complex. We also observed RIP1 ubiquitination in injured kidneys consistent with a physiological role of RIP1 ubiquitination in ischemia-reperfusion disease. Taken together, these data reveal that coordinated and interdependent RIP1 phosphorylation and ubiquitination within the necroptotic complex regulate necroptotic signaling and cell death.

NLRP3 recruitment by NLRC4 during Salmonella infection.

NLRC4 and NLRP3, of the NOD-like receptor (NLR) family of intracellular proteins, are expressed in innate immune cells and are thought to nucleate distinct inflammasome complexes that promote caspase-1 activation, secretion of the proinflammatory cytokines IL-1ß and IL-18, and a form of cell death termed pyroptosis. We show that NLRP3 associates with NLRC4 in macrophages infected with Salmonella typhimurium or transfected with flagellin. The significance of the interaction between the NLRC4 NACHT domain and NLRP3 was revealed when Nlrc4(S533A/S533A) bone marrow-derived macrophages (BMDMs) expressing phosphorylation site mutant NLRC4 S533A had only a mild defect in caspase-1 activation when compared with NLRC4-deficient BMDMs. NLRC4 S533A activated caspase-1 by recruiting NLRP3 and its adaptor protein ASC. Thus, Nlrc4(S533A/S533A) Nlrp3(-/-) BMDMs more closely resembled Nlrc4(-/-) BMDMs in their response to S. typhimurium or flagellin. The interplay between NLRP3 and NLRC4 reveals an unexpected overlap between what had been considered distinct inflammasome scaffolds.

Evolutionary divergence in the catalytic activity of the CAM-1, ROR1 and ROR2 kinase domains.

Receptor tyrosine kinase-like orphan receptors (ROR) 1 and 2 are atypical members of the receptor tyrosine kinase (RTK) family and have been associated with several human diseases. The vertebrate RORs contain an ATP binding domain that deviates from the consensus amino acid sequence, although the impact of this deviation on catalytic activity is not known and the kinase function of these receptors remains controversial. Recently, ROR2 was shown to signal through a Wnt responsive, ß-catenin independent pathway and suppress a canonical Wnt/ß-catenin signal. In this work we demonstrate that both ROR1 and ROR2 kinase domains are catalytically deficient while CAM-1, the C. elegans homolog of ROR, has an active tyrosine kinase domain, suggesting a divergence in the signaling processes of the ROR family during evolution. In addition, we show that substitution of the non-consensus residues from ROR1 or ROR2 into CAM-1 and MuSK markedly reduce kinase activity, while restoration of the consensus residues in ROR does not restore robust kinase function. We further demonstrate that the membrane-bound extracellular domain alone of either ROR1 or ROR2 is sufficient for suppression of canonical Wnt3a signaling, and that this domain can also enhance Wnt5a suppression of Wnt3a signaling. Based on these data, we conclude that human ROR1 and ROR2 are RTK-like pseudokinases.

Targeting interferon alpha subtypes in serum: a comparison of analytical approaches to the detection and quantitation of proteins in complex biological matrices.

The targeted detection and quantitation of proteins in complex biological fluids such as blood is as analytically challenging as it is crucial for biomedical research. Antibody-based techniques such as the ELISA are the current standards for such measurements, having in favorable cases high specificity and pg/mL detection limits. Long development timelines and susceptibility to cross reactivity have led researchers to investigate mass spectrometric alternatives. The literature contains diverse schemes for sample preparation and multiple platforms for mass spectrometric detection. Critical evaluations of competing technologies are, however, badly needed. Taking closely related subtypes of the pro-inflammatory cytokine interferon alpha as a test case, we compared a sample preparation workflow based on affinity enrichment to one based on generic multidimensional chromatography, and evaluated mass spectrometric techniques using tandem mass spectrometry on low resolution ion traps, high resolution "accurate mass tags," and triple quadrupole selective reaction monitoring. Each workflow and detection method proved capable of detecting and discriminating between these proteins at or below the ng/mL level in human serum. Quantitation by isotope dilution was evaluated using full length protein as the internal standard. Both triple quadrupole selected reaction monitoring and orbitrap selected ion monitoring produced linear calibration curves from 1 ng/mL to 1 microg/mL, with lower limits of quantitation below 5 and 50 ng/mL, respectively.

Association of C-terminal ubiquitin hydrolase BRCA1-associated protein 1 with cell cycle regulator host cell factor 1.

Protein ubiquitination provides an efficient and reversible mechanism to regulate cell cycle progression and checkpoint control. Numerous regulatory proteins direct the addition of ubiquitin to lysine residues on target proteins, and these are countered by an army of deubiquitinating enzymes (DUBs). BRCA1-associated protein-1 (Bap1) is a ubiquitin carboxy-terminal hydrolase and is frequently mutated in lung and sporadic breast tumors. Bap1 can suppress growth of lung cancer cells in athymic nude mice and this requires its DUB activity. We show here that Bap1 interacts with host cell factor 1 (HCF-1), a transcriptional cofactor found in a number of important regulatory complexes. Bap1 binds to the HCF-1 beta-propeller using a variant of the HCF-binding motif found in herpes simplex virus VP16 and other HCF-interacting proteins. HCF-1 is K48 and K63 ubiquitinated, with a major site of linkage at lysines 1807 and 1808 in the HCF-1(C) subunit. Expression of a catalytically inactive version of Bap1 results in the selective accumulation of K48 ubiquitinated polypeptides. Depletion of Bap1 using small interfering RNA results in a modest accumulation of HCF-1(C), suggesting that Bap1 helps to control cell proliferation by regulating HCF-1 protein levels and by associating with genes involved in the G(1)-S transition.