NRF2 mediates melanoma addiction to GCDH by modulating apoptotic signalling.

Tumour dependency on specific metabolic signals has been demonstrated and often guided numerous therapeutic approaches. We identify melanoma addiction to the mitochondrial protein glutaryl-CoA dehydrogenase (GCDH), which functions in lysine metabolism and controls protein glutarylation. GCDH knockdown induced cell death programmes in melanoma cells, an activity blocked by inhibition of the upstream lysine catabolism enzyme DHTKD1. The transcription factor NRF2 mediates GCDH-dependent melanoma cell death programmes. Mechanistically, GCDH knockdown induces NRF2 glutarylation, increasing its stability and DNA binding activity, with a concomitant transcriptional upregulation of ATF4, ATF3, DDIT3 and CHAC1, resulting in cell death. In vivo, inducible inactivation of GCDH effectively inhibited melanoma tumour growth. Correspondingly, reduced GCDH expression correlated with improved survival of patients with melanoma. These findings identify melanoma cell addiction to GCDH, limiting apoptotic signalling by controlling NRF2 glutarylation. Inhibiting the GCDH pathway could thus represent a therapeutic approach to treat melanoma.

Serine-Threonine Kinase TAO3-Mediated Trafficking of Endosomes Containing the Invadopodia Scaffold TKS5α Promotes Cancer Invasion and Tumor Growth.

Invadopodia are actin-based proteolytic membrane protrusions required for invasive behavior and tumor growth. In this study, we used our high-content screening assay to identify kinases whose activity affects invadopodia formation. Among the top hits selected for further analysis was TAO3, an STE20-like kinase of the GCK subfamily. TAO3 was overexpressed in many human cancers and regulated invadopodia formation in melanoma, breast, and bladder cancers. Furthermore, TAO3 catalytic activity facilitated melanoma growth in three-dimensional matrices and in vivo. A novel, potent catalytic inhibitor of TAO3 was developed that inhibited invadopodia formation and function as well as tumor cell extravasation and growth. Treatment with this inhibitor demonstrated that TAO3 activity is required for endosomal trafficking of TKS5α, an obligate invadopodia scaffold protein. A phosphoproteomics screen for TAO3 substrates revealed the dynein subunit protein LIC2 as a relevant substrate. Knockdown of LIC2 or expression of a phosphomimetic form promoted invadopodia formation. Thus, TAO3 is a new therapeutic target with a distinct mechanism of action. SIGNIFICANCE: An unbiased screening approach identifies TAO3 as a regulator of invadopodia formation and function, supporting clinical development of this class of target.

Identification and characterization of small molecule inhibitors of the ubiquitin ligases Siah1/2 in melanoma and prostate cancer cells.

Inhibition of ubiquitin ligases with small molecule remains a very challenging task, given the lack of catalytic activity of the target and the requirement of disruption of its interactions with other proteins. Siah1/2, which are E3 ubiquitin ligases, are implicated in melanoma and prostate cancer and represent high-value drug targets. We utilized three independent screening approaches in our efforts to identify small-molecule Siah1/2 inhibitors: Affinity Selection-Mass Spectrometry, a protein thermal shift-based assay and an in silico based screen. Inhibitors were assessed for their effect on viability of melanoma and prostate cancer cultures, colony formation, prolyl-hydroxylase-HIF1α signaling, expression of selected Siah2-related transcripts, and Siah2 ubiquitin ligase activity. Several analogs were further characterized, demonstrating improved efficacy. Combination of the top hits identified in the different assays demonstrated an additive effect, pointing to complementing mechanisms that underlie each of these Siah1/2 inhibitors.

Ectopic calcification in pseudoxanthoma elasticum responds to inhibition of tissue-nonspecific alkaline phosphatase.

Biallelic mutations in ABCC6 cause pseudoxanthoma elasticum (PXE), a disease characterized by calcification in the skin, eyes, and blood vessels. The function of ATP-binding cassette C6 (ABCC6) and the pathogenesis of PXE remain unclear. We used mouse models and patient fibroblasts to demonstrate genetic interaction and shared biochemical and cellular mechanisms underlying ectopic calcification in PXE and related disorders caused by defined perturbations in extracellular adenosine 5'-triphosphate catabolism. Under osteogenic culture conditions, ABCC6 mutant cells calcified, suggesting a provoked cell-autonomous defect. Using a conditional Abcc6 knockout mouse model, we excluded the prevailing pathogenic hypothesis that singularly invokes failure of hepatic secretion of an endocrine inhibitor of calcification. Instead, deficiency of Abcc6 in both local and distant cells was necessary to achieve the early onset and penetrant ectopic calcification observed upon constitutive gene targeting. ABCC6 mutant cells additionally had increased expression and activity of tissue-nonspecific alkaline phosphatase (TNAP), an enzyme that degrades pyrophosphate, a major inhibitor of calcification. A selective and orally bioavailable TNAP inhibitor prevented calcification in ABCC6 mutant cells in vitro and attenuated both the development and progression of calcification in Abcc6-/- mice in vivo, without the deleterious effects on bone associated with other proposed treatment strategies.

p97 Composition Changes Caused by Allosteric Inhibition Are Suppressed by an On-Target Mechanism that Increases the Enzyme's ATPase Activity.

The AAA ATPase p97/VCP regulates protein homeostasis using a diverse repertoire of cofactors to fulfill its biological functions. Here we use the allosteric p97 inhibitor NMS-873 to analyze its effects on enzyme composition and the ability of cells to adapt to its cytotoxicity. We found that p97 inhibition changes steady state cofactor-p97 composition, leading to the enrichment of a subset of its cofactors and polyubiquitin bound to p97. We isolated cells specifically insensitive to NMS-873 and identified a new mutation (A530T) in p97. A530T is sufficient to overcome the cytotoxicity of NMS-873 and alleviates p97 composition changes caused by the molecule but not other p97 inhibitors. This mutation does not affect NMS-873 binding but increases p97 catalytic efficiency through altered ATP and ADP binding. Collectively, these findings identify cofactor-p97 interactions sensitive to p97 inhibition and reveal a new on-target mechanism to suppress the cytotoxicity of NMS-873.

Ebselen inhibits QSOX1 enzymatic activity and suppresses invasion of pancreatic and renal cancer cell lines.

Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that is overexpressed in diverse tumor types. Its enzymatic activity promotes the growth and invasion of tumor cells and alters extracellular matrix composition. In a nude mouse-human tumor xenograft model, tumors containing shRNA for QSOX1 grew significantly more slowly than controls, suggesting that QSOX1 supports a proliferative phenotype in vivo. High throughput screening experiments identified ebselen as an in vitro inhibitor of QSOX1 enzymatic activity. Ebselen treatment of pancreatic and renal cancer cell lines stalled tumor growth and inhibited invasion through Matrigel in vitro. Daily oral treatment with ebselen resulted in a 58% reduction in tumor growth in mice bearing human pancreatic tumor xenografts compared to controls. Mass spectrometric analysis of ebselen-treated QSOX1 mechanistically revealed that C165 and C237 of QSOX1 covalently bound to ebselen. This report details the anti-neoplastic properties of ebselen in pancreatic and renal cancer cell lines. The results here offer a "proof-of-principle" that enzymatic inhibition of QSOX1 may have clinical relevancy.

Design of high-throughput screening assays and identification of a SUMO1-specific small molecule chemotype targeting the SUMO-interacting motif-binding surface.

Protein-protein interactions are generally challenging to target by small molecules. To address the challenge, we have used a multidisciplinary approach to identify small-molecule disruptors of protein-protein interactions that are mediated by SUMO (small ubiquitin-like modifier) proteins. SUMO modifications have emerged as a target with importance in treating cancer, neurodegenerative disorders, and viral infections. It has been shown that inhibiting SUMO-mediated protein-protein interactions can sensitize cancer cells to chemotherapy and radiation. We have developed highly sensitive assays using time-resolved fluorescence resonance energy transfer (TR-FRET) and fluorescence polarization (FP) that were used for high-throughput screening (HTS) to identify inhibitors for SUMO-dependent protein-protein interactions. Using these assays, we have identified a nonpeptidomimetic small molecule chemotype that binds to SUMO1 but not SUMO2 or 3. NMR chemical shift perturbation studies have shown that the compounds of this chemotype bind to the SUMO1 surface required for protein-protein interaction, despite the high sequence similarity of SUMO1 and SUMO2 and 3 at this surface.

pCAP-based peptide substrates: the new tool in the box of tyrosine phosphatase assays.

Robust, facile high throughput assays based on non-peptidic probes are available to detect the enzyme activity of protein tyrosine phosphatases. However, these assays cannot replace the use of peptide-based probes in many applications; for example when a closer mimic of the physiological target is desired or in substrate profiling expeditions. Phosphotyrosine peptides are often used in these assays, but their use is complicated by either poor sensitivity or the need for indirect detection methods, among other pitfalls. Novel peptide-based probes for protein tyrosine phosphatases are needed to replace phosphotyrosine peptides and accelerate the field of tyrosine phosphatase substrate profiling. Here we review a type of peptidic probe for tyrosine phosphatases, which is based on the incorporation of the phosphotyrosine-mimic phosphocoumaryl amino propionic acid (pCAP) into peptides. The resulting fluorogenic pCAP peptides are dephosphorylated by tyrosine phosphatases with similar efficiency as the homologous phosphotyrosine peptides. pCAP peptides outperform phosphotyrosine peptides, providing an assay that is as robust, sensitive and facile as the non-peptidic fluorogenic probes on the market. Finally the use of pCAP can expand the range of phosphatase assays, facilitating the investigation of multiphosphorylated peptides and providing an in-gel assay for phosphatase activity.

New activity assays for ENPP1 with physiological substrates ATP and ADP.

Existing assays monitoring ENPP1 activity are either not physiologically relevant or not suitable for high-throughput screening (HTS). Here, we describe the development and implementation of two new ENPP1 activity assays that address these drawbacks. These assays employ physiological substrates of ENPP1, ATP and ADP. They rely on detection of inorganic phosphate using a special modification of the malachite green-molybdate colorimetric procedure that ensures stability of acid-labile compounds, such as the ones containing phosphodiester bonds. The pyrophosphate generated in ENPP1 reaction is converted to inorganic phosphate in the presence of inorganic phosphatase; whereas, omission of this coupling enzyme enables detection of the inorganic phosphate generated by ENPP1. These new ENPP1 assays were miniaturized into high-density microplate formats. With minimal requirement for ENPP1 enzyme, low micromolar phosphate detection sensitivity, and simple protocol involving three to four simple liquid handling steps, these robust assays are suitable for HTS.

Mechanism of dephosphorylation of the SR protein ASF/SF2 by protein phosphatase 1.

SR proteins are essential splicing factors whose function is controlled by multi-site phosphorylation of a C-terminal domain rich in arginine-serine repeats (RS domain). The protein kinase SRPK1 has been shown to polyphosphorylate the N-terminal portion of the RS domain (RS1) of the SR protein ASF/SF2, a modification that promotes nuclear entry of this splicing factor and engagement in splicing function. Later, dephosphorylation is required for maturation of the spliceosome and other RNA processing steps. While phosphates are attached to RS1 in a sequential manner by SRPK1, little is known about how they are removed. To investigate factors that control dephosphorylation, we monitored region-specific mapping of phosphorylation sites in ASF/SF2 as a function of the protein phosphatase PP1. We showed that 10 phosphates added to the RS1 segment by SRPK1 are removed in a preferred N-to-C manner, directly opposing the C-to-N phosphorylation by SRPK1. Two N-terminal RNA recognition motifs in ASF/SF2 control access to the RS domain and guide the directional mechanism. Binding of RNA to the RNA recognition motifs protects against dephosphorylation, suggesting that engagement of the SR protein with exonic splicing enhancers can regulate phosphoryl content in the RS domain. In addition to regulation by N-terminal domains, phosphorylation of the C-terminal portion of the RS domain (RS2) by the nuclear protein kinase Clk/Sty inhibits RS1 dephosphorylation and disrupts the directional mechanism. The data indicate that both RNA-protein interactions and phosphorylation in flanking sequences induce conformations of ASF/SF2 that increase the lifetime of phosphates in the RS domain.

Adaptable molecular interactions guide phosphorylation of the SR protein ASF/SF2 by SRPK1.

The SR (arginine-serine rich) protein ASF/SF2 (also called human alternative splicing factor), an essential splicing factor, contains two functional modules consisting of tandem RNA recognition motifs (RRMs; RRM1-RRM2) and a C-terminal arginine-serine repeat region (RS domain, a domain rich in arginine-serine repeats). The SR-specific protein kinase (SRPK) 1 phosphorylates the RS domain at multiple serines using a directional (C-terminal-to-N-terminal) and processive mechanism--a process that directs the SR protein to the nucleus and influences protein-protein interactions associated with splicing function. To investigate how SRPK1 accomplishes this feat, the enzyme-substrate complex was analyzed using single-turnover and multiturnover kinetic methods. Deletion studies revealed that while recognition of the RS domain by a docking groove on SRPK1 is sufficient to initiate the processive and directional mechanism, continued processive phosphorylation in the presence of building repulsive charge relies on the fine-tuning of contacts with the RRM1-RRM2 module. An electropositive pocket in SRPK1 that stabilizes newly phosphorylated serines enhanced processive phosphorylation of later serines. These data indicate that SRPK1 uses stable, yet highly flexible protein-protein interactions to facilitate both early and late phases of the processive phosphorylation of SR proteins.

A sliding docking interaction is essential for sequential and processive phosphorylation of an SR protein by SRPK1.

The 2.9 A crystal structure of the core SRPK1:ASF/SF2 complex reveals that the N-terminal half of the basic RS domain of ASF/SF2, which is destined to be phosphorylated, is bound to an acidic docking groove of SRPK1 distal to the active site. Phosphorylation of ASF/SF2 at a single site in the C-terminal end of the RS domain generates a primed phosphoserine that binds to a basic site in the kinase. Biochemical experiments support a directional sliding of the RS peptide through the docking groove to the active site during phosphorylation, which ends with the unfolding of a beta strand of the RRM domain and binding of the unfolded region to the docking groove. We further suggest that the priming of the first serine facilitates directional substrate translocation and efficient phosphorylation.

Ordered multi-site phosphorylation of the splicing factor ASF/SF2 by SRPK1.

The human alternative splicing factor ASF/SF2, an SR (serine-arginine-rich) protein involved in mRNA splicing control, is activated by the multisite phosphorylation of its C-terminal RS domain, a segment containing numerous arginine-serine dipeptide repeats. The protein kinase responsible for this modification, SR-specific protein kinase 1 (SRPK1), catalyzes the selective phosphorylation of approximately a dozen serines in only the N-terminal portion of the RS domain (RS1). To gain insights into the nature of selective phosphate incorporation in ASF/SF2, region-specific phosphorylation in the RS domain was monitored as a function of reaction progress. Arg-to-Lys mutations were made at several positions to produce unique protease cleavage sites that separate the RS domain into identifiable N- and C-terminal phosphopeptides upon treatment with lysyl endoproteinase. These studies reveal that SRPK1 docks near the C-terminus of the RS1 segment and then moves in an N-terminal direction along the RS domain. Multiple quadruple Ser-to-Ala and deletion mutations did not disrupt the phosphorylation of other sites regardless of position, suggesting that the active site of SRPK1 docks in a flexible manner at the center of the RS domain. Taken together, these data suggest that SRPK1 uses a unique 'grab-and-pull' mechanism to control the regiospecific phosphorylation of its protein substrate.

Mass spectrometric and kinetic analysis of ASF/SF2 phosphorylation by SRPK1 and Clk/Sty.

Assembly of the spliceosome requires the participation of SR proteins, a family of splicing factors rich in arginine-serine dipeptide repeats. The repeat regions (RS domains) are polyphosphorylated by the SRPK and Clk/Sty families of kinases. The two families of kinases have distinct enzymatic properties, raising the question of how they may work to regulate the function of SR proteins in RNA metabolism in mammalian cells. Here we report the first mass spectral analysis of the RS domain of ASF/SF2, a prototypical SR protein. We found that SRPK1 was responsible for efficient phosphorylation of a short stretch of amino acids in the N-terminal portion of the RS domain of ASF/SF2 while Clk/Sty was able to transfer phosphate to all available serine residues in the RS domain, indicating that SR proteins may be phosphorylated by different kinases in a stepwise manner. Both kinases bind with high affinity and use fully processive catalytic mechanisms to achieve either restrictive or complete RS domain phosphorylation. These findings have important implications on the regulation of SR proteins in vivo by the SRPK and Clk/Sty families of kinases.