The complex lipid, SPPCT-800, reduces lung damage, improves pulmonary function and decreases pro-inflammatory cytokines in the murine LPS-induced acute respiratory distress syndrome (ARDS) model.

CONTEXT: Acute respiratory distress syndrome (ARDS) is a highly fatal, inflammatory condition of lungs with multiple causes. There is no adequate treatment. OBJECTIVE: Using the murine LPS-induced ARDS model, we investigate SPPCT-800 (a complex lipid) as treatment for ARDS. MATERIALS AND METHODS: C57B16/N mice received 50 µg of Escherichia coli O111:B4 lipopolysaccharide (LPS). SPPCT-800 was given as either: (1) 20 or 200 mg/kg dose 3 h after LPS; (2) 200 mg/kg (prophylactically) 30 min before LPS; or (3) eight 200 mg/kg treatments over 72 h. Controls received saline installations. RESULTS: At 48 and 72 h, SpO2 was 94% and 90% in controls compared to 97% and 94% in treated animals. Expiration times, at 24 and 48 h, were 160 and 137 msec for controls, but 139 and 107 msec with SPPCT-800. In BALF (24 h), cell counts were 4.7 × 106 (controls) and 2.9 × 106 (treated); protein levels were 1.5 mg (controls) and 0.4 mg (treated); and IL-6 was 942 ± 194 pg/mL (controls) versus 850 ± 212 pg/mL (treated) [at 72 h, 4664 ± 2591 pg/mL (controls) versus 276 ± 151 pg/mL (treated)]. Weight losses, at 48 and 72 h, were 20% and 18% (controls), but 14% and 8% (treated). Lung injury scores, at 24 and 72 h, were 1.4 and 3.0 (controls) and 0.3 and 2.2 (treated). DISCUSSION AND CONCLUSIONS: SPPCT-800 was effective in reducing manifestations of ARDS. SPPCT-800 should be further investigated as therapy for ARDS, especially in longer duration or higher cumulative dose studies.

Interactome Analysis of KIN (Kin17) Shows New Functions of This Protein.

KIN (Kin17) protein is overexpressed in a number of cancerous cell lines, and is therefore considered a possible cancer biomarker. It is a well-conserved protein across eukaryotes and is ubiquitously expressed in all cell types studied, suggesting an important role in the maintenance of basic cellular function which is yet to be well determined. Early studies on KIN suggested that this nuclear protein plays a role in cellular mechanisms such as DNA replication and/or repair; however, its association with chromatin depends on its methylation state. In order to provide a better understanding of the cellular role of this protein, we investigated its interactome by proximity-dependent biotin identification coupled to mass spectrometry (BioID-MS), used for identification of protein-protein interactions. Our analyses detected interaction with a novel set of proteins and reinforced previous observations linking KIN to factors involved in RNA processing, notably pre-mRNA splicing and ribosome biogenesis. However, little evidence supports that this protein is directly coupled to DNA replication and/or repair processes, as previously suggested. Furthermore, a novel interaction was observed with PRMT7 (protein arginine methyltransferase 7) and we demonstrated that KIN is modified by this enzyme. This interactome analysis indicates that KIN is associated with several cell metabolism functions, and shows for the first time an association with ribosome biogenesis, suggesting that KIN is likely a moonlight protein.

NTP42, a novel antagonist of the thromboxane receptor, attenuates experimentally induced pulmonary arterial hypertension.

BACKGROUND: NTP42 is a novel antagonist of the thromboxane prostanoid receptor (TP), currently in development for the treatment of pulmonary arterial hypertension (PAH). PAH is a devastating disease with multiple pathophysiological hallmarks including excessive pulmonary vasoconstriction, vascular remodelling, inflammation, fibrosis, in situ thrombosis and right ventricular hypertrophy. Signalling through the TP, thromboxane (TX) A2 is a potent vasoconstrictor and mediator of platelet aggregation. It is also a pro-mitogenic, pro-inflammatory and pro-fibrotic agent. Moreover, the TP also mediates the adverse actions of the isoprostane 8-iso-prostaglandin F2α, a free-radical-derived product of arachidonic acid produced in abundance during oxidative injury. Mechanistically, TP antagonists should treat most of the hallmarks of PAH, including inhibiting the excessive vasoconstriction and pulmonary artery remodelling, in situ thrombosis, inflammation and fibrosis. This study aimed to investigate the efficacy of NTP42 in the monocrotaline (MCT)-induced PAH rat model, alongside current standard-of-care drugs. METHODS: PAH was induced by subcutaneous injection of 60 mg/kg MCT in male Wistar-Kyoto rats. Animals were assigned into groups: 1. 'No MCT'; 2. 'MCT Only'; 3. MCT + NTP42 (0.25 mg/kg BID); 4. MCT + Sildenafil (50 mg/kg BID), and 5. MCT + Selexipag (1 mg/kg BID), where 28-day drug treatment was initiated within 24 h post-MCT. RESULTS: From haemodynamic assessments, NTP42 reduced the MCT-induced PAH, including mean pulmonary arterial pressure (mPAP) and right systolic ventricular pressure (RSVP), being at least comparable to the standard-of-care drugs Sildenafil or Selexipag in bringing about these effects. Moreover, NTP42 was superior to Sildenafil and Selexipag in significantly reducing pulmonary vascular remodelling, inflammatory mast cell infiltration and fibrosis in MCT-treated animals. CONCLUSIONS: These findings suggest that NTP42 and antagonism of the TP signalling pathway have a relevant role in alleviating the pathophysiology of PAH, representing a novel therapeutic target with marked benefits over existing standard-of-care therapies.

Upstream ORF-Encoded ASDURF Is a Novel Prefoldin-like Subunit of the PAQosome.

The PAQosome is an 11-subunit chaperone involved in the biogenesis of several human protein complexes. We show that ASDURF, a recently discovered upstream open reading frame (uORF) in the 5' UTR of ASNSD1 mRNA, encodes the 12th subunit of the PAQosome. ASDURF displays significant structural homology to ß-prefoldins and assembles with the five known subunits of the prefoldin-like module of the PAQosome to form a heterohexameric prefoldin-like complex. A model of the PAQosome prefoldin-like module is presented. The data presented here provide an example of a eukaryotic uORF-encoded polypeptide whose function is not limited to cis-acting translational regulation of downstream coding sequence and highlights the importance of including alternative ORF products in proteomic studies.

Posttranslational modification of proprotein convertase subtilisin/kexin type 9 is differentially regulated in response to distinct cardiometabolic treatments as revealed by targeted proteomics.

BACKGROUND: The proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted protein that interacts with the low-density lipoprotein (LDL) receptor at the surface of hepatocytes to regulate circulating LDL cholesterol levels. High circulating PCSK9 levels have been associated with elevated LDL cholesterol. Recently, the Food and Drug Administration of the United States approved new LDL cholesterol-lowering drugs that specifically target the inhibition of PCSK9. Similar to most human proteins, PCSK9 exists in multiple forms as it is the target of posttranslational modifications (PTMs) such as proteolytic cleavage, phosphorylation, and others, which can affect its biological activity. However, commercially available assays, such as enzyme-linked immunosorbent assays, do not discriminate between these forms. OBJECTIVE: To investigate, in 2 patient cohorts, the relationships between circulating levels of multiple forms of PCSK9 and cardiometabolic interventions or treatments known to reduce LDL cholesterol levels. METHODS: PCSK9 forms were measured in plasma: (1) in 20 patients before and 6 months after bariatric surgery and (2) in 132 patients before and 12 months after daily statin treatment. A series of specific peptides used as surrogates for various PCSK9 forms were quantified by a novel semiautomated proteomic assay termed protein affinity capture coupled to quantitative mass spectrometry. RESULTS: Bariatric surgery resulted in a decrease in the plasma level of PCSK9 prodomain (P < .05), but did not result in a significant change in other measured PCSK9 forms. Statin treatment resulted in an increase in all measured plasma PCSK9 peptides (P < .001), but a 25% decrease in the phosphorylated state of PCSK9 at S688 (P < .05). CONCLUSIONS: These unexpected findings indicate that measuring the circulating levels of the various domains and PTMs of PCSK9 provides more in depth information than total PCSK9 and that the prodomain and the phosphorylated state of S688 may represent novel biomarkers to explore in cardiometabolic diseases and response to treatment. In addition, our data generated new hypotheses on the function of PCSK9 PTMs in health and disease.

Evaluation of the effects of RP5063, a novel, multimodal, serotonin receptor modulator, as single-agent therapy and co-administrated with sildenafil, bosentan, and treprostinil in a monocrotaline-induced pulmonary arterial hypertension rat model.

Pulmonary arterial hypertension (PAH), a condition that is defined by pulmonary vasculature constriction and remodeling, involves dysfunctional signaling of the serotonin (5-HT) receptors, 5-HT2A/2B/7. In a rat model of monocrotaline (MCT)-induced PAH, the effectiveness of RP5063 (RP), a dopamine and 5-HT receptor modulator, was evaluated as monotherapy and as an adjunct to standard PAH treatments. After a single 60 mg/kg dose of MCT, rats received vehicle (MCT+Veh; gavage twice-daily [b.i.d.]), RP (10 mg/kg; gavage b.i.d.), bosentan (B; 100 mg/kg; gavage BID), sildenafil (S; 50 mg/kg; gavage, BID), treprostinil (T; 100 ng/kg/min over 24 h intravenous), RP+B, RP+S, and RP+T for 28 days. Single-agent RP limited the functional and structural effects of PAH seen in the MCT+Veh group, with significant improvements in pulmonary hemodynamics, right ventricular (RV) hypertrophy, SO2, and pulmonary blood vessel structural changes. These effects appeared comparable with those associated with B, S, and T. Adjunctive RP treatment resulted in significantly lower mean pulmonary arterial pressures, RV systolic pressure. It also improved SO2 measurements, as compared with MCT+Veh (P < 0.05), and diastolic pulmonary artery pressure (P < 0.05), as compared with single-agent B and S therapy (Bonferroni method adjusting for multiplicity). RP+S appeared to show the most consistent and extensive effects on pulmonary hemodynamics, respiratory parameters, and histopathologic changes. These results corroborate earlier preclinical findings supporting the efficacy of single-agent RP in PAH. RP, as mono and adjunctive therapy compared with induced-control, mitigated the functional and structural effects of MCT-induced PAH.

RP5063, a novel, multimodal, serotonin receptor modulator, prevents monocrotaline-induced pulmonary arterial hypertension in rats.

Pulmonary arterial hypertension (PAH), a condition characterized by pulmonary vasculature constriction and remodeling, involves dysregulation of the serotonin (5-HT) receptors 5-HT2A and 5-HT2B. A rat model of monocrotaline (MCT)-induced PAH was used to examine the potential beneficial effects of RP5063, a 5-HT receptor modulator. After a single 60mg/kg dose of MCT, rats were gavaged twice-daily (b.i.d.) with vehicle, RP5063 (1, 3, or 10mg/kg), or sildenafil (50mg/kg) for 28 days. RP5063 at a dose as low as 1mg/kg, b.i.d. reduced pulmonary resistance and increased systemic blood oxygen saturation. The highest dose of RP5063 (10mg/kg, b.i.d.) reduced diastolic, systolic, and mean pulmonary pressure, right systolic ventricular pressure, ventilatory pressure, and Fulton's index (ratio of right to left ventricular weight). Doses as low as 3mg/kg RP5063, b.i.d. also increased weight gain and body temperature, suggesting an improvement in overall health of MCT-treated animals. Similar reductions in pulmonary, right ventricular, and ventilatory pressure, pulmonary resistance, and Fulton's index as well as increased systemic blood oxygen saturation were observed in animals treated with the reference agent sildenafil at a higher dose (50mg/kg, b.i.d.). Histological examination revealed that RP5063 produced dose-dependent reductions in pulmonary blood vessel wall thickness and proportion of muscular vessels, similar to sildenafil. RP5063 completely blocked MCT-induced increases in the plasma cytokines TNFα, IL-1ß, and IL-6 at all doses. In summary, RP5063 improved pulmonary vascular pathology and hemodynamics, right ventricular pressure and hypertrophy, systemic oxygen saturation, and overall health of rats treated with MCT.

RP5063, a novel, multimodal, serotonin receptor modulator, prevents Sugen 5416-hypoxia-induced pulmonary arterial hypertension in rats.

RP5063, a multimodal dopamine (DA) and serotonin (5-HT) modulator with high affinity for DA2/3/4 and 5-HT2A/2B/7 receptors and moderate affinity for SERT, is a novel therapeutic of special interest in the treatment of pulmonary arterial hypertension (PAH). Evidence indicates that therapeutics targeting the 5-HT2A/2B receptors can influence the pathogenesis of PAH. However, the therapeutic effect of RP5063 in humans has yet to be investigated. A Sugen 5416-hypoxia (SuHx)-induced PAH model was used to evaluate twice-daily (b.i.d.) RP5063 at 10mg/kg (RP-10) and 20mg/kg (RP-20), as compared with positive (sildenafil 50mg/kg b.i.d.; Sil-50) and negative controls (SuHx+vehicle; SuHx+veh), in 24 adult male Wistar-Kyoto rats. RP5063 showed significantly lower systolic pulmonary arterial (both doses) and systolic right ventricular (RP-10) pressures, and improvement in oxygen saturation (RP-20). It significantly reduced small-vessel wall thickness (RP-20), lowered the percentage of muscular vessels (both doses). Both doses limited arterial obliteration due to endothelial cell proliferation, prevented plexiform lesion formation, and stemmed the release of leukotriene B4. Sildenafil showed statistically greater effects on vessel structure than that seen in both RP5063 groups and improved oxygen saturation. Additionally, Sildenafil did not demonstrate any significant effect on arterial obliteration, plexiform lesion development, or pulmonary arterial or right ventricular pressure. As PAH gains in severity, the impact of RP5063 inhibition of 5HT2B increases, preventing arterial constriction and improving pulmonary hemodynamics. Due to its functional, structural, and chemokine effects, RP5063 represents a promising candidate for investigation in late-phase PAH.

R2TP/Prefoldin-like component RUVBL1/RUVBL2 directly interacts with ZNHIT2 to regulate assembly of U5 small nuclear ribonucleoprotein.

The R2TP/Prefoldin-like (R2TP/PFDL) complex has emerged as a cochaperone complex involved in the assembly of a number of critical protein complexes including snoRNPs, nuclear RNA polymerases and PIKK-containing complexes. Here we report on the use of multiple target affinity purification coupled to mass spectrometry to identify two additional complexes that interact with R2TP/PFDL: the TSC1-TSC2 complex and the U5 small nuclear ribonucleoprotein (snRNP). The interaction between R2TP/PFDL and the U5 snRNP is mostly mediated by the previously uncharacterized factor ZNHIT2. A more general function for the zinc-finger HIT domain in binding RUVBL2 is exposed. Disruption of ZNHIT2 and RUVBL2 expression impacts the protein composition of the U5 snRNP suggesting a function for these proteins in promoting the assembly of the ribonucleoprotein. A possible implication of R2TP/PFDL as a major effector of stress-, energy- and nutrient-sensing pathways that regulate anabolic processes through the regulation of its chaperoning activity is discussed.

Absence of neurological abnormalities in mice homozygous for the Polr3a G672E hypomyelinating leukodystrophy mutation.

Recessive mutations in the ubiquitously expressed POLR3A gene cause one of the most frequent forms of childhood-onset hypomyelinating leukodystrophy (HLD): POLR3-HLD. POLR3A encodes the largest subunit of RNA Polymerase III (Pol III), which is responsible for the transcription of transfer RNAs (tRNAs) and a large array of other small non-coding RNAs. In order to study the central nervous system pathophysiology of the disease, we introduced the French Canadian founder Polr3a mutation c.2015G > A (p.G672E) in mice, generating homozygous knock-in (KI/KI) as well as compound heterozygous mice for one Polr3a KI and one null allele (KI/KO). Both KI/KI and KI/KO mice are viable and are able to reproduce. To establish if they manifest a motor phenotype, WT, KI/KI and KI/KO mice were submitted to a battery of behavioral tests over one year. The KI/KI and KI/KO mice have overall normal balance, muscle strength and general locomotion. Cerebral and cerebellar Luxol Fast Blue staining and measurement of levels of myelin proteins showed no significant differences between the three groups, suggesting that myelination is not overtly impaired in Polr3a KI/KI and KI/KO mice. Finally, expression levels of several Pol III transcripts in the brain showed no statistically significant differences. We conclude that the first transgenic mice with a leukodystrophy-causing Polr3a mutation do not recapitulate the childhood-onset HLD observed in the majority of human patients with POLR3A mutations, and provide essential information to guide selection of Polr3a mutations for developing future mouse models of the disease.

Curcumin-ER Prolonged Subcutaneous Delivery for the Treatment of Non-Small Cell Lung Cancer.

Non-small-cell lung cancer therapy is a challenge due to poor prognosis and low survival rate. There is an acute need for advanced therapies having higher drug efficacy, low immunogenicity and fewer side effects which will markedly improve patient compliance and quality of life of cancer patients. The purpose of this study was to develop a novel hybrid curcumin nanoformulation (Curcumin-ER) and evaluate the therapeutic efficacy of this formulation on a non-small cell lung cancer xenograft model. Use of curcumin, a natural anticancer agent, is majorly limited due to its poor aqueous solubility and hence it's low systemic bioavailability. In this paper, we carried out the nanoformulation of Curcumin-ER, optimized the formulation process and determined the anticancer effects of Curcumin-ER against human A549 non-small cell lung cancer using in vitro and in vivo studies. Xenograft tumors in nude mice were treated with 20 mg/kg subcutaneous injection of Curcumin-ER and liposomal curcumin (Lipocurc) twice a week for seven weeks. Results showed that tumor growth was suppressed by 52.1% by Curcumin-ER treatment and only 32.2% by Lipocurc compared to controls. Tumor sections were isolated from murine xenografts and histology and immunohistochemistry was performed. A decrease in expression of NFκB-p65 subunit and proliferation marker, Ki-67 was observed in treated tumors. In addition, a potent anti-angiogenic effect, characterized by reduced expression of annexin A2 protein, was observed in treated tumors. These results establish the effectiveness of Curcumin-ER in regressing human non-small cell lung cancer growth in the xenograft model using subcutaneous route of administration. The therapeutic efficacy of Curcumin-ER highlights the potential of this hybrid nanoformulation in treating patients with non-small cell lung cancer.

A semi-automated mass spectrometric immunoassay coupled to selected reaction monitoring (MSIA-SRM) reveals novel relationships between circulating PCSK9 and metabolic phenotypes in patient cohorts.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a key regulator of circulating low density lipoprotein cholesterol (LDL-C) levels. Besides its full-length mature form, multiple variants of PCSK9 have been reported such as forms that are truncated, mutated and/or with posttranslational modifications (PTMs). Previous studies have demonstrated that most of these variants affect PCSK9's function and thereby LDL-C levels. Commercial ELISA kits are available for quantification of PCSK9, but do not allow discrimination between the various forms and PTMs of the protein. To address this issue and given the complexity and wide dynamic range of the plasma proteome, we have developed a mass spectrometric immunoassay coupled to selected reaction monitoring (MSIA-SRM) for the multiplexed quantification of several forms of circulating PCSK9 in human plasma. Our MSIA-SRM assay quantifies peptides spanning the various protein domains and the S688 phosphorylation site. The assay was applied in two distinct cohorts of obese patients and healthy pregnant women stratified by their circulating LDL-C levels. Seven PCSK9 peptides were monitored in plasma samples: one in the prodomain prior to the autocleavage site at Q152, one in the catalytic domain prior to the furin cleavage site at R218, two in the catalytic domain following R218, one in the cysteine and histidine rich domain (CHRD) and the C-terminal peptide phosphorylated at S688 and unmodified. The latter was not detectable in sufficient amounts to be quantified in human plasma. All peptides were measured with high reproducibility and with LLOQ and LOD below the clinical range. The abundance of 5 of the 6 detectable PCSK9 peptides was higher in obese patients stratified with high circulating LDL-C levels as compared to those with low LDL-C (p < 0.05). The same 5 peptides showed good and statistically significant correlations with LDL-C levels (0.55 < r < 0.65; 0.0002 ⩽ p ⩽ 0.002), but not the S688 phosphorylated peptide. However, this phosphopeptide was significantly correlated with insulin resistance (r = 0.48; p = 0.04). In the pregnant women cohort, none of the peptides were associated to LDL-C levels. However, the 6 detectable PCSK9 peptides, but not PCSK9 measured by ELISA, were significantly correlated with serum triglyceride levels in this cohort. Our results also suggest that PCSK9 circulates with S688 phosphorylated at high stoichiometry. In summary, we have developed and applied a robust and sensitive MSIA-SRM assay for the absolute quantification of all PCSK9 domains and a PTM in human plasma. This assay revealed novel relationships between PCSK9 and metabolic phenotypes, as compared to classical ELISA assays.

Liposomes ameliorate Crizotinib- and Nilotinib-induced inhibition of the cardiac IKr channel and QTc prolongation.

Crizotinib (Xalkori®) and nilotinib (Tasigna®) are tyrosine kinase inhibitors approved for the treatment of non-small cell lung cancer and chronic myeloid leukemia, respectively. Both have been shown to result in electrocardiogram rate-corrected Q-wave T-wave interval (QTc) prolongation in humans and animals. Liposomes have been shown to ameliorate drug-induced effects on the cardiac-delayed rectifier K(+) current (IKr, KV11.1), coded by the human ether-a-go-go-related gene (hERG). This study was undertaken to determine if liposomes would also decrease the effect of crizotinib and nilotinib on the IKr channel. Crizotinib and nilotinib were tested in an in vitro IKr assay using human embryonic kidney (HEK) 293 cells stably transfected with the hERG. Dose-responses were determined and the 50% inhibitory concentrations (IC50s) were calculated. When the HEK 293 cells were treated with crizotinib or nilotinib that were mixed with liposomes, there was a significant decrease in the IKr channel inhibitory effects of these two drugs. When isolated, rabbit hearts were exposed to crizotinib or nilotinib, there were significant increases in QTc prolongation. Mixing either of the drugs with liposomes ameliorated the effects of the drugs. Rabbits dosed intravenously (IV) with crizotinib or nilotinib showed QTc prolongation. When liposomes were injected prior to crizotinib or nilotinib, the liposomes decreased the effects on the QTc interval. The use of liposomal encapsulated QT-prolongation agents, or giving liposomes in combination with drugs, may decrease their cardiac liability.

The CRAPome: a contaminant repository for affinity purification-mass spectrometry data.

Affinity purification coupled with mass spectrometry (AP-MS) is a widely used approach for the identification of protein-protein interactions. However, for any given protein of interest, determining which of the identified polypeptides represent bona fide interactors versus those that are background contaminants (for example, proteins that interact with the solid-phase support, affinity reagent or epitope tag) is a challenging task. The standard approach is to identify nonspecific interactions using one or more negative-control purifications, but many small-scale AP-MS studies do not capture a complete, accurate background protein set when available controls are limited. Fortunately, negative controls are largely bait independent. Hence, aggregating negative controls from multiple AP-MS studies can increase coverage and improve the characterization of background associated with a given experimental protocol. Here we present the contaminant repository for affinity purification (the CRAPome) and describe its use for scoring protein-protein interactions. The repository (currently available for Homo sapiens and Saccharomyces cerevisiae) and computational tools are freely accessible at http://www.crapome.org/.

Effects of injectable HPßCD-diclofenac on the human delayed rectifier potassium channel current in vitro and on proarrhythmic QTc in vivo.

BACKGROUND: Novel formulations and administration routes of established drugs may result in higher maximum concentrations or total exposures and potentially cause previously unrecognized adverse events. OBJECTIVE: This study evaluated the proarrhythmic potential of hydroxypropyl-ß-cyclodextrin (HPßCD)-diclofenac, a novel injectable diclofenac formulation solubilized with hydroxypropyl-ß-cyclodextrin (HPßCD), on ventricular electrical conduction in preclinical and clinical models. METHODS: We assessed the effects of diclofenac, HPßCD, and HPßCD-diclofenac on the human delayed rectifier potassium channel (IKr) using human embryonic kidney (HEK) 293 cells transfected with a human ether-à-go-go-related gene (hERG) using whole-cell patch-clamp. In a single-dose, active- and placebo-controlled, 4-period crossover, thorough QT in vivo study, 70 healthy volunteers (mean age, 23.3 years; range, 18-49 years; 55.75% male) received HPßCD-diclofenac at 37.5- and 75-mg doses, inactive vehicle (placebo), and an active control (moxifloxacin). RESULTS: In vitro, diclofenac produced no statistically significant effect on IKr. Significant, non-dose-dependent effects were observed in the presence of HPßCD or HPßCD-diclofenac of similar magnitude across the 300-fold dose range of concentrations tested, suggesting an artifact due to the detergent effect of HPßCD in this in vitro model. In vivo, neither HPßCD-diclofenac dose resulted in QTc prolongation ≥2 ms (≥5 ms is the threshold of clinical concern). No correlation was evident between changes in QTc and plasma concentrations of diclofenac or HPßCD. Confirming study sensitivity, moxifloxacin produced a mean QTc prolongation >10 ms. CONCLUSIONS: The findings from the present study suggest that HPßCD-diclofenac does not have a dose-dependent effect in the in vitro hERG assay system and does not produce proarrhythmic QTc prolongation in vivo. ClinicalTrials.gov identifier: NCT01812538.

Discovery of cell compartment specific protein-protein interactions using affinity purification combined with tandem mass spectrometry.

Affinity purification combined with tandem mass spectrometry (AP-MS/MS) is a well-established method used to discover interaction partners for a given protein of interest. Because most AP-MS/MS approaches are performed using the soluble fraction of whole cell extracts (WCE), information about the cellular compartments where the interactions occur is lost. More importantly, classical AP-MS/MS often fails to identify interactions that take place in the nonsoluble fraction of the cell, for example, on the chromatin or membranes; consequently, protein complexes that are less soluble are underrepresented. In this paper, we introduce a method called multiple cell compartment AP-MS/MS (MCC-AP-MS/MS), which identifies the interactions of a protein independently in three fractions of the cell: the cytoplasm, the nucleoplasm, and the chromatin. We show that this fractionation improves the sensitivity of the method when compared to the classical affinity purification procedure using soluble WCE while keeping a very high specificity. Using three proteins known to localize in various cell compartments as baits, the CDK9 subunit of transcription elongation factor P-TEFb, the RNA polymerase II (RNAP II)-associated protein 4 (RPAP4), and the largest subunit of RNAP II, POLR2A, we show that MCC-AP-MS/MS reproducibly yields fraction-specific interactions. Finally, we demonstrate that this improvement in sensitivity leads to the discovery of novel interactions of RNAP II carboxyl-terminal domain (CTD) interacting domain (CID) proteins with POLR2A.

Transcription factor IIS cooperates with the E3 ligase UBR5 to ubiquitinate the CDK9 subunit of the positive transcription elongation factor B.

Elongation of transcription by mammalian RNA polymerase II (RNAPII) is regulated by specific factors, including transcription factor IIS (TFIIS) and positive transcription elongation factor b (P-TEFb). We show that the E3 ubiquitin ligase UBR5 associates with the CDK9 subunit of positive transcription elongation factor b to mediate its polyubiquitination in human cells. TFIIS also binds UBR5 to stimulate CDK9 polyubiquitination. Co-localization of UBR5, CDK9, and TFIIS along specific regions of the γ fibrinogen (γFBG) gene indicates that a ternary complex involving these factors participates in the transcriptional regulation of this gene. In support of this notion, overexpression of TFIIS not only modifies the ubiquitination pattern of CDK9 in vivo but also increases the association of CDK9 with various regions of the γFBG gene. Notably, the TFIIS-mediated increase in CDK9 loading is obtained during both basal and activated transcription of the γFBG gene. This increased CDK9 binding is paralleled by an increase in the recruitment of RNAPII along the γFBG gene and the phosphorylation of the C-terminal domain of the RNAPII largest subunit RPB1 on Ser-2, a known target of CDK9. Together, these results identify UBR5 as a novel E3 ligase that regulates transcription and define an additional function of TFIIS in the regulation of CDK9.

The protein interaction network of the human transcription machinery reveals a role for the conserved GTPase RPAP4/GPN1 and microtubule assembly in nuclear import and biogenesis of RNA polymerase II.

RNA polymerase II (RNAPII), the 12-subunit enzyme that synthesizes all mRNAs and several non-coding RNAs in eukaryotes, plays a central role in cell function. Although multiple proteins are known to regulate the activity of RNAPII during transcription, little is known about the machinery that controls the fate of the enzyme before or after transcription. We used systematic protein affinity purification coupled to mass spectrometry (AP-MS) to characterize the high resolution network of protein interactions of RNAPII in the soluble fraction of human cell extracts. Our analysis revealed that many components of this network participate in RNAPII biogenesis. We show here that RNAPII-associated protein 4 (RPAP4/GPN1) shuttles between the nucleus and the cytoplasm and regulates nuclear import of POLR2A/RPB1 and POLR2B/RPB2, the two largest subunits of RNAPII. RPAP4/GPN1 is a member of a newly discovered GTPase family that contains a unique and highly conserved GPN loop motif that we show is essential, in conjunction with its GTP-binding motifs, for nuclear localization of POLR2A/RPB1 in a process that also requires microtubule assembly. A model for RNAPII biogenesis is presented.

Conformational coupling, bridge helix dynamics and active site dehydration in catalysis by RNA polymerase.

Molecular dynamics simulation of Thermus thermophilus (Tt) RNA polymerase (RNAP) in a catalytic conformation demonstrates that the active site dNMP-NTP base pair must be substantially dehydrated to support full active site closing and optimum conditions for phosphodiester bond synthesis. In silico mutant beta R428A RNAP, which was designed based on substitutions at the homologous position (Rpb2 R512) of Saccharomyces cerevisiae (Sc) RNAP II, was used as a reference structure to compare to Tt RNAP in simulations. Long range conformational coupling linking a dynamic segment of the bridge alpha-helix, the extended fork loop, the active site, and the trigger loop-trigger helix is apparent and adversely affected in beta R428A RNAP. Furthermore, bridge helix bending is detected in the catalytic structure, indicating that bridge helix dynamics may regulate phosphodiester bond synthesis as well as translocation. An active site "latch" assembly that includes a key trigger helix residue Tt beta' H1242 and highly conserved active site residues beta E445 and R557 appears to help regulate active site hydration/dehydration. The potential relevance of these observations in understanding RNAP and DNAP induced fit and fidelity is discussed.

High-resolution mapping of the protein interaction network for the human transcription machinery and affinity purification of RNA polymerase II-associated complexes.

Thirty years of research on gene transcription has uncovered a myriad of factors that regulate, directly or indirectly, the activity of RNA polymerase II (RNAPII) during mRNA synthesis. Yet many regulatory factors remain to be discovered. Using protein affinity purification coupled to mass spectrometry (AP-MS), we recently unraveled a high-density interaction network formed by RNAPII and its accessory factors from the soluble fraction of human cell extracts. Validation of the dataset using a machine learning approach trained to minimize the rate of false positives and false negatives yielded a high-confidence dataset and uncovered novel interactors that regulate the RNAPII transcription machinery, including a new protein assembly we named the RNAPII-Associated Protein 3 (RPAP3) complex.