👤 Molly McDonald

Intermittent hypoxia and hypercapnia (IHC), a hallmark of obstructive sleep apnea (OSA), accelerates atherosclerosis, yet the underlying mechanisms remain unclear. The gut microbiota and metabolites, specifically bile acids, change with IHC and thus the bile acid receptor farnesoid X receptor (FXR) might mediate IHC-induced atherosclerosis. In this study, Show less

To delineate organ-specific and systemic drivers of metabolic dysfunction-associated steatotic liver disease (MASLD), we applied integrative causal inference across clinical, imaging, and proteomic domains in individuals with and without type 2 diabetes (T2D). Bayesian network analyses and complementary two-sample Mendelian randomization were used to quantify causal pathways linking adipose distribution, glycemia, and insulin dynamics with liver fat in the IMI-DIRECT prospective cohort study. Data included frequently sampled metabolic challenge tests, MRI-derived abdominal and hepatic fat content, serological biomarkers, and Olink plasma proteomics from 331 adults with new-onset T2D and 964 adults without diabetes, with harmonized protocols enabling replication. High basal insulin secretion rate (BasalISR), estimated via C-peptide deconvolution, emerged as the primary potential causal driver of liver fat accumulation in both cohorts. BasalISR, a clearance-independent measure of β-cell insulin output distinct from peripheral insulin levels, was independently linked to hepatic steatosis. Visceral adipose tissue exhibited bidirectional associations with liver fat, suggesting a self-reinforcing metabolic loop. Of 446 analyzed proteins, 34 mapped to these metabolic networks (27 in the non-diabetes network, 18 in the T2D network, and 11 shared). Key proteins directly associated with liver fat included GUSB, ALDH1A1, LPL, IGFBP1/2, CTSD, HMOX1, FGF21, AGRP, and ACE2. Sex-stratified analyses identified GUSB in females and LEP in males as the strongest protein predictors of liver fat. BasalISR may better capture early β-cell-driven disturbances contributing to MASLD. These findings outline a multifactorial, sex- and disease stage-specific proteo-metabolic architecture of hepatic steatosis and identify potential biomarkers or therapeutic targets. Show less

Oxidative stress perturbs lipid homeostasis and contributes to metabolic diseases. Though ignored when compared with mitochondrial oxidation, the endoplasmic reticulum (ER) generates reactive oxygen species requiring antioxidant quality control. Using multi-organismal profiling featuring Drosophila, zebrafish, and mammalian hepatocytes, here we characterize the paraoxonase-like C20orf3/adipocyte plasma-membrane-associated protein (APMAP) as an ER-localized antioxidant that suppresses ER lipid oxidation to safeguard ER function. APMAP-depleted cells exhibit defective ER morphology, ER stress, and lipid peroxidation dependent on ER-oxidoreductase 1α (ERO1A), as well as sensitivity to ferroptosis and defects in ApoB-lipoprotein homeostasis. Similarly, organismal APMAP depletion in Drosophila and zebrafish perturbs ApoB-lipoprotein homeostasis. Strikingly, APMAP loss is rescued with chemical antioxidant N-acetyl-cysteine (NAC). Lipidomics identifies that APMAP loss elevates phospholipid peroxidation and boosts ceramides-signatures of lipid stress. Collectively, we propose that APMAP is an ER-localized antioxidant that promotes lipid and lipoprotein homeostasis in the ER network. Show less

Anti-HER2 antibodies are effective but often lead to resistance in patients with HER2+ breast cancer. Here, we report an epigenetic crosstalk with aberrant glycerophospholipid metabolism and inflammation as a key resistance mechanism of anti-HER2 therapies in HER2+ breast cancer. Histone reader ZMYND8 specifically confers resistance to cancer cells against trastuzumab and/or pertuzumab. Mechanistically, ZMYND8 enhances cPLA2α expression in resistant tumor cells through inducing c-Myc. cPLA2α inactivates phosphatidylcholine-specific phospholipase C to inhibit phosphatidylcholine breakdown into diacylglycerol, which diminishes protein kinase C activity leading to interleukin-27 secretion. Supplementation with interleukin-27 protein counteracts cPLA2α loss to reinforce trastuzumab resistance in HER2+ tumor cells and patient-derived organoids. Upregulation of ZMYND8, c-Myc, cPLA2α, and IL-27 is prevalent in HER2+ breast cancer patients following HER2-targeted therapies. Targeting c-Myc or cPLA2α effectively overcomes anti-HER2 therapy resistance in patient-derived xenografts. Collectively, this study uncovers a druggable signaling cascade that drives resistance to HER2-targeted therapies in HER2+ breast cancer. Show less

To delineate organ-specific and systemic drivers of metabolic dysfunction-associated steatotic liver disease (MASLD), we applied integrative causal inference across clinical, imaging, and proteomic domains in individuals with and without type 2 diabetes (T2D). We used Bayesian network analyses to quantify causal pathways linking adipose distribution, glycemia, and insulin dynamics with fatty liver using data from the IMI-DIRECT prospective cohort study. Measurements were made of glucose and insulin dynamics (using frequently-sampled metabolic challenge tests), MRI-derived abdominal and liver fat content, serological biomarkers, and Olink plasma proteomics from 331 adults with new-onset T2D and 964 adults free from diabetes at enrolment. The common protocols used in these two cohorts provided the opportunity for replication analyses to be performed. When the direction of the effect could not be determined with high probability through Bayesian networks, complementary two-sample Mendelian randomization (MR) was employed. High basal insulin secretion rate (BasalISR) was identified as the primary causal driver of liver fat accumulation in both diabetes and non-diabetes. Excess visceral adipose tissue (VAT) was bidirectionally associated with liver fat, indicating a self-reinforcing metabolic loop. Basal insulin clearance (Clinsb) worsened as a consequence of liver fat accumulation to a greater degree before the onset of T2D. Out of 446 analysed proteins, 34 mapped to these metabolic networks and 27 were identified in the non-diabetes network, 18 in the diabetes network, and 11 were common between the two networks. Key proteins directly associated with liver fat included GUSB, ALDH1A1, LPL, IGFBP1/2, CTSD, HMOX1, FGF21, AGRP, and ACE2. Sex-stratified analyses revealed distinct proteomic drivers: GUSB and LEP were most predictive of liver fat in females and males, respectively. Basal insulin hypersecretion is a modifiable, causal driver of MASLD, particularly prior to glycaemic decompensation. Our findings highlight a multifactorial, sex- and disease-stage-specific proteo-metabolic architecture of hepatic steatosis. Proteins such as GUSB, ALDH1A1, LPL, and IGFBPs warrant further investigation as potential biomarkers or therapeutic targets for MASLD prevention and treatment. Show less

During the ejection phase of the cardiac cycle, left ventricular (LV) cardiac myocytes undergo loaded shortening and generate power. However, few studies have measured sarcomere shortening during loaded contractions. Here, we simultaneously monitored muscle length (ML) and sarcomere length (SL) during isotonic contractions in rodent permeabilized LV cardiac myocyte preparations. In permeabilized cardiac myocyte preparations from rats, we found that ML and SL traces were closely matched, as SL velocities were within ∼77% of ML velocities during half-maximal Ca2+ activations. We next tested whether cardiac myosin binding protein-C (cMyBP-C) regulates loaded shortening and power output by modulating cross-bridge availability. We characterized force-velocity and power-load relationships in wildtype (WT) and cMyBP-C deficient (Mybpc3-/-) mouse permeabilized cardiac myocyte preparations, at both the ML and SL level, before and after treatment with the small molecule myosin inhibitor, mavacamten. We found that SL traces closely matched ML traces in both WT and Mybpc3-/- cardiac myocytes. However, Mybpc3-/- cardiac myocytes exhibited disproportionately high sarcomere shortening velocities at high loads. Interestingly, in Mybpc3-/- cardiac myocytes, 0.5 µM mavacamten slowed SL-loaded shortening across the force-velocity curve and normalized SL shortening velocity at high loads. Overall, these results suggest that cMyBP-C moderates sarcomere-loaded shortening, especially at high loads, at least in part, by modulating cross-bridge availability. Show less

Obesity is a major public health crisis associated with high mortality rates. Previous genome-wide association studies (GWAS) investigating body mass index (BMI) have largely relied on imputed data from European individuals. This study leveraged whole-genome sequencing (WGS) data from 88,873 participants from the Trans-Omics for Precision Medicine (TOPMed) Program, of which 51% were of non-European population groups. We discovered 18 BMI-associated signals (P < 5 × 10 Show less

Obesity is a major public health crisis associated with high mortality rates. Previous genome-wide association studies (GWAS) investigating body mass index (BMI) have largely relied on imputed data from European individuals. This study leveraged whole-genome sequencing (WGS) data from 88,873 participants from the Trans-Omics for Precision Medicine (TOPMed) Program, of which 51% were of non-European population groups. We discovered 18 BMI-associated signals ( Show less

Rice biofortification with Zinc (Zn) can improve the Zn status of rice-consuming populations. However, the metabolic impact in humans consuming Zn-biofortified rice is unknown. To determine the effects of Zn-biofortified rice on lipid metabolism in normolipidemic men. The men consumed a rice-based diet containing 6 mg Zn/d and 1.5 g phytate (phytate/Zn ratio = 44) for 2 wk followed by a 10-mg Zn/d diet without phytate for 4 wk. An ad libitum diet supplemented with 25 mg Zn/d was then fed for 3 wk. Fasting blood samples were taken at baseline and at the end of each metabolic period for measuring plasma zinc, glucose, insulin, triglyceride (TG), LDL and HDL cholesterol, fatty acids, oxylipins, and fatty acid desaturase activities. Statistical differences were assessed by linear mixed model. Fatty acid desaturase (FADS) 1 activity decreased by 29.1% (P = 0.007) when the 6-mg Zn/d diet was consumed for 2 wk. This change was associated with significant decreases in HDL and LDL cholesterol. The alterations in FADS1, HDL cholesterol, and TG remained unchanged when Zn intakes were increased to 10 mg/d for 4 wk. Supplementation with 25 mg Zn/d for 3 wk normalized these metabolic changes and significantly increased LDL cholesterol at the end of this metabolic period compared with baseline. FADS1 activity was inversely correlated with FADS2 (rmcorr = -0.52; P = 0.001) and TG (rmcorr = -0.55; P = 0.001) at all time points. A low-zinc, high-phytate rice-based diet reduced plasma HDL cholesterol concentrations and altered fatty acid profiles in healthy men within 2 wk. Consuming 10 mg Zn/d without phytate for 4 wk did not improve the lipid profiles, but a 25-mg Zn/d supplement corrects these alterations in lipid metabolism within 3 wk. Show less

Fibrosis is a leading cause of morbidity and mortality worldwide. Although fibrosis may involve different organ systems, transforming growth factor-β (TGFβ) has been established as a master regulator of fibrosis across organs. Pirfenidone and Nintedanib are the only currently-approved drugs to treat fibrosis, specifically idiopathic pulmonary fibrosis, but their mechanisms of action remain poorly understood. To identify novel drug targets and uncover potential mechanisms by which these drugs attenuate fibrosis, we performed an integrative 'omics analysis of transcriptomic and proteomic responses to TGFβ1-stimulated lung fibroblasts. Significant findings were annotated as associated with pirfenidone and nintedanib treatment in silico via Coremine. Integrative 'omics identified a co-expressed transcriptomic and proteomic module significantly correlated with TGFβ1 treatment that was enriched (FDR-p = 0.04) with genes associated with pirfenidone and nintedanib treatment. While a subset of genes in this module have been implicated in fibrogenesis, several novel TGFβ1 signaling targets were identified. Specifically, four genes (BASP1, HSD17B6, CDH11, and TNS1) have been associated with pirfenidone, while five genes (CLINT1, CADM1, MTDH, SYDE1, and MCTS1) have been associated with nintedanib, and MYDGF has been implicated with treatment using both drugs. Using the Clue Drug Repurposing Hub, succinic acid was highlighted as a metabolite regulated by the protein encoded by HSD17B6. This study provides new insights into the anti-fibrotic actions of pirfenidone and nintedanib and identifies novel targets for future mechanistic studies. Show less

Currently, no standardized system exists for evaluating and testing at-risk family members of decedents with abnormal post-mortem genetic testing in cases of sudden unexpected death (SUD). The goal of this study was to evaluate the outcomes of referrals made by an urban medical examiner's office to a multi-disciplinary cardiogenetics clinic. Relatives of decedents with pathogenic/likely pathogenic (P/LP) variants or variants of unknown significance (VUS) in genes known to be associated with cardiomyopathies and/or arrhythmias were identified by the New York City Office of Chief Medical Examiner and referred to the Cardiogenetics Clinic at Montefiore Medical Center. Familial referrals of 15 decedents (median 15 years, range 2 days to 57 years) were evaluated. Variants in 13 genes were identified among decedents (9 arrhythmia, 5 cardiomyopathy). P/LP variants were identified in both arrhythmia (RYR2, SCN5A) and cardiomyopathy syndrome (MYBPC3 (2), MYH7) genes. Thirty-two family members were referred, and 14 variants were detected. One pathogenic (MYBPC3) and two likely pathogenic (RYR2, MYH7) mutations were identified. Referral of at-risk family members of decedents who experienced SUD based on informative post-mortem genetic testing for cardiac and genetic evaluation is warranted, as family studies help to reclassify variants and prevent additional sudden death. Show less

Biofortification is a novel method for improving the nutritional value of grains. Wheat is widely consumed worldwide. Thus, wheat zinc biofortification may improve the zinc status of populations. We determined the effect of consuming zinc-biofortified wheat on plasma zinc concentrations and biomarkers of zinc-dependent functions in a controlled feeding study. Thirty-six healthy adult men, aged 18 to 51 y, participated in a 10-wk zinc-controlled feeding trial. After a 2-wk run-in period [metabolic period (MP) 1] (9.3 mg zinc/d and 2.1 g total phytate/d) to standardize zinc status, the participants consumed bread made from zinc-biofortified wheat (10.9 mg zinc/d) with no additional phytate (0.6 g/d total phytate) for 6 wk (MP2). During the final 2 wk (MP3), half of the men took a 25-mg zinc supplement daily to determine if the supplement further altered zinc status biomarkers. Repeated-measures linear regression methods were used to compare plasma zinc concentrations, fatty acid desaturase (FADS) activities, glutathione (GSH) concentrations, and DNA strand breaks assessed at enrollment and the end of each metabolic period. Plasma zinc concentrations did not change throughout the study. From the end of MP1 to the end of MP2, the conversion of linoleic acid to γ-linolenic acid (FADS2 activity) increased from 0.020 to 0.025 (P = 0.02), and the conversion of dihomo-γ-linolenic acid to arachidonic acid (FADS1 activity) decreased from 6.37 to 5.53 (P = 0.01). GSH concentrations and DNA strand breaks did not change. Zinc supplementation (25 mg/d) in MP3 did not alter any of the endpoints. In healthy adult men, a 1.6-mg/d increase in dietary zinc from biofortified wheat modified FADS2 and FADS1 activities without changing DNA damage, plasma zinc, or GSH concentrations, demonstrating that FADS activities are more sensitive to small changes in zinc consumed with a meal. This trial was registered at clinicaltrials.gov as NCT03451214. Show less

Mutations in melanocortin receptor (MC4R) are the most common cause of monogenic obesity in children of European ancestry, but little is known about their prevalence in children from the minority populations in the United States. This study aims to identify the prevalence of MC4R mutations in children with severe early-onset obesity of African American or Latino ancestry. Participants were recruited from the weight management clinics at two hospitals and from the institutional biobank at a third hospital. Sequencing of the MC4R gene was performed by whole exome or Sanger sequencing. Functional testing was performed to establish the surface expression of the receptor and cAMP response to its cognate ligand α-melanocyte-stimulating hormone. Three hundred twelve children (1 to 18 years old, 50% girls) with body mass index (BMI) >120% of 95th percentile of Centers for Disease Control and Prevention 2000 growth charts at an age <6 years, with no known pathological cause of obesity, were enrolled. Eight rare MC4R mutations (2.6%) were identified in this study [R7S, F202L (n = 2), M215I, G252D, V253I, I269N, and F284I], three of which were not previously reported (G252D, F284I, and R7S). The pathogenicity of selected variants was confirmed by prior literature reports or functional testing. There was no significant difference in the BMI or height trajectories of children with or without MC4R mutations in this cohort. Although the prevalence of MC4R mutations in this cohort was similar to that reported for obese children of European ancestry, some of the variants were novel. Show less

Limited knowledge exists of the extent of epigenetic alterations, such as DNA methylation, in heart failure (HF). We conducted targeted DNA methylation sequencing to identify DNA methylation alterations in coding and noncoding RNA (ncRNA) across different etiological subtypes of HF. A targeted bisulfite sequence capture sequencing platform was applied to DNA extracted from cardiac interventricular septal tissue of 30 male HF patients encompassing causes including hypertrophic obstructive cardiomyopathy, ischemic cardiomyopathy, dilated cardiomyopathy, and 9 control patients with nonfailing hearts. We detected 62 678 differentially methylated regions in the studied HF cohort. By comparing each HF subgroup to the nonfailing control group, we identified 195 unique differentially methylated regions: 5 in hypertrophic obstructive cardiomyopathy, 151 in dilated cardiomyopathy, and 55 in ischemic cardiomyopathy. These translated to 4 genes/1 ncRNA in hypertrophic obstructive cardiomyopathy, 131 genes/17 ncRNA in dilated cardiomyopathy, and 51 genes/5 ncRNA in ischemic cardiomyopathy. Subsequent gene/ncRNA expression analysis was assessed using quantitative reverse transcription polymerase chain reaction and revealed 6 genes: 4 hypermethylated ( HEY2, MSR1, MYOM3, and COX17), 2 hypomethylated ( CTGF and MMP2); and 2 microRNA: 1 hypermethylated (miR-24-1), 1 hypomethylated (miR-155) with significantly upregulated or downregulated expression levels consistent with the direction of methylation in the particular HF subgroup. For the first time DNA methylation alterations and associated gene expression changes were identified in etiologically variant pathological HF tissue. The methylation-sensitive and disease-associated genes/ncRNA identified from this study represent a unique cohort of loci that demonstrate a plausible potential as a novel diagnostic and therapeutic target in HF and warrant further investigation. Show less

Deregulation of the cyclin-dependent kinases (CDKs) has been implicated in the pathogenesis of multiple cancer types. Consequently, CDKs have garnered intense interest as therapeutic targets for the treatment of cancer. We describe herein the molecular and cellular effects of CCT068127, a novel inhibitor of CDK2 and CDK9. Optimized from the purine template of seliciclib, CCT068127 exhibits greater potency and selectivity against purified CDK2 and CDK9 and superior antiproliferative activity against human colon cancer and melanoma cell lines. X-ray crystallography studies reveal that hydrogen bonding with the DFG motif of CDK2 is the likely mechanism of greater enzymatic potency. Commensurate with inhibition of CDK activity, CCT068127 treatment results in decreased retinoblastoma protein (RB) phosphorylation, reduced phosphorylation of RNA polymerase II, and induction of cell cycle arrest and apoptosis. The transcriptional signature of CCT068127 shows greatest similarity to other small-molecule CDK and also HDAC inhibitors. CCT068127 caused a dramatic loss in expression of DUSP6 phosphatase, alongside elevated ERK phosphorylation and activation of MAPK pathway target genes. MCL1 protein levels are rapidly decreased by CCT068127 treatment and this associates with synergistic antiproliferative activity after combined treatment with CCT068127 and ABT263, a BCL2 family inhibitor. These findings support the rational combination of this series of CDK2/9 inhibitors and BCL2 family inhibitors for the treatment of human cancer. Show less

Our aim is to characterize predicted protein-truncating variants (PTVs) in MYBPC3, the gene most commonly associated with hypertrophic cardiomyopathy (HCM), found in a series of autopsied HCM cases after sudden unexpected cardiac death. All cases underwent death scene investigation, gross and microscopic autopsies, toxicological testing, a review of medical records, and a molecular analysis of 95 cardiac genes. We found four pathogenic PTVs in MYBPC3 among male decedents. All variants were previously submitted to ClinVar without phenotype details. Two PTVs were located in the cardiac-specific myosin S2-binding (M) motif at the N-terminus of the MYBPC3-encoded cMyBP-C protein, and two PTVs were in the non-cardiac-specific C-terminus of the protein. The carriers of two cardiac-specific M-motif PTVs died at age 38 years; their heart weight (HW, g) and body mass index (BMI, kg/m Show less

The contribution of polymorphisms associated with adult lipids in early life is unknown. We studied 158 adult lipid polymorphisms in 1440 participants (544 children, 544 mothers and 324 fathers) of the Family Atherosclerosis Monitoring In early life (FAMILY) birth cohort. Total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) measurements were collected at birth, 3 and 5 years of age. Polymorphisms were genotyped using the Illumina Cardio-Metabochip array. Genotype scores (GS) were calculated for TC, HDL-C, LDL-C and TG. Linear and mixed-effects regressions adjusted for sex, age and population stratification were performed. The GS was associated with LDL-C level at 3 and 5 years (β = 0.017 ± 0.003, P = 2.9 × 10 Show less

Mechanisms underlying changes in HDL composition caused by obesity are poorly defined, partly because mice lack expression of cholesteryl ester transfer protein (CETP), which shuttles triglyceride and cholesteryl ester between lipoproteins. Because menopause is associated with weight gain, altered glucose metabolism, and changes in HDL, we tested the effect of feeding a high-fat diet (HFD) and ovariectomy (OVX) on glucose metabolism and HDL composition in CETP transgenic mice. After OVX, female CETP-expressing mice had accelerated weight gain with HFD-feeding and impaired glucose tolerance by hyperglycemic clamp techniques, compared with OVX mice fed a low-fat diet (LFD). Sham-operated mice (SHAM) did not show HFD-induced weight gain and had less glucose intolerance than OVX mice. Using shotgun HDL proteomics, HFD-feeding in OVX mice had a large effect on HDL composition, including increased levels of apoA2, apoA4, apoC2, and apoC3, proteins involved in TG metabolism. These changes were associated with decreased hepatic expression of SR-B1, ABCA1, and LDL receptor, proteins involved in modulating the lipid content of HDL. In SHAM mice, there were minimal changes in HDL composition with HFD feeding. These studies suggest that the absence of ovarian hormones negatively influences the response to high-fat feeding in terms of glucose tolerance and HDL composition. CETP-expressing mice may represent a useful model to define how metabolic changes affect HDL composition and function. Show less

After injury to the central nervous system intrinsic factors such as myelin associated inhibitory factors inhibit cellular and axonal regeneration resulting in permanent disability. Three of these factors (Nogo-A, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein) bind to a common receptor: the Nogo-66 receptor (NgR1). NgR1 is expressed mainly on neurons and is usually associated in a trimolecular complex. The second member of the complex, LINGO-1, is often connected to NgR1 function and is further found to function independently as a negative regulator of oligodendrocyte proliferation and differentiation. The third member of the NgR complex is either the p75 neurotrophin receptor, TROY, or an as yet unidentified co-receptor. Targeting of factors contained in this complex has been described to lead to the promotion of neurite outgrowth, oligodendrocyte proliferation and differentiation and inhibition of cell death. In the current review, we aim to describe the mechanisms of action of the chemical and biological compounds used in targeting NgR1 and LINGO-1. This will be achieved using three examples: blocking of ligand binding to NgR1 in treatment of spinal cord injury, antibody-mediated inhibition of LINGO-1 to promote oligodendrocyte differentiation in multiple sclerosis, and the use of soluble NgR1 to sequester Abeta peptide in the periphery in Alzheimer's disease. Show less

In the budding yeast Saccharomyces cerevisiae, cell cycle initiation is prompted during G(1) phase by Cln3/cyclin-dependent protein kinase-mediated transcriptional activation of G(1)-specific genes. A recent screening performed to reveal novel interactors of SCB-binding factor (SBF) and MCB-binding factor (MBF) identified, in addition to the SBF-specific repressor Whi5 and the MBF-specific corepressor Nrm1, a pair of homologous proteins, Msa1 and Msa2 (encoded by YOR066w and YKR077w), as interactors of SBF and MBF, respectively. MSA1 is expressed periodically during the cell cycle with peak mRNA levels occurring at the late M/early G(1) phase and peak protein levels occurring in early G(1). Msa1 associates with SBF- and MBF-regulated target promoters consistent with a role in G(1)-specific transcriptional regulation. Msa1 affects cell cycle initiation by advancing the timing of transcription of G(1)-specific genes. Msa1 binds to SBF- and MBF-regulated promoters and binding is maximal during the G(1) phase. Binding depends upon the cognate transcription factor. Msa1 overexpression advances the timing of SBF-dependent transcription and budding, whereas depletion delays both indicators of cell cycle initiation. Similar effects on MBF-regulated transcription are observed. Based upon these results, we conclude that Msa1 acts to advance the timing of G(1)-specific transcription and cell cycle initiation. Show less

Mitogen-activated protein kinase phosphatase (MKP)-1 is a dual-specificity phosphatase that negatively regulates the activity of mitogen-activated kinases and that is overexpressed in human tumors. Contemporary studies suggest that induction of MKP-1 during chemotherapy may limit the efficacy of clinically used antineoplastic agents. Thus, MKP-1 is a rational target to enhance anticancer drug activity, but suitable small-molecule inhibitors of MKP-1 are currently unavailable. Here, we have used a high-content, multiparameter fluorescence-based chemical complementation assay for MKP activity in intact mammalian cells to evaluate the cellular MKP-1 and MKP-3 inhibitory activities of four previously described, quinone-based, dual-specificity phosphatase inhibitors, that is, NSC 672121, NSC 95397, DA-3003-1 (NSC 663284), and JUN-1111. All compounds induced formation of reactive oxygen species in mammalian cells, but only one (NSC 95397) inhibited cellular MKP-1 and MKP-3 with an IC(50) of 13 mumol/L. Chemical induction of MKP-1 by dexamethasone protected cells from paclitaxel-induced apoptosis but had no effect on NSC 95397. NSC 95397 phenocopied the effects of MKP-1 small inhibitory RNA by reversing the cytoprotective effects of dexamethasone in paclitaxel-treated cells. Isobologram analysis revealed synergism between paclitaxel and NSC 95397 only in the presence of dexamethasone. The data show the power of a well-defined cellular assay for identifying cell-active inhibitors of MKPs and support the hypothesis that small-molecule inhibitors of MKP-1 may be useful as antineoplastic agents under conditions of high MKP-1 expression. Show less

G1-specific transcriptional activation by Cln3/CDK initiates the budding yeast cell cycle. To identify targets of Cln3/CDK, we analyzed the SBF and MBF transcription factor complexes by multidimensional protein interaction technology (MudPIT). Whi5 was identified as a stably bound component of SBF but not MBF. Inactivation of Whi5 leads to premature expression of G1-specific genes and budding, whereas overexpression retards those processes. Whi5 inactivation bypasses the requirement for Cln3 both for transcriptional activation and cell cycle initiation. Whi5 associates with G1-specific promoters via SBF during early G1 phase, then dissociates coincident with transcriptional activation. Dissociation of Whi5 is promoted by Cln3 in vivo. Cln/CDK phosphorylation of Whi5 in vitro promotes its dissociation from SBF complexes. Mutation of putative CDK phosphorylation sites, at least five of which are phosphorylated in vivo, strongly reduces SBF-dependent transcription and delays cell cycle initiation. Like mammalian Rb, Whi5 is a G1-specific transcriptional repressor antagonized by CDK. Show less

The epithelial Na(+) channel (ENaC) is a critical component of the pathway maintaining salt and water balance. The channel is regulated by members of the Nedd4 family of ubiquitin-protein ligases, which bind to channel subunits and catalyze channel internalization and degradation. ENaC mutations that abolish this interaction cause Liddle's syndrome, a genetic form of hypertension. Here, we test the hypothesis that WW domain-containing protein 2 (WWP2), a member of the Nedd4 family of ubiquitin-protein ligases, is a candidate to regulate ENaC. Consistent with this hypothesis, we found that WWP2 is expressed in epithelial tissues that express ENaC, as well as in a wide variety of other tissues. WWP2 contains four WW domains, three of which bound differentially to ENaC subunits. In contrast, all four human Nedd4-2 WW domains bound to ENaC. WWP2 inhibited ENaC when coexpressed in epithelia, requiring a direct interaction between the proteins; mutation of the ENaC PY motifs abolished inhibition. Thus expression, binding, and functional data all suggest that WWP2 is a candidate to regulate ENaC-mediated Na(+) transport in epithelia. Show less