The leucine zipper domain of the transcriptional repressor Opi1 underlies a signal transduction mechanism regulating lipid synthesis.

In Saccharomyces cerevisiae, the transcriptional repressor Opi1 regulates the expression of genes involved in phospholipid synthesis responding to the abundance of the phospholipid precursor phosphatidic acid at the endoplasmic reticulum. We report here the identification of the conserved leucine zipper (LZ) domain of Opi1 as a hot spot for gain of function mutations and the characterization of the strongest variant identified, Opi1N150D. LZ modeling posits asparagine 150 embedded on the hydrophobic surface of the zipper and specifying dynamic parallel homodimerization by allowing electrostatic bonding across the hydrophobic dimerization interface. Opi1 variants carrying any of the other three ionic residues at amino acid 150 were also repressing. Genetic analyses showed that Opi1N150D variant is dominant, and its phenotype is attenuated when loss of function mutations identified in the other two conserved domains are present in cis. We build on the notion that membrane binding facilitates LZ dimerization to antagonize an intramolecular interaction of the zipper necessary for repression. Dissecting Opi1 protein in three polypeptides containing each conserved region, we performed in vitro analyses to explore interdomain interactions. An Opi11-190 probe interacted with Opi1291-404, the C terminus that bears the activator interacting domain (AID). LZ or AID loss of function mutations attenuated the interaction of the probes but was unaffected by the N150D mutation. We propose a model for Opi1 signal transduction whereby synergy between membrane-binding events and LZ dimerization antagonizes intramolecular LZ-AID interaction and transcriptional repression.

A mouse model of inherited choline kinase ß-deficiency presents with specific cardiac abnormalities and a predisposition to arrhythmia.

The CHKB gene encodes choline kinase ß, which catalyzes the first step in the biosynthetic pathway for the major phospholipid phosphatidylcholine. Homozygous loss-of-function variants in human CHKB are associated with a congenital muscular dystrophy. Dilated cardiomyopathy is present in some CHKB patients and can cause heart failure and death. Mechanisms underlying a cardiac phenotype due to decreased CHKB levels are not well characterized. We determined that there is cardiac hypertrophy in Chkb-/- mice along with a decrease in left ventricle size, internal diameter, and stroke volume compared with wildtype and Chkb+/- mice. Unlike wildtype mice, 60% of the Chkb+/- and all Chkb-/- mice tested displayed arrhythmic events when challenged with isoproterenol. Lipidomic analysis revealed that the major change in lipid level in Chkb+/- and Chkb-/- hearts was an increase in the arrhythmogenic lipid acylcarnitine. An increase in acylcarnitine level is also associated with a defect in the ability of mitochondria to use fatty acids for energy and we observed that mitochondria from Chkb-/- hearts had abnormal cristae and inefficient electron transport chain activity. Atrial natriuretic peptide (ANP) is a hormone produced by the heart that protects against the development of heart failure including ventricular conduction defects. We determined that there was a decrease in expression of ANP, its receptor NPRA, as well as ventricular conduction system markers in Chkb+/- and Chkb-/- mice.

Bi-allelic variants in CHKA cause a neurodevelopmental disorder with epilepsy and microcephaly.

The Kennedy pathways catalyse the de novo synthesis of phosphatidylcholine and phosphatidylethanolamine, the most abundant components of eukaryotic cell membranes. In recent years, these pathways have moved into clinical focus because four of ten genes involved have been associated with a range of autosomal recessive rare diseases such as a neurodevelopmental disorder with muscular dystrophy (CHKB), bone abnormalities and cone-rod dystrophy (PCYT1A) and spastic paraplegia (PCYT2, SELENOI). We identified six individuals from five families with bi-allelic variants in CHKA presenting with severe global developmental delay, epilepsy, movement disorders and microcephaly. Using structural molecular modelling and functional testing of the variants in a cell-based Saccharomyces cerevisiae model, we determined that these variants reduce the enzymatic activity of CHKA and confer a significant impairment of the first enzymatic step of the Kennedy pathway. In summary, we present CHKA as a novel autosomal recessive gene for a neurodevelopmental disorder with epilepsy and microcephaly.

Genetic diseases of the Kennedy pathways for membrane synthesis.

The two branches of the Kennedy pathways (CDP-choline and CDP-ethanolamine) are the predominant pathways responsible for the synthesis of the most abundant phospholipids, phosphatidylcholine and phosphatidylethanolamine, respectively, in mammalian membranes. Recently, hereditary diseases associated with single gene mutations in the Kennedy pathways have been identified. Interestingly, genetic diseases within the same pathway vary greatly, ranging from muscular dystrophy to spastic paraplegia to a childhood blinding disorder to bone deformations. Indeed, different point mutations in the same gene (PCYT1; CCTα) result in at least three distinct diseases. In this review, we will summarize and review the genetic diseases associated with mutations in genes of the Kennedy pathway for phospholipid synthesis. These single-gene disorders provide insight, indeed direct genotype-phenotype relationships, into the biological functions of specific enzymes of the Kennedy pathway. We discuss potential mechanisms of how mutations within the same pathway can cause disparate disease.

Both CLIC4 and CLIC5A activate ERM proteins in glomerular endothelium.

The chloride intracellular channel (CLIC) 5A is expressed at very high levels in renal glomeruli, in both endothelial cells (EC) and podocytes. CLIC5A stimulates Rac1- and phosphatidylinositol (4,5)-bisphosphate-dependent ERM (ezrin, radixin, moesin) activation. ERM proteins, in turn, function in lumen formation and in the development of actin-based cellular projections. In mice lacking CLIC5A, ERM phosphorylation is profoundly reduced in podocytes, but preserved in glomerular EC. Since glomerular EC also express CLIC4, we reasoned that, if CLIC4 activates ERM proteins like CLIC5A, then CLIC4 could compensate for the CLIC5A loss in glomerular EC. In glomeruli of CLIC5-deficient mice, CLIC4 expression was upregulated and colocalized with moesin and ezrin in glomerular EC, but not in podocytes. In cultured glomerular EC, CLIC4 silencing reduced ERM phosphorylation and cytoskeletal association, and expression of exogenous CLIC4 or CLIC5A rescued ERM de-phosphorylation due to CLIC4 silencing. In mice lacking either CLIC4 or CLIC5, ERM phosphorylation was retained in glomerular EC, but, in mice lacking both CLIC4 and CLIC5, glomerular EC ERM phosphorylation was profoundly reduced. Although glomerular EC fenestrae developed normally in dual CLIC4/CLIC5-deficient mice, the density of fenestrae declined substantially by 8 mo of age, along with the deposition of subendothelial electron-lucent material. The dual CLIC4/CLIC5-deficient mice developed spontaneous proteinuria, glomerular cell proliferation, and matrix deposition. Thus CLIC4 stimulates ERM activation and can compensate for CLIC5A in glomerular EC. The findings indicate that CLIC4/CLIC5A-mediated ERM activation is required for maintenance of the glomerular capillary architecture.

The chloride intracellular channel 5A stimulates podocyte Rac1, protecting against hypertension-induced glomerular injury.

Glomerular capillary hypertension elicits podocyte remodeling and is a risk factor for the progression of glomerular disease. Ezrin, which links podocalyxin to actin in podocytes, is activated through the chloride intracellular channel 5A (CLIC5A)-dependent phosphatidylinositol 4,5 bisphosphate (PI[4,5]P2) accumulation. Because Rac1 is involved in podocyte actin remodeling and can promote PI[4,5]P2 production we determined whether CLIC5A-dependent PI[4,5]P2 generation and ezrin activation are mediated by Rac1. In COS7 cells, CLIC5A expression stimulated Rac1 but not Cdc42 or Rho activity. CLIC5A also stimulated phosphorylation of the Rac1 effector Pak1 in COS7 cells and in cultured mouse podocytes. CLIC5A-induced PI[4,5]P2 accumulation and Pak1 and ezrin phosphorylation were all Rac1 dependent. In DOCA/Salt hypertension, phosphorylated Pak increased in podocytes of wild-type, but not CLIC5-deficient mice. In DOCA/salt hypertensive mice lacking CLIC5, glomerular capillary microaneurysms were more frequent and albuminuria was greater than in wild-type mice. Thus, augmented hypertension-induced glomerular capillary injury in mice lacking CLIC5 results from abrogation of Rac1-dependent Pak and ezrin activation, perhaps reducing the tensile strength of the podocyte actin cytoskeleton.

Defects in integrin complex formation promote CHKB-mediated muscular dystrophy.

Phosphatidylcholine (PC) is the major membrane phospholipid in most eukaryotic cells. Bi-allelic loss of function variants in CHKB, encoding the first step in the synthesis of PC, is the cause of a rostrocaudal muscular dystrophy in both humans and mice. Loss of sarcolemma integrity is a hallmark of muscular dystrophies; however, how this occurs in the absence of choline kinase function is not known. We determine that in Chkb -/- mice there is a failure of the α7ß1 integrin complex that is specific to affected muscle. We observed that in Chkb -/- hindlimb muscles there is a decrease in sarcolemma association/abundance of the PI(4,5)P2 binding integrin complex proteins vinculin, and α-actinin, and a decrease in actin association with the sarcolemma. In cells, pharmacological inhibition of choline kinase activity results in internalization of a fluorescent PI(4,5)P2 reporter from discrete plasma membrane clusters at the cell surface membrane to cytosol, this corresponds with a decreased vinculin localization at plasma membrane focal adhesions that was rescued by overexpression of CHKB.

Atrial natriuretic peptide signaling co-regulates lipid metabolism and ventricular conduction system gene expression in the embryonic heart.

It has been shown that atrial natriuretic peptide (ANP) and its high affinity receptor (NPRA) are involved in the formation of ventricular conduction system (VCS). Inherited genetic variants in fatty acid oxidation (FAO) genes are known to cause conduction abnormalities in newborn children. Although the effect of ANP on energy metabolism in noncardiac cell types is well documented, the role of lipid metabolism in VCS cell differentiation via ANP/NPRA signaling is not known. In this study, histological sections and primary cultures obtained from E11.5 mouse ventricles were analyzed to determine the role of metabolic adaptations in VCS cell fate determination and maturation. Exogenous treatment of E11.5 ventricular cells with ANP revealed a significant increase in lipid droplet accumulation, FAO and higher expression of VCS marker Cx40. Using specific inhibitors, we further identified PPARγ and FAO as critical downstream regulators of ANP-mediated regulation of metabolism and VCS formation.

Computer-aided drug design to generate a unique antibiotic family.

The World Health Organization has identified antibiotic resistance as one of the three greatest threats to human health. The need for antibiotics is a pressing matter that requires immediate attention. Here, computer-aided drug design is used to develop a structurally unique antibiotic family targeting holo-acyl carrier protein synthase (AcpS). AcpS is a highly conserved enzyme essential for bacterial survival that catalyzes the first step in lipid synthesis. To the best of our knowledge, there are no current antibiotics targeting AcpS making this drug development program of high interest. We synthesize a library of > 700 novel compounds targeting AcpS, from which 33 inhibit bacterial growth in vitro at ≤ 2 µg/mL. We demonstrate that compounds from this class have stand-alone activity against a broad spectrum of Gram-positive organisms and synergize with colistin to enable coverage of Gram-negative species. We demonstrate efficacy against clinically relevant multi-drug resistant strains in vitro and in animal models of infection in vivo including a difficult-to-treat ischemic infection exemplified by diabetic foot ulcer infections in humans. This antibiotic family could form the basis for several multi-drug-resistant antimicrobial programs.

Compact and automated eDNA sampler for in situ monitoring of marine environments.

Using environmental DNA (eDNA) to monitor biodiversity in aquatic environments is becoming an efficient and cost-effective alternative to other methods such as visual and acoustic identification. Until recently, eDNA sampling was accomplished primarily through manual sampling methods; however, with technological advances, automated samplers are being developed to make sampling easier and more accessible. This paper describes a new eDNA sampler capable of self-cleaning and multi-sample capture and preservation, all within a single unit capable of being deployed by a single person. The first in-field test of this sampler took place in the Bedford Basin, Nova Scotia, Canada alongside parallel samples taken using the typical Niskin bottle collection and post-collection filtration method. Both methods were able to capture the same aquatic microbial community and counts of representative DNA sequences were well correlated between methods with R[Formula: see text] values ranging from 0.71-0.93. The two collection methods returned the same top 10 families in near identical relative abundance, demonstrating that the sampler was able to capture the same community composition of common microbes as the Niskin. The presented eDNA sampler provides a robust alternative to manual sampling methods, is amenable to autonomous vehicle payload constraints, and will facilitate persistent monitoring of remote and inaccessible sites.

ErbB2/Her2-dependent downregulation of a cell death-promoting protein BLNK in breast cancer cells is required for 3D breast tumor growth.

A significant proportion of breast cancers are driven by ErbB2/Her2 oncoprotein that they overexpress. These malignancies are typically treated with various ErbB2-targeted drugs, but many such cancers develop resistance to these agents and become incurable. Conceivably, treatment of ErbB2-positive cancers could be facilitated by use of agents blocking oncogenic signaling mechanisms downstream of ErbB2. However, current understanding of these mechanisms is limited. The ability of solid tumor cells to resist anoikis, cell death triggered by cell detachment from the extracellular matrix (ECM), is thought to be critical for 3D tumor growth. In an effort to understand the mechanisms of ErbB2-driven breast cancer cell anoikis resistance we found that detachment of non-malignant breast epithelial cells from the ECM upregulates a cell death-promoting tumor suppressor adapter protein BLNK and that ErbB2 blocks this upregulation by reducing tumor cell levels of transcription factor IRF6. We further observed that trastuzumab, a therapeutic anti-ErbB2 antibody, upregulates BLNK in human trastuzumab-sensitive but not trastuzumab-resistant ErbB2-positive breast cancer cells. Moreover, we established that BLNK promotes anoikis by activating p38 MAP kinase and that ErbB2-dependent BLNK downregulation blocks breast cancer cell anoikis. In search for pharmacological approaches allowing to upregulate BLNK in tumor cells we found that clinically approved proteasome inhibitor bortezomib upregulates IRF6 and BLNK in human breast cancer cells and inhibits their 3D growth in a BLNK-dependent manner. In addition, we found that BLNK upregulation in human ErbB2-positive breast cancer cells blocks their ability to form tumors in mice. Furthermore, we used publicly available data on mRNA levels in multiple breast cancers to demonstrate that increased BLNK mRNA levels correlate with increased relapse-free survival in a cohort of approximately 400 patients with ErbB2-positive breast cancer. In summary, we discovered a novel mechanism of ErbB2-driven 3D breast tumor growth mediated by ErbB2-dependent BLNK downregulation.

Mechanism of action and therapeutic route for a muscular dystrophy caused by a genetic defect in lipid metabolism.

CHKB encodes one of two mammalian choline kinase enzymes that catalyze the first step in the synthesis of the membrane phospholipid phosphatidylcholine. In humans and mice, inactivation of the CHKB gene (Chkb in mice) causes a recessive rostral-to-caudal muscular dystrophy. Using Chkb knockout mice, we reveal that at no stage of the disease is phosphatidylcholine level significantly altered. We observe that in affected muscle a temporal change in lipid metabolism occurs with an initial inability to utilize fatty acids for energy via mitochondrial ß-oxidation resulting in shunting of fatty acids into triacyglycerol as the disease progresses. There is a decrease in peroxisome proliferator-activated receptors and target gene expression specific to Chkb-/- affected muscle. Treatment of Chkb-/- myocytes with peroxisome proliferator-activated receptor agonists enables fatty acids to be used for ß-oxidation and prevents triacyglyerol accumulation, while simultaneously increasing expression of the compensatory choline kinase alpha (Chka) isoform, preventing muscle cell injury.

Development and analytical validation of a novel bioavailable 25-hydroxyvitamin D assay.

BACKGROUND: Bioavailable 25-hydroxyvitamin D (25OHD) may be a better indicator of vitamin D sufficiency than total 25OHD. This report describes a novel assay for measuring serum bioavailable 25OHD. METHODS: We developed an assay for 25OHD % bioavailability based on competitive binding of 25OHD tracer between vitamin D-binding protein (DBP)-coated affinity chromatography beads and serum DBP. Bioavailable 25OHD, total 25OHD, albumin, and DBP protein concentrations were measured in 89 samples from hospitalized patients and 42 healthy controls to determine how the DBP binding assay responds to differences in concentrations of DBP and compares to calculated bioavailable 25OHD values. RESULTS: DBP binding assay showed a linear relationship between DBP-bound 25OHD tracer recovered from bead supernatant and DBP calibrator concentrations (y = 0.0017x +0.731, R2 = 0.9961, p<0.001). Inversion of this relationship allowed interpolation of DBP binding equivalents based upon 25OHD tracer recovered. The relationship between DBP binding equivalents and % bioavailability fits a non-linear curve, allowing calculation of % bioavailable 25OHD from DBP binding equivalents (y = 10.625x-0.817, R2 = 0.9961, p<0.001). In hospitalized patient samples, there were linear relationships between DBP protein concentrations and DBP binding equivalents (y = 0.7905x + 59.82, R2 = 0.8597, p<0.001), between measured vs. calculated % bioavailability (y = 0.9528 + 0.0357, R2 = 0.7200, p<0.001), and between absolute concentrations of measured vs. calculated bioavailable 25OHD (y = 1.2403 + 0.1221, R2 = 0.8913, p<0.001). CONCLUSIONS: The DBP-binding assay for bioavailable 25OHD shows expected changes in 25OHD % bioavailability in response to changes in DBP concentrations and concordance with calculated bioavailable 25OHD concentrations.

Soluble fms-Like Tyrosine Kinase 1 Localization in Renal Biopsies of CKD.

INTRODUCTION: Soluble fms-like tyrosine kinase 1 (sFLT1) is a splice variant of the vascular endothelial growth factor (VEGF) receptor lacking the transmembrane and cytoplasmic domains and acts as a powerful antagonist of VEGF signaling. Plasma sFLT1 levels are higher in patients with chronic kidney disease (CKD) and correlate with renal dysfunction. The source of plasma sFLT1 in CKD is unclear. METHODS: Fifty-two renal biopsies were studied for sFLT1 expression using immunohistochemistry and evaluated on a 0-4 grading scale of positive cells within inflammatory infiltrates. These included drug-induced interstitial nephritis (6); allografts (12), with polyomavirus nephritis (3); diabetes mellitus (10); lupus glomerulonephritis (6); pauci-immune vasculitis (7); IgA nephropathy (6); and miscellaneous CKD (5). RESULTS: Forty-seven biopsies had inflammatory infiltrates of which 37 had sFLT1-positive cells: of these biopsies, 3 were grade 4, i.e., had cells that constituted more than 50% of the inflammatory infiltrate, 9 were grade 3 (25%-50%), 5 were grade 2 (10%-25%), 3 were grade 1 (10%), and 17 were grade 0.5 (<10%). There was a robust correlation (r2 = 0.89) between degree of inflammation and sFLT1-positive cells. CD68/sFLT1 co-immunostaining studies indicated that sFLT1-positive cells were histiocytes. The surrounding capillary network was reduced. CONCLUSION: sFLT1-positive histiocytes are generally part of the inflammatory infiltrates noted in CKD and are particularly abundant in forms of interstitial nephritis. Their presence promotes an anti-angiogenic state locally in the tubulointerstitium that could inhibit capillary repair, contribute to peritubular capillary loss, and enhance fibrosis in CKD.

A time course analysis of cyclooxygenase-2 suggests a role in spatial memory retrieval in rats.

We previously showed a role for COX-2 in spatial memory retention. In that study we investigated the effects of post-training intrahippocampal infusion of celecoxib as a COX-2-specific inhibitor on spatial memory retention. Those infusions impaired spatial memory retention in the Morris water maze. In the present study a time course analysis of role of COX-2 in spatial memory was conducted. Here stereotaxic surgery was employed for the bilateral implantation of guide cannulas into the CA1 region of the hippocampus. Training trials were started after recovery of the animals. Immediately after last trial of training on third day, the celecoxib (0.1M) was infused bilaterally and testing trials, were performed 1, 2, 3, and 7 days after celecoxib infusions. Significant alterations were observed in escape latency and traveled distance 2 and 3 days after celecoxib infusions. The maximum impairment was obtained 72 h after the infusions. The data suggests that the effect of celecoxib is transient and that its effect on performance is likely caused by a problem in memory retrieval. Quantification analyses of the immunostaining of COX-2-containing neurons in the dorsal hippocampus show that celecoxib infusions significantly reduced (P<0.05) COX-2 immunoreactivity for the animals that were tested 3 days after the drug infusion. Results from the behavioral study along with the findings from immunohistochemical analyses suggest that COX-2 has significant role in spatial memory retrieval. Moreover, the memory deficits induced by the infusions continuously persists for 3 days.

Inflammation alters angiotensin converting enzymes (ACE and ACE-2) balance in rat heart.

Angiotensin converting enzymes (ACE) and more recently discovered ACE-2 are important proteins involved in the renin-angiotensin system. The balance between ACE and ACE-2 is important for the regulation of blood pressure and electrolyte homeostasis. Inflammatory diseases like rheumatoid arthritis are associated with increased risk for cardiovascular complications. We studied the effect of inflammation on the expression levels of ACE and ACE-2 in two groups (n = 4/group) of adjuvant arthritis (AA) and healthy (control) rats. The AA group received 0.2 ml of 50 mg ml(-1) of Mycobacterium butyricum suspended in squalene into the tail base. On day 12, rats were euthanized and their organs (hearts, liver, kidney, and intestine) were excised. The mRNA of ACE and ACE-2 were determined by real-time polymerase chain reaction. ACE and ACE-2 protein expression in rat heart was determined by Western blot. Inflammation resulted in 80% reduction of ACE-2 gene expression in rat heart. ACE-2/ACE expression ratio was significantly reduced from 0.7 ± 0.4 in control rats to 0.07 ± 0.09 in AA. Similarly, ACE-2/ACE protein expression ratio was also disrupted with a significant reduction in AA animals (6.7 ± 4.8 vs. 0.9 ± 05 in control and AA, respectively). ACE-2 has been found to provide negative feedback of renin-angiotensin system and protection of the heart and kidneys. Disruption of the balance between ACE and ACE-2 observed in inflammation may be, at least in part, involved in the cardiovascular complications seen in patients with inflammatory diseases.

Post-training intrahippocampal infusion of nicotine prevents spatial memory retention deficits induced by the cyclo-oxygenase-2-specific inhibitor celecoxib in rats.

Recently, we demonstrated that intrahippocampal infusion of the cyclo-oxygenase (COX)-2-specific inhibitor celecoxib impaired spatial memory retention in the Morris water maze. In the present work, we investigated the effects of nicotine, infused in the rat dorsal hippocampus several minutes after infusion of celecoxib, on memory retention in the Morris water maze. Rats were trained for 3 days; each day included two blocks, and each block contained four trials. Test trials were conducted 48 h after surgery. As expected, bilateral intrahippocampal infusion of celecoxib (19 microg/side; 0.1 m) increased escape latency and travel distance in rats, indicating significant impairment of spatial memory retention. We also examined the effects of bilateral infusion of nicotine (0.5, 1.0 and 2.0 microg/side) on memory retention. Infusion of 1 microg nicotine significantly decreased escape latency and travel distance but not swimming speed, compared with controls, suggesting memory retention enhancement by nicotine at this concentration. In separate experiments, bilateral infusion of nicotine, infused 5 min after 0.1 m (19 microg/side) celecoxib infusion, was associated with escape latency, travel distance and swimming speed profiles very similar to those in control animals. Brain tissue sections from several of these animals were subjected to immunohistochemical staining analysis with anti-COX-2 antibodies. Quantification analysis by optical density measurements showed that the celecoxib infusion reduced the immunoreactivity of COX-2-containing neurons in the CA1 area of the hippocampus compared with controls, although this reduction was not significant. However, infusion of a combination of celecoxib and nicotine significantly increased this immunoreactivity compared with levels in control and celecoxib-infused groups. These results suggest that nicotine prevented or reversed the adverse effects of celecoxib on spatial memory retention and protected or restored the immunostaining pattern of COX-2 neurons in the rat dorsal hippocampus.