Ettickan Boopathi

Bimodal Protein Targeting through Activation of Cryptic Mitochondrial Targeting Signals by an Inducible Cytosolic Endoprotease

Bimodal targeting of the endoplasmic reticular protein, cytochrome P4501A1 (CYP1A1), to mitochondria involves activation of a cryptic mitochondrial targeting signal through endoprotease processing of the protein. Here, we characterized the endoprotease that regulates mitochondrial targeting of CYP1A1. The endoprotease, which was induced by beta-naphthoflavone, was a dimer of 90 kDa and 40 kDa subunits, each containing Ser protease domains. The purified protease processed CYP1A1 in a sequence-specific manner, leading to its mitochondrial import. The glucocorticoid receptor, retinoid X receptor, and p53 underwent similar processing-coupled mitochondrial transport. The inducible 90 kDa subunit was a limiting factor in many cells and some tissues and, thus, regulates the mitochondrial levels of these proteins. A number of other mitochondria-associated proteins with noncanonical targeting signals may also be substrates of this endoprotease. Our results describe a new mechanism of mitochondrial protein import that requires an inducible cytoplasmic endoprotease for activation of cryptic mitochondrial targeting signals.

Accumulation of Mitochondrial P450MT2, NH2-terminal Truncated Cytochrome P4501A1 in Rat Brain during Chronic Treatment with β-Naphthoflavone A ROLE IN THE METABOLISM OF NEUROACTIVE DRUGS

The biochemical and molecular characteristics of cytochrome P4501A1 targeted to rat brain mitochondria was studied to determine the generality of the targeting mechanism previously described for mitochondrial cytochrome P450MT2 (P450MT2) from rat liver. In rat brain and C6 glioma cells chronically exposed to β-naphoflavone (BNF), P450MT2 content reached 50 and 95% of the total cellular pool, respectively. P450MT2 from 10 days of BNF-treated rat brain was purified to over 85% purity using hydrophobic chromatography followed by adrenodoxin affinity binding. Purified brain P450MT2 consisted of two distinct molecular species with NH2 termini identical to liver mitochondrial forms. These results confirm the specificity of endoprotease-processing sites. The purified P450MT2 showed a preference for adrenodoxin + adrenodoxin reductase electron donor system and exhibited high erythromycinN-demethylation activity. Brain mitoplasts from 10-day BNF-treated rats and also purified P450MT2 exhibited highN-demethylation activities for a number of neuroactive drugs, including trycyclic anti-depressants, anti-convulsants, and opiates. At 10 days of BNF treatment, the mitochondrial metabolism of these neuroactive drugs represented about 85% of the total tissue activity. These results provide new insights on the role of P450MT2 in modulating the pharmacological potencies of different neuroactive drugs in chronically exposed individuals.

Alterations in caveolin expression and ultrastructure after bladder smooth muscle hypertrophy.

PURPOSE Partial bladder outlet obstruction in male rabbits causes detrusor smooth muscle hypertrophy and voiding dysfunction similar to that observed in men with benign prostate hyperplasia. Using this model, we analyzed the protein expression and ultrastructure of caveolae and the intermediate size filament in detrusor smooth muscle following partial bladder outlet obstruction induced hypertrophy. MATERIALS AND METHODS Detrusor smooth muscle sections from bladder body were processed for immunofluorescence and electron microscopy. Western analysis was performed to determine the expression of caveolin isoform-1, 2 and 3, and intermediate size filament proteins. RESULTS Detrusor smooth muscle cells from both normal and hypertrophied bladders contain orderly arrays of thick and thin myofilaments, interspersed with dense bodies. In addition, there was an increase in intermediate size filaments in the hypertrophic detrusor smooth muscle cells. The dense plaques in the inner membrane of hypertrophied detrusor smooth muscle were longer than those of the control. Detrusor smooth muscle from hypertrophied bladder revealed a decreased number of caveolae and a lack of their orderly distribution at the plasma membrane. Western blotting showed decreased expression of caveolin-1, 2 and 3 in hypertrophied detrusor smooth muscle. CONCLUSIONS Caveolae serve as platforms for proteins and receptors that have a role in signal transduction. The decreased number of caveolae and caveolin protein expression in hypertrophied detrusor smooth muscle might contribute to alterations in signal transduction pathways that regulate the downstream effects of agonist induced contraction, including calcium sensitization, observed in obstructed bladder. In addition, the increased number of intermediate size filaments in the hypertrophied detrusor smooth muscle is likely to alter the cytoskeletal structure and affect the cellular transmission of passive and/or active force.

Role of Protein Kinase C-mediated Protein Phosphorylation in Mitochondrial Translocation of Mouse CYP1A1, Which Contains a Non-canonical Targeting Signal

A large number of mitochondrial proteins lack canonical mitochondrial-targeting signals. The bimodal transport of cytochromes P450 (CYPs) to endoplasmic reticulum and mitochondria (MT), reported previously by us, likely represents one mode of non-canonical protein targeting to MT. Herein, we have studied the mechanism of mouse MT-CYP1A1 targeting to gain insight into the regulatory features and evolutionary conservation of bimodal targeting mechanism. Mouse MT-CYP1A1 consists of two NH2-terminal-truncated molecular species, +91A1 and +331A1. Mutations Pro-2 → Leu and Tyr-5 → Leu, which increase the signal recognition particle (SRP) binding, diminished MT targeting of the protein in intact cells. By contrast, mutations Leu-7 → Asn and Leu-17 → Asn, which decreased SRP-binding affinity, enhanced MT targeting, thus suggesting that SRP binding is an important regulatory step that modulates bimodal targeting. Protein kinase C (PKC)-mediated phosphorylation of nascent chains at Thr-35 vastly decreased affinity for SRP binding suggesting an important regulatory step. In support of these results, COS cell transfection experiments show that phosphomimetic mutation Thr-35 → Asp or induced cellular PKC caused increased CYP1A1 targeting to MT and correspondingly lower levels to the endoplasmic reticulum. Results suggest evolutionary conservation of chimeric signals and bimodal targeting of CYP1A1 in different species. The mouse MT-CYP1A1 is an extrinsic membrane protein, which exhibited high FDX1 plus FDXR-mediated N-demethylation of a number of tricyclic antidepressants, pain killers, anti-psychotics, and narcotics that are poor substrates for microsomal CYP1A1.

Transcriptional repression of Caveolin-1 (CAV1) gene expression by GATA-6 in bladder smooth muscle hypertrophy in mice and human beings.

Hypertrophy occurs in urinary bladder wall smooth muscle (BSM) in men with partial bladder outlet obstruction (PBOO) caused by benign prostatic hyperplasia (BPH) and in animal models of PBOO. Hypertrophied BSM from the rabbit model exhibits down-regulation of caveolin-1, a structural and functional protein of caveolae that function as signaling platforms to mediate interaction between receptor proteins and adaptor and effector molecules to regulate signal generation, amplification, and diversification. Caveolin-1 expression is diminished in PBOO-induced BSM hypertrophy in mice and in men with BPH. The proximal promoter of the human and mouse caveolin-1 (CAV1) gene was characterized, and it was observed that the transcription factor GATA-6 binds this promoter, causing reduced expression of caveolin-1. Furthermore, caveolin-1 expression levels inversely correlate with the abundance of GATA-6 in BSM hypertrophy in mice and human beings. Silencing of GATA6 gene expression up-regulates caveolin-1 expression, whereas overexpression of GATA-6 protein sustains the transcriptional repression of caveolin-1 in bladder smooth muscle cells. Together, these data suggest that GATA-6 acts as a transcriptional repressor of CAV1 gene expression in PBOO-induced BSM hypertrophy in men and mice. GATA-6-induced transcriptional repression represents a new regulatory mechanism of CAV1 gene expression in pathologic BSM, and may serve as a target for new therapy for BPH-induced bladder dysfunction in aging men.

Regulation of Murine Cytochrome c Oxidase Vb Gene Expression during Myogenesis YY-1 AND HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN D-LIKE PROTEIN (JKTBP1) RECIPROCALLY REGULATE TRANSCRIPTION ACTIVITY BY PHYSICAL INTERACTION WITH THE BERF-1/ZBP-89 FACTOR

A transcription suppressor element (sequence –481 to –320) containing a G-rich motif (designated GTG) and a newly identified CAT-rich motif (designated CATR) was previously shown to modulate expression of the mouse cytochrome c oxidase Vb gene during myogenesis. Here, we show that the GTG element is critical for transcription activation in both undifferentiated and differentiated myocytes. Mutations of the CATR motif abolished transcription repression in myoblasts while limiting transcription activation in differentiated myotubes, suggesting contrasting functional attributes of this DNA motif at different stages of myogenesis. Results show that the activity of the transcription suppressor motif is modulated by an orchestrated interplay between ubiquitous transcription factors: ZBP-89, YY-1, and a member of the heterogeneous nuclear ribonucleoprotein D-like protein (also known as JKTBP1) family. In undifferentiated muscle cells, GTG motif-bound ZBP-89 physically and functionally interacted with CATR motif-bound YY-1 to mediate transcription repression. In differentiated myotubes, heterogeneous nuclear ribonucleoprotein D-like protein/JKTBP1 bound to the CATR motif exclusive of YY-1 and interacted with ZBP-89 in attenuating repressor activity, leading to transcription activation. Our results show a novel mechanism of protein factor switching in transcription regulation of the cytochrome c oxidase Vb gene during myogenesis.

Inherent sex‐dependent regulation of human hepatic CYP3A5

Expression of hepatic cytochromes P450 (CYP) in all species examined, including humans, is generally sexually dimorphic. We examined the sex‐dependent expression of CYP3A5 and the hormone‐regulated molecular mechanism(s) responsible for any dimorphism.

GATA-6 and NF-κB activate CPI-17 gene transcription and regulate Ca2+ sensitization of smooth muscle contraction.

ABSTRACT Protein kinase C (PKC)-potentiated inhibitory protein of 17 kDa (CPI-17) inhibits myosin light chain phosphatase, altering the levels of myosin light chain phosphorylation and Ca2+ sensitivity in smooth muscle. In this study, we characterized the CPI-17 promoter and identified binding sites for GATA-6 and nuclear factor kappa B (NF-κB). GATA-6 and NF-κB upregulated CPI-17 expression in cultured human and mouse bladder smooth muscle (BSM) cells in an additive manner. CPI-17 expression was decreased upon GATA-6 silencing in cultured BSM cells and in BSM from NF-κB knockout (KO) mice. Moreover, force maintenance by BSM strips from KO mice was decreased compared with the force maintenance of BSM strips from wild-type mice. GATA-6 and NF-κB overexpression was associated with CPI-17 overexpression in BSM from men with benign prostatic hyperplasia (BPH)-induced bladder hypertrophy and in a mouse model of bladder outlet obstruction. Thus, aberrant expression of NF-κB and GATA-6 deregulates CPI-17 expression and the contractile function of smooth muscle. Our data provide insight into how GATA-6 and NF-κB mediate CPI-17 transcription, PKC-mediated signaling, and BSM remodeling associated with lower urinary tract symptoms in patients with BPH.

Regulation of miR106b cluster through the RB pathway: mechanism and functional targets.

The RB pathway plays a critical role in proliferation control that is commonly subverted in tumor development. However, restoration of RB pathway function can be elicited in many tumor cells by the inhibition of CDK4/6 activity that leads to dephosphorylation of RB and subsequent repression of E2F-mediated transcription. In this context, active RB/E2F complexes inhibit the expression of a critical program of coding genes that promote cell cycle progression. However, the non-coding RNA target genes downstream from RB that could be relevant for tumor biology remain obscure. Here, miRNA gene expression profiling identified the miR106b cluster as being efficiently repressed with CDK4/6 inhibition in an E2F and RB-dependent manner. Importantly, the miR106B-cluster is intragenic of MCM7, and through a series of functional studies, the basis of MCM7 regulation and concordant expression of the miRNA species within the 106b cluster was determined. Importantly, RB-mediated repression of the 106b cluster enhances the transcript levels of p21Cip1 and PTEN. These data provide a mechanistic basis for cross-talk between the RB pathway and p21 and PTEN through the regulation of the MCM7/miR106b locus.

RB Loss Promotes Prostate Cancer Metastasis

RB loss occurs commonly in neoplasia but its contributions to advanced cancer have not been assessed directly. Here we show that RB loss in multiple murine models of cancer produces a prometastatic phenotype. Gene expression analyses showed that regulation of the cell motility receptor RHAMM by the RB/E2F pathway was critical for epithelial-mesenchymal transition, motility, and invasion by cancer cells. Genetic modulation or pharmacologic inhibition of RHAMM activity was sufficient and necessary for metastatic phenotypes induced by RB loss in prostate cancer. Mechanistic studies in this setting established that RHAMM stabilized F-actin polymerization by controlling ROCK signaling. Collectively, our findings show how RB loss drives metastatic capacity and highlight RHAMM as a candidate therapeutic target for treating advanced prostate cancer. Cancer Res; 77(4); 982-95. ©2016 AACR.