Search results for "Isoform"

tappAS: a comprehensive computational framework for the analysis of the functional impact of differential splicing

2019

AbstractTraditionally, the functional analysis of gene expression data has used pathway and network enrichment algorithms. These methods are usually gene rather than transcript centric and hence fall short to unravel functional roles associated to posttranscriptional regulatory mechanisms such as Alternative Splicing (AS) and Alternative PolyAdenylation (APA), jointly referred here as Alternative Transcript Processing (AltTP). Moreover, short-read RNA-seq has serious limitations to resolve full-length transcripts, further complicating the study of isoform expression. Recent advances in long-read sequencing open exciting opportunities for studying isoform biology and function. However, there…

Semi-automatic quantitative RT-PCR to measure CYP induction by drugs in human hepatocytes

2003

An assay has been developed for the quantitative measurement of CYP mRNA content of the major human isoforms (1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5) in human hepatocytes. The method is based on the conversion of mRNAs into their corresponding cDNAs, followed by PCR amplification using appropriate primers. Making use of appropriate internal and external standards it is possible to estimate changes in CYP mRNA content of hepatocytes. The technique has been standardised to run semi-automatically. This procedure can be used to assess the CYP induction potential of new pharmaceuticals at a pre-clinical stage of development. To this aim, human hepatocytes obtained from functional l…

2013

Clusterin, also known as apolipoprotein J, is expressed from a variety of tissues and implicated in pathological disorders such as neurodegenerative diseases, ischemia and cancer. In contrast to secretory clusterin (sCLU), which acts as an extracellular chaperone, the synthesis, subcellular localization and function(s) of intracellular CLU isoforms is currently a matter of intense discussion. By investigating human CLU mRNAs we here unravel mechanisms leading to the synthesis of distinct CLU protein isoforms and analyze their subcellular localization and their impact on apoptosis and on NF-κB-activity. Quantitative PCR-analyses revealed the expression of four different stress-inducible CLU …

Interaction mechanism of endogenous PP2A inhibitor protein ENSA with PP2A

2022

The vast diversity of protein phosphatase 2A (PP2A) holoenzyme composition ensures its multifaceted role in the regulation of cellular growth and signal transduction. In several pathological conditions, such as cancer, PP2A is inhibited by endogenous inhibitor proteins. Several PP2A inhibitor proteins have been identified, one of which is α-endosulfine (ENSA). ENSA inhibits PP2A activity when it is phosphorylated at Ser67 by Greatwall (Gwl) kinase. The role of ENSA in PP2A inhibition is rather well characterized, but knowledge of the mechanism of inhibition is scarce. In this study, we have performed comprehensive structural characterization of ENSA, and its interaction with PP2A A- and var…

Efficient Expression and Crystallization System of Cancer-Associated Carbonic Anhydrase Isoform IX.

2015

Human carbonic anhydrase IX (CA IX) is overexpressed in a number of solid tumors and is considered to be a marker for cellular hypoxia that it is not produced in most normal tissues. CA IX contributes to the acidification of the extracellular matrix, which, in turn, favors tumor growth and metastasis. Therefore, CA IX is considered to be a promising anti-cancer drug target. However, the ability to specifically target CA IX is challenging due to the fact that the human genome encodes 15 different carbonic anhydrase isoforms that have a high degree of homology. Furthermore, structure-based drug design of CA IX inhibitors so far has been largely unsuccessful due to technical difficulties regar…

5-Substituted-benzylsulfanyl-thiophene-2-sulfonamides with effective carbonic anhydrase inhibitory activity: Solution and crystallographic investigat…

2017

Abstract A series of 5-substituted-benzylsulfanyl-thiophene-2-sulfonamides was prepared by reacting 5-bromo-thiophene-2-sulfonamide with 5-substituted-benzyl mercaptans. The new compounds were investigated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. The cytosolic human (h) isoforms hCA I was poorly inhibited by the new sulfonamides (KIs in the range of 683–4250 nM), whereas hCA II, and the transmembrane, tumor associated isoforms hCA IX and XII were effectively inhibited in the subnanomolar–nanomolar range. A high resolution X-ray crystal structure of the adduct of hCA II with one of the new sulfonamides allowed us to rationalize the excellent inhibitory activity of these heterocycli…

Model of a six immunoglobulin-like domain fragment of filamin A (16-21) built using residual dipolar couplings.

2012

Filamins are actin-binding proteins that participate in a wide range of cell functions, including cell morphology, locomotion, membrane protein localization, and intracellular signaling. The three filamin isoforms found in humans, filamins A, B, and C, are highly homologous, and their roles are partly complementary. In addition to actin, filamins interact with dozens of other proteins that have roles as membrane receptors and channels, enzymes, signaling intermediates, and transcription factors. Filamins are composed of an N-terminal actin-binding domain and 24 filamin-type immunoglobulin-like domains (FLN) that form tail-to-tail dimers with their C-terminal FLN domain. Many of the filamin …

Identification of Cysteine Residues in Human Cationic Amino Acid Transporter hCAT-2A That Are Targets for Inhibition by N-Ethylmaleimide

2013

In most cells, cationic amino acids such as l-arginine, l-lysine, and l-ornithine are transported by cationic (CAT) and y(+)L (y(+)LAT) amino acid transporters. In human erythrocytes, the cysteine-modifying agent N-ethylmaleimide (NEM) has been shown to inhibit system y(+) (most likely CAT-1), but not system y(+)L (Devés, R., Angelo, S., and Chávez, P. (1993) J. Physiol. 468, 753-766). We thus wondered if sensitivity to NEM distinguishes generally all CAT and y(+)LAT isoforms. Transport assays in Xenopus laevis oocytes established that indeed all human CATs (including the low affinity hCAT-2A), but neither y(+)LAT isoform, are inhibited by NEM. hCAT-2A inhibition was not due to reduced tran…

Nitric Oxide: Biological Synthesis and Functions

2012

The pluripotent gaseous messenger molecule nitric oxide (NO) controls vital functions such as neurotransmission or vascular tone (via activation of soluble guanylyl cyclase), gene transcription, mRNA translation (via iron-responsive elements), and post-translational modifications of proteins (via ADP-ribosylation). In higher concentrations, NO is capable of destroying parasites and tumor cells by inhibiting iron-containing enzymes or directly interacting with the DNA of these cells. In view of this multitude of functions of NO, it is important to understand the mechanisms by which cells accomplish and regulate the production of this molecule. In mammals, three isozymes of NO synthase (NOS; …

Dendritic localization of mammalian neuralized mRNA encoding a protein with transcription repression activities.

2002

Drosophila neurogenic gene neuralized (neu) is required for the maintenance of neuroblast cell fate and differentiation. In the present study we have characterized a mouse and a rat homologue of Drosophila neu. Mammalian neu1 encodes several C-terminal RING zinc finger proteins with one or two neuralized homology repeat (NHR) domains. Mammalian neu1 mRNAs are predominantly expressed in the nervous system and in the skeletal muscle with the highest levels in the adult. In the nervous system neu1 mRNAs are expressed in neurons and dendritically localized in several brain regions, suggesting a role of neu1 in the regulation of synaptic function. Mammalian neu1 isoforms exhibit transcription re…