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Results for “Protein Isoforms”

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PubMed1992

Role of protein kinase C in the modulation of multidrug resistance: expression of the atypical gamma isoform of protein kinase C does not confer increased resistance to doxorubicin.

Cross-resistance to anticancer drugs, termed multidrug resistance (MDR), is functionally associated with the expression of a plasma membrane, energy-dependent, drug efflux pump termed P-glycoprotein (PGP), the product of the mdr1 gene. We have shown previously that MCF-7 breast carcinoma cells transfected with the human mdr1 gene (BC-19 cells) exhibit greater MDR when stably transfected with protein kinase C alpha (PKC alpha). We now demonstrate that transfection of BC-19 cells with the gamma isoform of PKC (BC-19/PKC gamma cells), which is not normally present in BC-19 cells, does not confer increased resistance to doxorubicin, despite a 19-fold increase in PKC activity. All of the increased PKC activity is accounted for by PKC gamma and it is rapidly down-regulated by phorbol dibutyrate, within 15 min of treatment. Endogenous PKC alpha and PKC epsilon activities are not affected by phorbol dibutyrate. The cytotoxicity of doxorubicin was similar in BC-19/neo or BC-19/PKC gamma cells after either 2-hr or continuous drug exposure, and co-treatment with phorbol dibutyrate increased resistance to doxorubicin 4-fold in both cell lines. Phosphorylation of PGP was similar in both cell lines and drug accumulation was not affected by overexpression of PKC gamma. These results demonstrate that transfection of PGP-expressing cells with an atypical isoform of PKC does not confer increased MDR, and they suggest that the regulation of PGP is phenotype specific with respect to the isoform of PKC.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1992

Human erythrocyte protein 4.2: isoform expression, differential splicing, and chromosomal assignment.

Human protein 4.2 (P4.2) is a major membrane skeletal protein in erythrocytes. Individuals with P4.2 deficiency exhibit spherocytosis and experience various degrees of hemolytic anemia, suggesting a role for this protein in maintaining stability and integrity of the membrane. Molecular cloning of P4.2 cDNAs showed that P4.2 is a transglutaminaselike molecule in erythrocytes but lacks the essential cysteine for cross-linking activity. Two cDNA isoforms have been identified from a human reticulocyte cDNA library, with the long isoform containing a 90-base pair (bp) in-frame insertion encoding an extra 30 amino acids near the N-terminus. Characterization of the P4.2 gene suggests differential splicing as the mechanism for generating these two cDNA isoforms. The donor site for the short isoform (P4.2S) agrees better with the consensus than the donor site for the long isoform (P4.2L) does. Expression of P4.2L was detected by a long-isoform-specific antibody raised against a peptide within the 30-amino acid insert. Western blot analyses showed P4.2L to be a minor membrane skeletal protein in human erythrocytes with an apparent molecular weight (mol wt) of approximately 3 Kd larger than the major protein 4.2, P4.2S. By in situ hybridization of a full-length 2.4-kilobase (kb) cDNA to human metaphase chromosomes, the gene for P4.2 was mapped to bands q15-q21 of chromosome 15, and it is not linked to the gene for coagulation factor XIIIa (plasma transglutaminase, TGase).

Amino Acid Sequence↗

PubMed2006

Enhanced expression of metallothionein isoform 3 protein in tumor heterotransplants derived from As+3- and Cd+2-transformed human urothelial cells.

This laboratory has proposed that the third isoform of the metallothionein gene family (MT-3) might be a biomarker for the development of human bladder cancer. Immunohistochemical staining of MT-3 on archival diagnostic specimens showed that only 2 of 63 (3.17%) benign bladder specimens had even weak reactivity for the MT-3 protein. In contrast, 103 of 107 (96.26%) high-grade urothelial cancers and 17 of 17 (100%) specimens of carcinoma in situ stained positive for the MT-3 protein. For low-grade bladder cancer it was shown that 30 of 48 specimens (62.5%) expressed the MT-3 protein. Using a cell culture model (UROtsa), it was demonstrated that expression of the MT-3 protein was not required for malignant transformation of urothelial cells by either Cd(+2) or As(+3). In contrast, it was shown that the cells transformed by Cd(+2) and As(+3) that did not express the MT-3 gene in cell culture, gained expression of MT-3 when grown as heterotransplants in nude mice. The gain in MT-3 expression when cells were grown as heterotransplants was also shown to occur for the MCF-7, T-47D, Hs 578t, MDA-MB-231 breast cancer, and the PC-3 prostate cancer cell lines. An analysis of MT-3 mRNA and protein expression suggested that a posttranscriptional mechanism was responsible for accumulation of the MT-3 protein. The results provide strong evidence that MT-3 could be a biomarker for the development of high-grade bladder cancer and that the expression of the MT-3 gene is not involved in the in vitro malignant transformation of UROtsa cells by Cd(+2) and As(+3).

Animals↗

PubMed1998

Functional comparison of two human monocyte chemotactic protein-2 isoforms, role of the amino-terminal pyroglutamic acid and processing by CD26/dipeptidyl peptidase IV.

Human Monocyte Chemotactic Protein (MCP)-2 has originally been isolated from stimulated osteosarcoma cells as a chemokine coproduced with MCP-1 and MCP-3. Here, a 5'-end extended MCP-2 cDNA was cloned from a human testis cDNA library. It encoded a 76 residue MCP-2 protein, but differed from the reported bone marrow-derived MCP-2 cDNA sequence in codon 46, which coded for a Lys instead of a Gln. This MCP-2Lys46 variant, caused by a single nucleotide polymorphism (SNP), was biologically compared with MCP-2Gln46. The coding regions were subcloned into the bacterial expression vector pHEN1, and after transformation of Escherichia coli, the two MCP-2 protein variants were recovered from the periplasm. The recombinant proteins were purified to homogeneity by heparin-Sepharose affinity chromatography and reversed-phase HPLC. Edman degradation revealed a Gln residue at the NH2 terminus instead of a pGlu. To evaluate the influence of the cyclization, this Gln was chemically converted into pGlu in both MCP-2 variants. The conversion was confirmed by electrospray mass spectrometry. rMCP-2Gln46 and rMCP-2Lys46 and the NH2-terminal cyclic counterparts were tested on monocytic cells in calcium mobilization and chemotaxis assays. No significant difference in biological activity was observed between the rMCP-2Gln46 and rMCP-2Lys46 isoforms. However, for both MCP-2 variants the NH2-terminal pyroglutamate was shown to be essential for chemotaxis, but not for calcium mobilization. NH2-terminal truncation of rMCP-2Lys46 by the serine protease CD26/dipeptidyl peptidase IV (CD26/DPP IV) resulted in the cleavage of the NH2-terminal Gln-Pro dipeptide, whereas synthetic MCP-2 with an amino-terminal pGlu remained unaffected. CD26/DPP IV-clipped rMCP-2Lys46(3-76) was almost completely inactive in both chemotaxis and signaling assays. These observations indicate that the NH2-terminal pGlu in MCP-2 is necessary for chemotactic activity but also that it protects the protein against degradation by CD26/DPP IV.

Alleles↗

PubMed2004

Differential expression of activator protein-2 isoforms in renal cell carcinoma.

OBJECTIVES: To investigate the expression of activator protein-2 (AP-2) in renal cell carcinoma (RCC) by immunohistochemistry. Three AP-2 isoforms alpha (alpha), beta (beta), and gamma (gamma) are known to exhibit a highly homologous structure; however, their functions are considered to be different. AP-2 has been implicated to play a role in carcinogenesis, as well as in the development of the kidney. METHODS: The expression of the three AP-2 isoforms, alpha, beta, and gamma, was determined in 58 patients with RCC by immunohistochemistry. Epidermal growth factor receptor and erbB2 expression in 42 patients with RCC was also evaluated to investigate the correlation with AP-2 isoforms. RESULTS: AP-2 isoforms are differentially expressed in normal renal tubules. Of 58 RCC tissue specimens, 15 (25.9%) demonstrated nuclear and cytoplasmic expression of AP-2alpha. Clear cell RCC had a significantly greater rate of AP-2alpha expression than the nonclear subtypes (14 of 41 clear versus 1 of 17 nonclear subtypes). Of the 58 specimens, 8 (13.8%) showed nuclear staining for AP-2beta; notably, localized small cases had a significantly greater rate of nuclear staining for AP-2beta (5 of 13 in pT1a versus 3 of 45 in pT1b or greater). In addition, only 2 cases (3.5%) demonstrated nuclear staining for AP-2gamma. Epidermal growth factor receptor and erbB2 expression did not correlate with expression of the AP-2 isoforms. CONCLUSIONS: AP-2 isoforms were differentially expressed in RCC, as well as in the adult normal kidney. AP-2alpha was dominantly expressed in clear cell RCC. AP-2beta expression was observed in the low-stage subtypes of RCC, and this transcription factor may be related to early carcinogenesis.

Adenocarcinoma↗

PubMed2004

Expression of different isoforms of protein kinase C in the rat hippocampus after pilocarpine-induced status epilepticus with special reference to CA1 area and the dentate gyrus.

At 4 h during pilocarpine-induced status epilepticus (DPISE) in rat, protein kinase C (PKC)beta1, PKCbeta2, and PKCgamma were induced at the border between the stratum oriens and alveus (O/A border) of CA1 in the hippocampus. Induced PKCgamma was colocalized with metabotropic glutamate receptor alpha (mGluR alpha). By intracerebroventricular injection of mGluR1alpha antagonists, (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), PKCbeta1, PKCbeta2, and PKCgamma immunoreactive products decreased dramatically; however, intracerebroventricular injection of saline did not change the expression of PKCbeta1, PKCbeta2, and PKCgamma, suggesting that these three PKC isoforms might be involved in mGluR1alpha-related excitoneurotoxicity. One day after pilocarpine-induced status epilepticus (APISE), PKCdelta was induced in microglial cells. At this time point, both PKCgamma and PKCepsilon immunopositive products decreased in the inner molecular layer of upper blade of the stratum granulosum. At 7-31 days APISE, induced PKCbeta1, PKCdelta, PKCeta, and PKCzeta positive astrocytes were demonstrated in all parts of hippocampus, suggesting that they may be involved in gliosis. By this time, both PKCgamma and PKCepsilon immunopositive products in the inner molecular layer had almost disappeared, suggesting that they may be involved in the inhibition of granule cells by controlling neurotransmitter release presynaptically in the dentate gyrus of normal rats.

Animals↗

PubMed1995

Regulated expression of heat shock factor 1 isoforms with distinct leucine zipper arrays via tissue-dependent alternative splicing.

HSF1 mediates the stress induced expression of heat shock proteins, referred to as the cellular stress response. Previous results indicated that mammalian cells express two distinct HSF1 protein isoforms, with molecular sizes of 69 kDa (HSF1-beta) and 71 kDa (HSF1-alpha). The purpose of this study was to determine the mechanism by which these two HSF1 protein isoforms are generated. Our results show that mammalian cells express two distinct HSF1 mRNA isoforms which arise via alternative splicing of the HSF1 pre-mRNA. The two HSF1 mRNA isoforms differ by a single 66 bp exon of the HSF1 gene which is spliced into the HSF1-alpha mRNA isoform but skipped in the HSF1-beta mRNA isoform. This 66 bp exon encodes a 22 amino acid sequence, whose molecular weight (2.3 kDa) matches the difference in size between the HSF1-beta and HSF1-alpha protein isoforms (69 and 71 kDa). Further analysis reveals that this extra 22 amino acid sequence, whose insertion site in the HSF1-alpha isoform is located immediately adjacent to a C-terminal leucine zipper motif (leucine zipper 4) previously shown to be involved in maintenance of HSF1 in the non-DNA-binding control form, contains an additional, previously unidentified leucine zipper motif (leucine zipper 5). Our results also show that the levels of the two HSF1 isoforms are regulated in a tissue dependent manner, with testis expressing higher levels of the HSF1-beta isoform while heart and brain express higher levels of the HSF1-alpha isoform. These results demonstrate a new mechanism by which HSF1 expression is regulated in mammalian cells and suggest a potential role for the HSF1 isoforms in mediating tissue-dependent regulation of the cellular stress response.

Alternative Splicing↗

PubMed1996

Analysis of phosphorylated isoforms of the p53 tumor suppressor protein in human lung carcinoma cells undergoing apoptosis.

The expression of p53 tumor suppressor protein isoforms in H460a cells induced to undergo apoptosis by 2-methoxyestradiol was investigated by two-dimensional gel electrophoresis. Whole-cell proteins from control and apoptotic H460a cells were separated by two-dimensional electrophoresis and were transferred to nitrocellulose. The p53 isoforms were detected by immunoblotting using p53 monoclonal antibody Bp53-12. Four isoforms of p53 (2, 3, 5, and 6) differing in phosphorylation state were detected in control cells. Three additional isoforms (1, 4, and 7) were observed to be expressed at significant levels only in apoptotic cells. The differential expression of isoforms 1, 4, and 7 in apoptotic cells suggests that one or more specific phosphorylation events generating these forms of p53 could play a role in regulating the function of p53 in apoptosis.

2-Methoxyestradiol↗

PubMed1999

spen encodes an RNP motif protein that interacts with Hox pathways to repress the development of head-like sclerites in the Drosophila trunk.

Drosophila has eight Hox proteins, and they require factors acting in parallel to regulate different segmental morphologies. Here we find that the Drosophila gene split ends (spen), has a homeotic mutant phenotype, and appears to encode such a parallel factor. Our results indicate that spen plays two important segment identity roles. One is to promote sclerite development in the head region, in parallel with Hox genes; the other is to cooperate with Antennapedia and teashirt to suppress head-like sclerite development in the thorax. Our results also indicate that without spen and teashirt functions, Antennapedia loses its ability to specify thoracic identity in the epidermis. spen transcripts encode extraordinarily large protein isoforms (approx. 5,500 amino acids), which are concentrated in embryonic nuclei. Both Spen protein isoforms and Spen-like proteins in other animals possess a clustered repeat of three RNP (or RRM) domains, as well as a conserved motif of 165 amino acids (SPOC domain) at their C-termini. Spen is the only known homeotic protein with RNP binding motifs, which indicates that splicing, transport, or other RNA regulatory steps are involved in the diversification of segmental morphology. Previous studies by Dickson and others (Dickson, B. J., Van Der Straten, A., Dominguez, M. and Hafen, E. (1996). Genetics 142, 163-171) identified spen as a gene that acts downstream of Raf to suppress Raf signaling in a manner similar to the ETS transcription factor Aop/Yan. This raises the intriguing possibility that the Spen RNP protein might integrate signals from both the Raf and Hox pathways.

Amino Acid Motifs↗

PubMed1996

The UK epidemic of BSE: slow virus or chronic pesticide-initiated modification of the prion protein? Part 1: Mechanisms for a chemically induced pathogenesis/transmissibility.

It is proposed that exposure of the bovine embryo to specific high-dose lipophilic formulations of organophosphate insecticide (containing phthalimide) applied exclusively in the UK during the 1980s/early 1990s was the primary trigger that initiated the UK's bovine spongioform encephalopathy epidemic. Multi-site binding organophosphate toxic metabolites penetrate the fetus, covalently binding with, phosphorylating and ageing serine, tyrosine or histidine active sites on fetal central nervous system prion protein. An abnormal negative charge corrupts prion protein molecular surface, which blocks both proteases and chaperones from accessing their cleavage/bonding sites. This impairs normal degradation and folding of prion protein respectively. Once the abnormally phosphorylated abnormal prion protein isoform agent is initiated, any stress event ensuing in adult life induces a nerve growth factor-mediated synthesis of normal cellular prion protein isoform that aggregates to abnormally phosphorylated abnormal prion protein isoform, thereby becoming 'infected'/transformed into the same; due to the vicious circle of positive feedback invoked by the blocking of a prion protein-specific kinase. Prion protein could therefore serve as a hitherto unrecognized critical link in a chain of delayed neuroexcitotoxic proteins that are triggered off by chronic exposure to specific classes of chemical/metal that 'hit and run' during the vulnerable in utero period, producing spongioform encephalopathy disease years later.

Amino Acid Sequence↗

PubMed1995

Differential expression of the alpha- and beta-isoforms of protein kinase C in peripheral blood T and B cells from young and elderly adults.

The expression of alpha- and beta-isoforms of protein kinase C (PKC) was analyzed in the peripheral blood T and B cells from 11 elderly and young humans. Immunoblot analysis with isoenzyme specific antibodies showed that T cells from five of 11 elderly subjects exhibited selective reductions in PKC alpha which was < 60% of those in young subjects whereas the levels of PKC beta were comparable to T cells of young subjects. No age-related reductions of PKC alpha or beta were observed in B cells. Among individual elderly subjects, the reductions in T cell PKC alpha were not associated with lower levels of PKC beta thereby resulting in only approximately 60-70% reductions of combined PKC alpha plus PKC beta. In addition, the functional properties of PKC in stimulated T cells of elderly subjects with respect to activation/translocation were comparable to T cells of young subjects. These results suggest that selective alterations in PKC isoenzymes can occur in human T cells during aging which may not be readily apparent in standard enzymatic assays and may contribute to aberrancies in intracellular signal transduction.

Adult↗

PubMed2003

Differentiation stage-specific activation of p38 mitogen-activated protein kinase isoforms in primary human erythroid cells.

p38alpha, p38beta, p38gamma, and p38delta are four isoforms of p38 mitogen-activated protein (MAP) kinase (MAPK) involved in multiple cellular functions such as cell proliferation, differentiation, apoptosis, and inflammation response. In the present study, we examined the mRNA expression pattern of each of the four isoforms during erythroid differentiation of primary erythroid progenitors. We show that p38alpha and p38gamma transcripts are expressed in early hematopoietic progenitors as well as in late differentiating erythroblasts, whereas p38delta mRNA is only expressed and active during the terminal phase of erythroid differentiation. On the other hand, p38beta is minimally expressed in early CD34(+) hematopoietic progenitors but not expressed in lineage-committed erythroid progenitors. We also determined the phosphorylation/activation of p38alpha, MAPK kinase 3/6, and MAPKAP-2 in response to erythropoietin and stem cell factor. We found that phosphorylation of p38alpha, MAPK kinase kinase 3/6 and MAPKAP-2 occurs only upon growth factor withdrawal in primary erythroid progenitors. Moreover, our data indicate that activation of p38alpha does not induce apoptosis or promote proliferation of erythroid progenitors. On the other hand, under steady-state culture conditions, both p38alpha and p38delta isoforms are increasingly phosphorylated activated in the terminal phase of differentiation. This increased phosphorylation/activity was accompanied by up-regulation of heat shock protein 27 phosphorylation. Finally, we demonstrate that tumor necrosis factor alpha, an inflammatory cytokine that is modulated by p38alpha, is expressed by differentiating erythroblasts and inhibition of p38alpha or tumor necrosis factor alpha results in reduction in differentiation. Taken together, our data demonstrate that both p38alpha and delta isoforms function to promote the late-stage differentiation of primary erythroid progenitors and are likely to be involved in functions related to erythrocyte membrane remodeling and enucleation.

Base Sequence↗

PubMed2003

Molecular characterization and allergenic activity of Lyc e 2 (beta-fructofuranosidase), a glycosylated allergen of tomato.

Until now, only a small amount of information is available about tomato allergens. In the present study, a glycosylated allergen of tomato (Lycopersicon esculentum), Lyc e 2, was purified from tomato extract by a two-step FPLC method. The cDNA of two different isoforms of the protein, Lyc e 2.01 and Lyc e 2.02, was cloned into the bacterial expression vector pET100D. The recombinant proteins were purified by electroelution and refolded. The IgE reactivity of both the recombinant and the natural proteins was investigated with sera of patients with adverse reactions to tomato. IgE-binding to natural Lyc e 2 was completely inhibited by the pineapple stem bromelain glycopeptide MUXF (Man alpha 1-6(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3)GlcNAc). Accordingly, the nonglycosylated recombinant protein isoforms did not bind IgE of tomato allergic patients. Hence, we concluded that the IgE reactivity of the natural protein mainly depends on the glycan structure. The amino acid sequences of both isoforms of the allergen contain four possible N-glycosylation sites. By application of MALDI-TOF mass spectrometry the predominant glycan structure of the natural allergen was identified as MMXF (Man alpha 1-6(Man alpha 1-3)(Xyl beta 1-2)Man beta 1-4GlcNAc beta 1-4(Fuc alpha 1-3) GlcNAc). Natural Lyc e 2, but not the recombinant protein was able to trigger histamine release from passively sensitized basophils of patients with IgE to carbohydrate determinants, demonstrating that glycan structures can be important for the biological activity of allergens.

Adolescent↗

PubMed2001

The epsilon isoform of protein kinase C is involved in regulation of the LTD(4)-induced calcium signal in human intestinal epithelial cells.

We investigated the potential roles of specific isoforms of protein kinase C (PKC) in the regulation of leukotriene D(4)-induced Ca(2+) signaling in the intestinal epithelial cell line Int 407. RT-PCR and Western blot analysis revealed that these cells express the PKC isoforms alpha, betaII, delta, epsilon, zeta, and mu, but not betaI, gamma, eta, or theta;. The inflammatory mediator leukotriene D(4) (LTD(4)) caused the TPA-sensitive PKC isoforms alpha, delta, and epsilon, but not betaII, to rapidly translocate to a membrane-enriched fraction. The PKC inhibitor GF109203X at 30 microM but not 2 microM significantly impaired the LTD(4)-induced Ca(2+) signal, indicating that the response involves a novel PKC isoform, such as delta or epsilon, but not alpha. LTD(4)-induced Ca(2+) signaling was significantly suppressed in cells pretreated with TPA for 15 min and was abolished when the pretreatment was prolonged to 2 h. Immunoblot analysis revealed that the reduction in the LTD(4)-induced calcium signal coincided with a reduction in the cellular content of PKCepsilon and, to a limited extent, PKCdelta. LTD(4)-induced Ca(2+) signaling was also markedly suppressed by microinjection of antibodies against PKCepsilon but not PKCdelta. These data suggest that PKCepsilon plays a unique role in regulation of the LTD(4)-dependent Ca(2+) signal in intestinal epithelial cells.

Antibodies↗

PubMed2004

Two fast-type fibers in claw closer and abdominal deep muscles of the Australian freshwater crustacean, Cherax destructor, differ in Ca2+ sensitivity and troponin-I isoforms.

One type of fast fiber and two types of slow (slow-twitch, S1 and slow-tonic, S2) fibers are found in decapod crustacean skeletal muscles that differ in contractile properties and myofibrillar protein isoform compositions. In this study the structural characteristics, protein isoform compositions, and Ca2+-activation properties of fast fibers in the claw closer (F1) and abdominal deep flexor (F2) muscles of Cherax destructor were analyzed. For comparison, myofibrillar protein isoform compositions of slow (long-sarcomere) fibers from claw and abdomen were also determined; our results indicate that the slow fibers in the claw closer were the slow-twitch (S1) type and those in the abdominal superficial flexor were primarily slow-tonic (S2) type. F1 fibers had shorter resting sarcomere lengths (2.93 microm in unstretched fibers and 3.06 microm in stretched fibers) and smaller fiber diameter (256 microm) than F2 fibers (sarcomere lengths 3.48 microm in unstretched and 3.46 microm in stretched; 747 microm diameter). Moreover, F1 fibers showed a narrower range in sarcomere lengths than F2 fibers (2.81 to 3.28 microm vs. 2.47 to 4.05 micro m in unstretched fibers). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that the fast fibers from claw and abdomen differed in troponin-I composition; F1 fibers expressed two isoforms of troponin-I (TnI1 and TnI2) in approximately equal amounts, whereas F2 fibers expressed primarily TnI3 and lower levels of TnI1. F1 fibers were more sensitive to Ca2+, as shown by higher pCa values at threshold activation (pCa(10)=6.50+/-0.07) and at 50% maximum force (pCa(50)=6.43+/-0.07) than F2 fibers (pCa(10)=6.12+/-0.04 and pCa(50)=5.88+/-0.03, respectively). F1 fibers also had a greater degree of co-operativity in Ca2+ activation, as shown by a higher maximum slope of the force-pCa curve (n(Ca)=12.98+/-2.27 vs. 4.34+/-0.64). These data indicate that there is a greater fast fiber-type diversity in crustacean muscles than was previously supposed. Moreover, the differences in activation properties suggest that the TnI isoform composition influences the Ca2+ sensitivity of the contractile mechanism.

Animals↗

PubMed2005

Inhibitory interaction of the 14-3-3{epsilon} protein with isoform 4 of the plasma membrane Ca(2+)-ATPase pump.

The isoform-specific interaction of plasma membrane Ca(2+)-ATPase (PMCA) pumps with partner proteins has been explored using a yeast two-hybrid technique. The 90 N-terminal residues of two pump isoforms (PMCA2 and PMCA4), which have a low degree of sequence homology, have been used as baits. Screening of 5 x 10(6) clones of a human brain cDNA library yielded approximately 100 LEU2- and galactoside-positive clones for both pumps. A clone obtained with the PMCA4 bait specified the epsilon-isoform of the 14-3-3 protein, whereas no 14-3-3epsilon clone was obtained with the PMCA2 bait. The 14-3-3epsilon protein immunoprecipitated with PMCA4 (not with PMCA2) when expressed in HeLa cells. Overexpression of 14-3-3epsilon in HeLa cells together with targeted aequorins showed that the ability of the cells to export Ca(2+) was impaired; stimulation with histamine, an inositol 1,4,5-trisphosphate-producing agonist, generated higher cytosolic [Ca(2+)] transients, higher post-transient plateaus of the cytosolic [Ca(2+)], and higher Ca(2+) levels in the endoplasmic reticulum lumen and in the subplasmalemmal domain. Thus, the interaction with 14-3-3epsilon inhibited PMCA4. Silencing of the 14-3-3epsilon gene by RNA interference significantly reduced the expression of 14-3-3epsilon, substantially decreasing the height of the histamine-induced cytosolic [Ca(2+)] transient and of the post-transient cytosolic [Ca(2+)] plateau.

14-3-3 Proteins↗

PubMed2024

CLN3 transcript complexity revealed by long-read RNA sequencing analysis.

BACKGROUND: Batten disease is a group of rare inherited neurodegenerative diseases. Juvenile CLN3 disease is the most prevalent type, and the most common pathogenic variant shared by most patients is the "1-kb" deletion which removes two internal coding exons (7 and 8) in CLN3. Previously, we identified two transcripts in patient fibroblasts homozygous for the 1-kb deletion: the 'major' and 'minor' transcripts. To understand the full variety of disease transcripts and their role in disease pathogenesis, it is necessary to first investigate CLN3 transcription in "healthy" samples without juvenile CLN3 disease. METHODS: We leveraged PacBio long-read RNA sequencing datasets from ENCODE to investigate the full range of CLN3 transcripts across various tissues and cell types in human control samples. Then we sought to validate their existence using data from different sources. RESULTS: We found that a readthrough gene affects the quantification and annotation of CLN3. After taking this into account, we detected over 100 novel CLN3 transcripts, with no dominantly expressed CLN3 transcript. The most abundant transcript has median usage of 42.9%. Surprisingly, the known disease-associated 'major' transcripts are detected. Together, they have median usage of 1.5% across 22 samples. Furthermore, we identified 48 CLN3 ORFs, of which 26 are novel. The predominant ORF that encodes the canonical CLN3 protein isoform has median usage of 66.7%, meaning around one-third of CLN3 transcripts encode protein isoforms with different stretches of amino acids. The same ORFs could be found with alternative UTRs. Moreover, we were able to validate the translational potential of certain transcripts using public mass spectrometry data. CONCLUSION: Overall, these findings provide valuable insights into the complexity of CLN3 transcription, highlighting the importance of studying both canonical and non-canonical CLN3 protein isoforms as well as the regulatory role of UTRs to fully comprehend the regulation and function(s) of CLN3. This knowledge is essential for investigating the impact of the 1-kb deletion and rare pathogenic variants on CLN3 transcription and disease pathogenesis.

Humans↗

PubMed1994

Mouse cells with null p53 mutation have all p53 isoforms deleted and lose negative growth control.

Embryonic mouse cells containing a disrupted p53 gene (-/-) were compared to the heterozygote (+/-) and homozygote control (+/+) for growth characteristics and the presence of p53 protein isoforms. There were considerable morphological differences between the null cells and homozygote, with the null cells having irregular shapes and sizes, and densely staining pleomorphic nuclei. Growth curves showed the null cells to have essentially remained in log phase growth during the course of these studies, losing contact inhibition. Losses of all protein isoforms indicate a single locus origination, and suggest that the multiple protein isoforms observed are due to different net charges on the protein as a result of post-translational modification. These results confirm deactivation of the p53 gene by site-specific disruption of exon 5 as described by Donehower (Donehower et al., 1992).

Animals↗