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PubMed2000

Identification and characterization of multiple isoforms of a mouse ribosome receptor.

We isolated multiple cDNA clones encoding various isoforms of a mouse ribosome receptor protein (mRRp). The cDNAs were isolated from a 13.5-day-old mouse conceptus cDNA library by polymerase chain reaction-based screening. The predicted proteins encoded by these cDNAs showed significant homology with ribosome receptors present in dogs (83%), humans (80%), and chickens (45%). The cDNA isoforms had highly identical N- and C-terminal sequences but differed in their central sequences, suggesting that these cDNA isoforms may be derived by alternative splicing from a single gene. Genomic Southern blot analysis confirmed the existence of only a single mouse ribosome receptor gene. Alignments of the deduced amino acid sequences of the mRRp cDNA isoforms revealed that they differ in the number of decapeptide repeats present in the central domain of the protein. These repeats have been previously suggested to mediate ribosome binding and thus differences in repeat number may translate to different ribosome binding abilities. The longest mRRp isoform had 61 tandem repeats. This is of interest because in the human and canine ribosome receptor proteins there are only 54 tandem repeats, suggesting that humans and dogs may also have larger ribosome receptor protein isoforms. Surprisingly, mRRp has a very short basic C-terminal sequence of only 35 amino acids, while in contrast, the known human and canine forms of this protein have acidic C-terminal regions comprised of 803 and 798 amino acid residues, respectively. Although the function of the C-terminal region is currently unknown, it may be that those C-terminal sequences that are present in human and canine RRp proteins but missing in mRRp do not play critical roles in RRp function. The cDNA of the ES/130 isoform, which lacks tandem repeats and presumably are unable to bind ribosomes, could be isolated by reverse transcriptase-PCR from E 13.5 mouse embryos. mRRp mRNAs were expressed in all tissues examined but expression levels of each isoform differed between tissues. The identification of multiple mRRp isoforms in the mouse will allow us to study the regulation and function of ribosome receptors on a genetic level.

Amino Acid Sequence↗
PubMed2003

Isoform specific control of gene activity in vivo by the Drosophila ecdysone receptor.

The steroid hormone 20-hydroxyecdysone induces metamorphosis in insects. The receptor for the hormone is the ecdysone receptor, a heterodimer of two nuclear receptors, EcR and USP. In Drosophila the EcR gene encodes 3 isoforms (EcR-A, EcR-B1 and EcR-B2) that vary in their N-terminal region but not in their DNA binding and ligand binding domains. The stage and tissue specific distribution of the isoforms during metamorphosis suggests distinct functions for the different isoforms. By over-expressing the three isoforms in animals we present results supporting this hypothesis. We tested for the ability of the different isoforms to rescue the lack of dendritic pruning that is characteristic of mutants lacking both EcR-B1 and EcR-B2. By expressing the different isoforms specifically in the affected neurons, we found that both EcR-B isoforms were able to rescue the neuronal defect cell autonomously, but that EcR-A was less effective. We also analyzed the effect of over-expressing the isoforms in a wild-type background. We determined a sensitive period when high levels of either EcR-B isoform were lethal, indicating that the low levels of EcR-B at this time are crucial to ensure normal development. Over-expressing EcR-A in contrast had no detrimental effect. However, high levels of EcR-A expressed in the posterior compartment suppressed puparial tanning, and resulted in down-regulation of some of the tested target genes in the posterior compartment of the wing disc. EcR-B1 or EcR-B2 over-expression had little or no effect.

Animals↗
PubMed2000

Erbb4 and its isoforms: selective regulation of growth factor responses by naturally occurring receptor variants.

ErbB4 is a member of the epidermal growth factor receptor (EGFR, ErbB) family that mediates responses to neuregulins and other EGF-like growth factors. ErbB4 is a central regulator of cardiovascular and neural development as well as differentiation of the mammary gland. A role for ErbB4 has also been implicated in malignancies and heart diseases. Four structurally and functionally distinct ErbB4 isoforms have recently been identified. One pair of isoforms differs within their extracellular juxtamembrane domains. These juxtamembrane ErbB4 isoforms are either susceptible or resistant to proteolytic processing that release a soluble receptor ectodomain. Another pair of ErbB4 isoforms differs within their cytoplasmic tails. Analysis of the intracellular signal transduction pathways indicates that both cytoplasmic ErbB4 isoforms can couple to the Shc-MAPK signaling pathway, while the other one is incapable of coupling to the phosphoinositide 3-kinase (PI3-K)-Akt pathway. The differences in the activation of signaling cascades are reflected in the cellular responses stimulated via the cytoplasmic isoforms. Both cytoplasmic ErbB4 isoforms can stimulate proliferation, but the isoform that cannot activate PI3-K is defective in stimulating cellular survival and chemotaxis. Together these four naturally occurring receptor variants provide a new level of diversity to the control of growth factor-stimulated cellular responses. Thus, the ErbB4 isoforms may have distinct and specific roles in the regulation of various developmental and pathological processes.

Animals↗
PubMed2002

Molecular cloning and functional characterization of chicken brain tau: isoforms with up to five tandem repeats.

Tau is a major microtubule-associated protein in mammalian brain, where it exists as multiple isoforms that are produced from a single gene by alternative mRNA splicing. Here we present the first report on the structure and function of tau protein from a nonmammalian vertebrate. In the adult chicken brain, five main tau isoforms are expressed. One isoform has three tandem repeats, two isoforms have four repeats each, and two isoforms have five repeats each. Similar to mammalian tau, some chicken tau isoforms contain an amino-terminal insert of 53 amino acids. Unlike mammalian tau, a 34 amino acid insert in the proline-rich region upstream of the repeats is alternatively spliced in chicken tau. It is preceded by a constitutively expressed sequence of 17 amino acids that is absent in tau from human and rodent brains. The expression of chicken tau isoforms and their phosphorylation are developmentally regulated, similar to what has been described in mammalian brain. Functionally, chicken tau isoforms with five repeats have the greatest ability to promote microtubule assembly, followed by isoforms with four and three repeats, respectively. The 34 amino acid insert positively influences both the rate and the extent of microtubule assembly, whereas the 53 amino acid insert only influences the extent of assembly.

Aging↗
PubMed2000

Coordinated regulation of two TRAIL-R2/KILLER/DR5 mRNA isoforms by DNA damaging agents, serum and 17beta-estradiol in human breast cancer cells.

A search of the Genebank database revealed that there are two distinct gene sequences with the common name of TRAIL-R2/Killer/DR5. Using reverse transcription-polymerase chain reaction (RT-PCR), we confirmed the existence of two isoforms of TRAIL-R2/Killer/DR5 mRNA, which we have designated the long and short isoforms based on their electrophoretic mobility. We found that both the long and short mRNA isoforms are ubiquitously expressed in human tissues and cell lines. The long form generally predominates, but the proportion of the two isoforms varies depending on the tissue type. Treatment of MCF-7 human breast cancer cells with the DNA damaging drugs adriamycin, campthothecin, or etoposide causes a coordinated up-regulation of both isoforms. Treatment of the p53-mutant T-47D breast cancer cell line with adriamycin also results in up-regulation of both isoforms, suggesting that adriamycin up-regulates TRAIL-R2/Killer/DR5 expression independent of functional p53. The expression of both mRNA isoforms are increased in MCF-7 cells cultured in charcoal-stripped fetal bovine serum compared to normal serum, suggesting that sex steroid hormones may play a role in the negative regulation of their expression. This was confirmed in MCF-7 cells cultured in stripped serum supplemented with 17beta-estradiol, which also resulted in a decrease in the mRNA expression of both isoforms. These results demonstrate that the TRAIL-R2/Killer/DR5 gene gives rise to two distinct forms of mRNA, and that these two forms are coordinately regulated by DNA damage and 17beta-estradiol in human breast cancer cells. The functional significance of the two isoforms remains to be determined.

Antineoplastic Agents↗
PubMed2000

Splice isoforms of alpha(1a)-adrenoceptor in rabbit.

Two splice isoforms of rabbit alpha(1a)-adrenergic receptor (AR), (named alpha(1a)-OCU.2-AR and alpha(1a)-OCU.3-AR) have been isolated from the liver cDNA library in addition to the previously reported isoform (alpha(1a)-OCU.1-AR). Although they have the identical splice position with human alpha(1a)-AR isoforms, the C-terminal sequences are distinct from those of human isoforms. Among these rabbit alpha(1a)-AR isoforms, there are no significant differences in pharmacological properties: high affinity for prazosin, WB4101, KMD-3213 and YM617 and low affinity for BMY7378, using COS-7 cells expressing each isoform by radioligand binding assay. Competitive reverse transcription-polymerase chain reaction (RT - PCR) analysis revealed that mRNA of alpha(1a)-ARs was expressed in liver, thoracic aorta, brain stem and thalamus of rabbit. The splice isoforms exhibited a distinct distribution pattern in rabbit; alpha(1a)-OCU. 1-AR was expressed most abundantly in those tissues. CHO clones, stably expressing each isoforms with receptor density 740 fmol mg(-1) protein in alpha(1a)-OCU.1-AR, 1200 fmol mg(-1) in alpha(1a)-OCU.2-AR and 570 fmol mg(-1) in alpha(1a)-OCU.3-AR, respectively, showed a noradrenaline-induced increase in inositol trisphosphate which was suppressed by prazosin. Noradrenaline elicited a concentration-dependent increase in extracellular acidification rate (EAR) in the CHO clones with pEC(50) values of 6. 19 for alpha(1a)-OCU.1-AR, 6.49 for alpha(1a)-OCU.2-AR and 6.58 for alpha(1a)-OCU.3-AR, respectively. Noradrenaline caused a concentration-dependent increase in intracellular Ca(2+) concentration ([Ca(2+)]i) in the CHO clones with pEC(50) values of 6. 14 for alpha(1a)-OCU.1-AR, 7.25 for alpha(1a)-OCU.2-AR and 7.70 for alpha(1a)-OCU.3-AR, respectively. In conclusion, the present study shows the occurrence of three splice isoforms of rabbit alpha(1a)-AR, which are unique in C-terminal sequence and in tissue distribution. They show similar pharmacological profiles in binding studies but alpha(1a)-OCU.3-AR had the highest potency of noradrenaline in functional studies in spite of the lowest receptor density. These findings suggest that the structure of C-terminus of alpha(1a)-ARs may give the characteristic functional profile.

Alternative Splicing↗
PubMedDate not supplied

Proteomic analysis of cytokeratin isoforms uncovers association with survival in lung adenocarcinoma.

Cytokeratins (CK) are intermediate filaments whose expression is often altered in epithelial cancer. Systematic identification of lung adenocarcinoma proteins using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry has uncovered numerous CK isoforms. In this study, 93 lung adenocarcinomas (64 stage I and 29 stage III) and 10 uninvolved lung samples were quantitatively examined for protein expression. Fourteen of 21 isoforms of CK 7, 8, 18, and 19 occurred at significantly higher levels (P < .05) in tumors compared to uninvolved adjacent tissue. Specific isoforms of the four types of CK identified correlated with either clinical outcome or individual clinical-pathological parameters. All five of the CK7 isoforms associated with patient survival represented cleavage products. Two of five CK7 isoforms (nos. 2165 and 2091), one of eight CK8 isoforms (no. 439), and one of three CK19 isoforms (no. 1955) were associated with survival and significantly correlated to their mRNA levels, suggesting that transcription underlies overexpression of these CK isoforms. Our data indicate substantial heterogeneity among CK in lung adenocarcinomas resulting from posttranslational modifications, some of which correlated with patient survival and other clinical parameters. Therefore, specific isoforms of individual CK may have utility as diagnostic or predictive markers in lung adenocarcinomas.

Adenocarcinoma↗
PubMed1999

Isoforms of c-KIT differ in activation of signalling pathways and transformation of NIH3T3 fibroblasts.

Alternate splicing of mRNA encoding c-KIT results in isoforms which differ in the presence or absence of four amino acids (GNNK) in the juxtamembrane region of the extracellular domain of the receptor. In this study we show that these isoforms of human c-KIT, expressed at similar levels in NIH3T3 cells, display differential effects on various attributes of transformation. The GNNK- isoform strongly promoted anchorage independent growth (colony formation in semi-solid medium), loss of contact inhibition (focus formation), and led to tumorigenicity in nude mice. In contrast, the GNNK+ isoform elicited colony formation but relatively poor focus formation and no tumorigenicity. Saturation binding analysis indicated that the isoforms do not differ significantly in their affinity for the KIT ligand, Steel Factor (SLF). Negligible ligand-independent receptor phosphorylation was observed in either case but, after ligand stimulation, the GNNK- isoform displayed more rapid and extensive tyrosine autophosphorylation and faster internalization. Both isoforms recruited the p85 subunit of phosphatidylinositol 3-kinase and led to similar phosphorylation of its downstream effector c-Akt, but the GNNK- isoform gave rise to more MAP kinase phosphorylation. Thus the c-KIT isoforms display different signalling characteristics and have different transforming activity in NIH3T3 cells.

3T3 Cells↗
PubMed2000

Expression of thyroid receptor isoforms in the human fetal central nervous system and the effects of intrauterine growth restriction.

BACKGROUND: Congenital hypothyroidism is known to be associated with mental retardation which, if recognized promptly, is largely prevented by thyroid hormone replacement. Intrauterine growth restriction (IUGR) is a major cause of perinatal mortality and morbidity, and is also associated with neurodevelopmental delay. Fetuses with IUGR have reduced circulating concentrations of free thyroxine (T4) and free triiodothyronine (T3), leading to the hypothesis that a reduction in the tissue effects of thyroid hormones in the central nervous system (CNS) may contribute to neurodevelopmental morbidity. Since thyroid hormone effects are mediated through binding to specific nuclear thyroid hormone receptors (TRs), we have defined the pattern of TR isoform expression in the CNS throughout normal human development and have compared TR expression in the CNS of normal fetuses with those affected by IUGR. METHODS: Samples of normal human fetal brain from first and second trimesters were obtained at surgical termination of pregnancy. Appropriately grown and third trimester fetuses affected by Intrauterine growth restriction (IUGR) were also investigated after unexplained stillbirth at post mortem examination. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to examine the expression of TR isoform mRNAs in frozen cerebral cortex from 10 to 16 weeks gestation. TR protein expression in human fetal brains (both cerebral hemispheres and cerebellum) was also examined in formalin fixed sections and expression of TR alpha1, alpha2, beta1 and beta2 isoforms being defined using semiquantitative immunocytochemistry. RESULTS: RT-PCR revealed the presence of mRNAs encoding TR alpha1, beta1 and beta2 isoforms and the nonfunctional TRalpha2 variant in the fetal cerebral cortex from week 10 of human pregnancy. Immunostaining of the fetal brain revealed TR alpha1 and alpha2 protein from week 11 of human gestation. Expression of all TR isoform proteins was largely confined to the pyramidal neurones of the cerebral cortex and the Purkinje cells of the cerebellum with increasing receptor expression evident with gestational age. Semiquantitative observer scoring showed that by the second trimester, there was a marked increase in the proportion of pyramidal and Purkinje cells expressing TR isoforms, while by the third trimester, all these cells immunostained. Comparison of TR immunostaining in the cerebral cortex and cerebellum from IUGR fetuses (n = 18) matched for gestational age to normal fetuses revealed a lower intensity of expression of all the TR isoforms confirmed by observer scoring and quantification using TR protein immunofluoresence (P<0.01). CONCLUSIONS: Our findings indicate both pre- and post-translational expression of TR alpha and beta isoforms in the cerebral cortex of first trimester fetuses. These findings support the view that the transplacental passage of thyroid hormone in early gestation may be critical to neurological development. Our finding that in severe IUGR the expression of TR isoforms in the human fetal cerebral cortex and cerebellum was significantly reduced, in association with reduced circulating thyroid hormone concentrations indicate that changes in free ligand and receptors may affect CNS development. These findings should prompt further investigation of the potential therapeutic role of peripartum thyroid hormone treatment.

Case-Control Studies↗
PubMed2000

Pressure denaturation of beta-lactoglobulin. Different stabilities of isoforms A and B, and an investigation of the Tanford transition.

Beta-lactoglobulin, the main whey protein in bovine milk, exists in several isoforms of which the most abundant are isoforms A and B. We have previously reported the denaturation of beta-lactoglobulin A by hydrostatic pressure [Valente-Mesquita, V.L., Botelho, M.M. & Ferreira, S.T. (1998) Biophys. J. 75, 471-476]. Here, we compare the pressure stabilities of isoforms A and B. These isoforms differ by two amino-acid substitutions: Asp64 and Val118 in isoform A are replaced by glycine and alanine, respectively, in isoform B. Replacement of the buried Val118 residue by the smaller alanine side-chain is not accompanied by significant structural rearrangements of the neighbouring polypeptide chain and creates a cavity in the core of beta-lactoglobulin. Pressure denaturation experiments revealed different stabilities of the two isoforms. Standard volume changes (DeltaVunf) of - 49 +/- 8 mL.mol-1 and -75 +/- 3 mL.mol-1, and unfolding free energy changes (DeltaGunf) of 8.5 +/- 1.3 kJ.mol-1 and 11.3 +/- 0.4 kJ.mol-1 were obtained for isoforms A and B, respectively. The volume occupied by the two methyl groups of Val118 removed in the V118A substitution is approximately 40 A3 per monomer of beta-lactoglobulin, in excellent agreement with the experimentally measured difference in DeltaVunf for the two isoforms (DeltaDeltaVunf = 26 mL.mol-1, corresponding to approximately 43 A3 per monomer). Thus, the existence of a core cavity in beta-lactoglobulin B may explain its enhanced pressure sensitivity relative to beta-lactoglobulin A. beta-Lactoglobulin undergoes a reversible pH-induced conformational change around pH 7, known as the Tanford transition. We have compared the pressure denaturation of beta-lactoglobulin A at pH 7 and 8. Unfolding free energy changes of 8.5 +/- 1.3 and 8.3 +/- 0.3 kJ.mol-1 were obtained at pH 7 and 8, respectively, showing that the thermodynamic stability of beta-lactoglobulin is identical at these pH values. Interestingly, DeltaVunf was dependent on pH, and varied from -49 +/- 8 mL.mol-1 to -68 +/- 2 mL.mol-1 at pH 7 and 8, respectively. The large increase in DeltaVunf at pH 8 relative to pH 7 appears to be associated with an overall expansion of the protein structure and could explain the increased pressure sensitivity of beta-lactoglobulin at alkaline pH.

Animals↗
PubMed1999

Interaction of a kinesin-like protein with calmodulin isoforms from Arabidopsis.

In Arabidopsis and other plants there are multiple calmodulin isoforms. However, the role of these isoforms in regulating the activity of target proteins is obscure. Here, we analyzed the interaction between a kinesin-like calmodulin-binding motor protein (Reddy, A. S. N., Safadi, F., Narasimhulu, S. B., Golovkin, M., and Hu, X. (1996) J. Biol. Chem. 271, 7052-7060) and three calmodulin isoforms (calmodulin-2, -4, and -6) from Arabidopsis using different approaches. Gel mobility and fluorescence shift assays revealed that the motor binds to all calmodulin isoforms in a calcium-dependent manner. Furthermore, all calmodulin isoforms were able to activate bovine calcium/calmodulin-dependent phosphodiesterase. However, the concentration of calmodulin-2 required for half-maximal activation of phosphodiesterase is 2- and 6-fold lower compared with calmodulin-4 and -6, respectively. The dissociation constants of the motor to calmodulin-2, -4, and -6 are 12.8, 27.0, and 27.8 nM, respectively, indicating that calmodulin-2 has 2-fold higher affinity for the motor than calmodulin-4 and -6. Similar results were obtained using another assay that involves the binding of (35)S-labeled calmodulin isoforms to the motor. The binding saturation curves of the motor with calmodulin isoforms have confirmed that calmodulin-2 has 2-fold higher affinity to the motor. However, the affinity of calmodulin-4 and -6 isoforms for the motor was about the same. Based on these studies, we conclude that all calmodulin isoforms bind to the motor protein but with different affinities.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1999

Interdomain interaction of merlin isoforms and its influence on intermolecular binding to NHE-RF.

Merlin, the neurofibromatosis 2 tumor suppressor protein, has two major isoforms with alternate C termini and is related to the ERM (ezrin, radixin, moesin) proteins. Regulation of the ERMs involves intramolecular and/or intermolecular head-to-tail associations between family members. We have determined whether merlin undergoes similar interactions, and our findings indicate that the C terminus of merlin isoform 1 is able to associate with its N-terminal domain in a head-to-tail fashion. However, the C terminus of isoform 2 lacks this property. Similarly, the N terminus of merlin can also associate with C terminus of moesin. We have also explored the effect of merlin self-association on binding to the regulatory cofactor of Na(+)-H(+) exchanger (NHE-RF), an interacting protein for merlin and the ERMs. Merlin isoform 2 captures more NHE-RF than merlin isoform 1 in affinity binding assays, suggesting that in full-length merlin isoform 1, the NHE-RF binding site is masked because of the self-interactions of merlin. Treatment with a phospholipid known to decrease self-association of ERMs enhances the binding of merlin isoform 1 to NHE-RF. Thus, although isoform 1 resembles the ERM proteins, which transition between inactive (closed) and active (open) states, isoform 2 is distinct, existing only in the active (open) state and presumably constitutively more available for interaction with other protein partners.

Animals↗
PubMed2000

Distinct isoforms of the cofactor BAG-1 differentially affect Hsc70 chaperone function.

In the mammalian cytosol and nucleus the activity of the molecular chaperone Hsc70 is regulated by chaperone cofactors that modulate ATP binding and hydrolysis by Hsc70. Among such cofactors is the anti-apoptotic protein BAG-1. Remarkably, BAG-1 is expressed as multiple isoforms, which are distinguished by their amino termini. We investigated whether distinct isoforms differ with respect to their Hsc70-regulating activity. By comparing the mainly cytosolic isoforms BAG-1M and BAG-1S, opposite effects of the two isoforms were observed in chaperone-assisted folding reactions. Whereas BAG-1M was found to inhibit the Hsc70-mediated refolding of nonnative polypeptide substrates, the BAG-1S isoform stimulated Hsc70 chaperone activity. The opposite effects are not due to differences in the regulation of the ATPase activity of Hsc70 by the two isoforms. Both isoforms stimulated ATP hydrolysis by Hsc70 in an Hsp40-dependent manner through an acceleration of ADP-ATP exchange. Our results reveal that the different amino termini of the distinct BAG-1 isoforms determine the outcome of an Hsc70-mediated folding event, most likely by transiently interacting with the polypeptide substrate. Employing isoforms of a cofactor with different substrate binding properties appears to provide the means to influence the chaperone function of Hsc70 in addition to modulating its ATPase cycle.

Amino Acid Sequence↗
PubMed2001

Functional analyses of two alternative isoforms of the transcription factor Pax-5.

The Pax-5 gene plays a central role in B cell development, activation, and differentiation. At least four different isoforms have been identified, of which isoform Pax-5a has been extensively studied, while functions for alternative isoforms were previously unknown. Here, using a transient transfection system, we provide evidence that alternative isoform Pax-5d acts as a dominant-negative regulator by suppressing activity of Pax-5a in a dose-dependent manner. In contrast, co-expression in the presence of alternative isoform Pax-5e causes an increase in Pax-5a activity. Protein studies on Pax-5e using Western blot analysis revealed that this 19-kDa isoform migrates as a 27-kDa species on SDS-polyacrylamide electrophoresis gels, while a mutant Pax-5e form in which a C-terminal cysteine residue has been mutated, runs at the expected 19 kDa. Using both Western blot and immunoprecipitation assays, we further provide evidence that this size discrepancy may be caused by a tight association between Pax-5e and a thioredoxin-like factor. Comparison of various B cell lines as well as resting and lipopolysaccharide-activated mature B lymphocytes shows that increased B cell proliferation correlates with increased levels of Pax-5e/thioredoxin, whereas increased Pax-5d amounts correlate with inhibition of cell growth. Together, our results suggest that during activation and differentiation of B lymphocytes, Pax-5a function is modulated by two alternative spliced isoforms: the dominant negative Pax-5d isoform may mediate inhibition of Pax-5a activity in resting B cells, while alternative isoform Pax-5e associated with thioredoxin may increase Pax-5a activity through an unknown (redox) mechanism.

3T3 Cells↗
PubMed2025

IsoBayes: a Bayesian approach for single-isoform proteomics inference.

MOTIVATION: Studying protein isoforms is an essential step in biomedical research; at present, the main approach for analyzing proteins is via bottom-up mass spectrometry proteomics, which return peptide identifications, that are indirectly used to infer the presence of protein isoforms. However, the detection and quantification processes are noisy; in particular, peptides may be erroneously detected, and most peptides, known as shared peptides, are associated to multiple protein isoforms. As a consequence, studying individual protein isoforms is challenging, and inferred protein results are often abstracted to the gene-level or to groups of protein isoforms. RESULTS: Here, we introduce IsoBayes, a novel statistical method to perform inference at the isoform level. Our method enhances the information available, by integrating mass spectrometry proteomics and transcriptomics data in a Bayesian probabilistic framework. To account for the uncertainty in the measurement process, we propose a two-layer latent variable approach: first, we sample if a peptide has been correctly detected (or, alternatively filter peptides); second, we allocate the abundance of such selected peptides across the protein(s) they are compatible with. This enables us, starting from peptide-level data, to recover protein-level data; in particular, we: (i) infer the presence/absence of each protein isoform (via a posterior probability), (ii) estimate its abundance (and credible interval), and (iii) target isoforms where transcript and protein relative abundances significantly differ. We benchmarked our approach in simulations, and in two multi-protease real datasets: our method displays good sensitivity and specificity when detecting protein isoforms, its estimated abundances highly correlate with the ground truth, and can detect changes between protein and transcript relative abundances. AVAILABILITY AND IMPLEMENTATION: IsoBayes is freely distributed as a Bioconductor R package, and is accompanied by an example usage vignette.

Proteomics↗
PubMed2005

Expression of divalent metal transporter 1 (DMT1) isoforms in first trimester human placenta and embryonic tissues.

BACKGROUND: Divalent metal transporter 1 (DMT1) is a transmembrane glycoprotein which mediates the proton-coupled transport of a variety of divalent metal ions. Two isoforms, which differ by the presence (DMT1-IRE) or absence (DMT1-nonIRE) of an iron-responsive element (IRE) in their 3' untranslated region, are implicated in apical iron transport and endosomal iron transport respectively. Although the expression pattern of DMT1 isoforms is tissue specific in adult, data regarding its expression in embryonic tissues are lacking. METHODS: Semiquantitative RT-PCR and immunohistochemistry were used to study the mRNA and protein expression of both DMT1 isoforms in embryonic tissues between 8 and 14 weeks gestational age. RESULTS: DMT1-IRE and DMT1-nonIRE expressions were ubiquitous in embryonic tissues examined. In the lung, statistically significant correlations were found between the levels of DMT1 isoform expression and gestational age. In the placenta, DMT1-IRE was the predominantly expressed isoform. Both isoform proteins were localized in embryonic epithelial cellular membrane. CONCLUSION: Both DMT1 isoforms are ubiquitously expressed in embryonic tissues in the first trimester. Predominant DMT1-IRE isoform expression in placenta suggests an iron-regulatory mechanism reminiscent of that in the adult duodenum. Epithelial distributions of both DMT1 isoforms are associated with the absorptive or excretory functions of the expressed tissues.

Adult↗
PubMed1999

FSH-induced resumption of meiosis in mouse oocytes: effect of different isoforms.

The ability of different isoforms of follicle stimulating hormone (FSH) to induce the resumption of meiosis in cultured mouse oocytes was evaluated. Oocytes were cultured in the presence of hypoxanthine to prevent spontaneous resumption of meiosis. Using serial dilutions of the isoform fractions representing less acidic isoforms (pI 6. 43-5.69), mid-acidic (pI 5.62-4.96) and acidic (pI 4.69-3.75), the concentration which caused 50% of the oocytes to resume meiosis and undergo germinal vesicle breakdown (GVBD) after a culture period of 24 h (i.e. ED(50%) GVBD) was determined. The FSH concentration of the isoform fractions was determined by radioimmunoassay, radio-receptor assay or through cAMP release in a Chinese hamster ovary-cell line expressing the human FSH-receptor. Determined by radioimmunoassay, the (ED(50%) GVBD) values were: less acidic 6.4 +/- 0.3 IU/l (mean +/- SD), mid-acidic 6.1 +/- 0.7 IU/l and acidic 12.2 +/- 0.7 IU/l. The less and mid-acidic isoforms were significant lower than the acidic (P < 0.0005). Similar relationships between the isoform fractions were obtained by the two other FSH assays. The results demonstrate that FSH isoforms with a pI of >5.0 induced resumption of meiosis significantly more efficiently than acidic isoforms. Less and mid-acidic isoforms may exert an important physiological function by inducing the resumption of meiosis in oocytes from pre-ovulatory follicles during the mid-cycle gonadotrophin surge.

Animals↗
PubMed2003

Lipoprotein (a) levels in those with high molecular weight apo (a) isoforms may remain low in a significant proportion of patients with end-stage renal disease.

BACKGROUND: Studies have reported an increase in median Lipoprotein (Lp) (a) in patients with high molecular weight (HMW) apolipoprotein (apo) (a) isoforms and renal impairment. Some studies identify Lp (a) levels as a risk factor for vascular disease in renal failure whilst others have demonstrated an association with apo (a) isoform type and vascular disease. METHODS: A total of 239 patients at end-stage renal failure (ESRF) were studied prior to the initiation of dialysis. Blood was taken for Lp (a) levels and apo (a) isoforms. Clinical vascular disease (CVD) was assessed on the basis of clinical history and Rose questionnaire. The control group for Lp (a) levels consisted of 228 healthy volunteers. RESULTS: Despite a higher median Lp (a) level in those with HMW isoforms, 30% of patients had Lp (a) levels <10 mg/dl. Overall, 49% patients were identified as having CVD. Diabetes, smoking history and Lp (a) levels were significantly associated with CVD in logistic regression analysis, although when patients with low molecular weight (LMW) and HMW isoforms were analysed separately, Lp (a) levels were not significantly associated with CVD in those with LMW isoforms. The rates of CVD in those with HMW isoform and low Lp (a) levels were significantly lower than those with HMW isoforms and elevated Lp (a) levels, 34 vs 57% (P < 0.01). CONCLUSIONS: Although median Lp (a) levels in those patients at ESRF with HMW isoforms are higher than controls, in a third of such patients Lp (a) levels remain relatively low. These patients have lower rates of CVD than those with high levels of Lp (a).

Aged↗