Protein Isoform

Two SDF-1 protein isoforms, SDF-1 α and SDF-1 β, that arise from alternative mRNA splicing were originally characterized in the process of cloning the chemokine [27].

From: Handbook of Biologically Active Peptides, 2006

Female Reproduction

Rong Li, Francesco J. DeMayo, in Encyclopedia of Reproduction (Second Edition), 2018

Isoforms

Protein isoforms are generated from the same gene but with distinct amino acid sequences and biological roles (Gunning, 2006). Alternative promoter, splicing and translation initiation sites are common mechanisms of producing protein isoforms (Chen and Manley, 2009; Pal et al., 2011; Kochetov, 2008), and are identified in SRs.

Multiple promoters reside at SR genes. At a minimum, the number of promoters is eleven for ESR1 (Kos et al., 2001; Zou et al., 2009; Okuda et al., 2003), four for ESR2 (Hirata et al., 2001; Shoda et al., 2002; Smith et al., 2010; Lee et al., 2013), five for PGR (Yamanaka et al., 2002; Hirata et al., 2000, 2002; Saner et al., 2003; Kastner et al., 1990), nine for GR (Palma-Gudiel et al., 2015; Breslin et al., 2001; Turner and Muller, 2005; Presul et al., 2007) and two for MR (Zennaro et al., 1995), but only one identified for AR so far (Ahrens-Fath et al., 2005; Dehm et al., 2008; Tilley et al., 1990).

Coupling with the multiple promoters, alternative splicing produces dozens of transcription isoforms by intron/exon skipping, inserting, or replicating. Many SR transcription isoforms have been demonstrated with physiological and/or pathological functions, such as ERβcx (Herynk and Fuqua, 2004), PGR-C, -M, -S (Wei et al., 1990; Cork et al., 2008), AR-V7, -V9 (Lu et al., 2015; Dehm and Tindall, 2011), GRγ, GR-A and GR-P(Moalli et al., 1993; Ray et al., 1996), and MR248, MR260, MR+4 (Bloem et al., 1995; Zennaro et al., 2001) etc.

Alternative translation further increases the number of protein isoforms. For example, 8 alternative translation initiation sites (TIS) have been identified in GR, thus 8 translation isoforms could be generated from a single GR transcription isoform, such as GRα and β (Lu and Cidlowski, 2005). In the same paper, the authors predict more species conserved TIS at the N-terminal of ESR1, PGR, AR, and MR (Lu and Cidlowski, 2005). Several of these internal TIS have already been identified as truly functional alternative translational start sites (Pascual-Le Tallec et al., 2004; Barraille et al., 1999; Wilson and McPhaul, 1994; Chantalat et al., 2016).

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The human pituitary proteome: clinical applications

XIANQUAN ZHAN, ... DOMINIC M. DESIDERIO, in Medical Applications of Mass Spectrometry, 2008

3.2.4 Analysis of protein isoforms

Protein isoforms result from PTMs, splicing variants, etc. Each protein isoform has its own pI and Mr values; 2DGE, or 2DGE coupled with corresponding protein antibodies, is an effective method to array those different isoforms of each protein. MS, especially MS / MS, plays an important role in the characterization of each PTM and splicing variant. We found that prolactin had multiple isoforms in human pituitary control tissues—six 2D gel spots that contained prolactin were identified [32; Evans et al., in preparation]. Further experiments are needed to determine whether the ratio of each prolactin isoform changes in NF pituitary adenomas and prolactinomas.

Twenty-four 2D gel spots that contained human GH were found in human pituitary control tissues [34]. Those hGHs in the 24 2D gel spots were classified into the four types of hGH splicing isoforms, 1–4. The expression proportion of those four isoforms was isoform 1 (87.5%) > isoform 2 (8.1%) > isoform 3 (3.3%) > isoform 4 (1.1%); a significant statistical difference was found among those isoforms. PTM analysis demonstrated that, among those 24 GH spots, some spots had a measurably different pI, but the same Mr; that result could be due to the deamidation of asparagine to aspartate that was identified with MALDI–TOF MS. That deamidation caused a change in charge. Other spots had a measurably different Mr, but the same apparent pI; that result could be due to N-glycosylation, polymer formation, or proteolysis. MS/MS data demonstrated that the hGH in 1 of the 24 2D-gel spots was a phosphoprotein with three phosphate groups (Ser-77, Ser-132, and Ser-176). Further study is required to determine whether the ratio of GH isoform changes in human pituitary adenoma compared to controls.

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Regulation of Drug-Metabolizing Enzymes and Drug Metabolism by Inflammatory Responses

E.T. Morgan, in Drug Metabolism in Diseases, 2017

Downregulation of Positive Transcription Factors

C/EBP isoforms and HNFs are transcription factors that contribute to the basal expression of many P450 genes. In rodent livers, HNF1α, HNF3β, and HNF4α DNA binding activities are rapidly downregulated in response to LPS injection (Chen et al., 1995; Li and Klaassen, 2004). Repression of a CYP3A4 reporter transgene in mice with extrahepatic cancers was associated with decreased levels of both the LIP and active forms of C/EBPβ (Kacevska et al., 2011). One would predict that the downregulation of multiple positive factors is likely to contribute to downregulation. Studies in HNF1α knockout mice demonstrated the importance of reduced HNF1α binding for downregulation of the drug transporter (Li and Klaassen, 2004).

Nuclear receptors PXR and CAR mediate the selective induction of subsets of DME genes by xenobiotics, and retinoid X receptor-α (RXRα) is their obligatory dimerization partner. Numerous studies have demonstrated the downregulation of each of these receptors by inflammatory stimuli (Beigneux et al., 2000; Pascussi et al., 2000; Assenat et al., 2004; Ghose et al., 2008, 2011; Ghaffari et al., 2011; Kacevska et al., 2011) and that this causes suppression of drug-induced expression of target genes (Muntane-Relat et al., 1995; Pascussi et al., 2000; Assenat et al., 2004; Moriya et al., 2012). IL-1 downregulates CAR in an NF-κB-dependent mechanism (Assenat et al., 2004). The degree to which downregulation (or inhibition, see later discussion) of nuclear receptors contributes to the downregulation of DME expression in the absence of drug inducers is less clear. In PXR−/− or CAR−/− mice, downregulation of DME genes by LPS was not impaired (Richardson and Morgan, 2005; Shah et al., 2014). However, the downregulation of Cyp3a11 and some drug transporters by IL-6 injection was attenuated (Teng and Piquette-Miller, 2005) in PXR−/− mice, and the downregulation of DME genes by the TLR2 ligand lipoteichoic acid was attenuated in CAR−/− mice (Shah et al., 2014). In human hepatocytes, downregulation of CYP3A4 could be inhibited by knockdown of PXR with small interfering RNA (Yang et al., 2010a). Taken together, these results suggest that modulation of nuclear receptor expression or activity contributes to the downregulation of constitutive DME expression, the magnitude of the contribution depending on the nature of the inflammatory stimulus. Diet may also affect their contributions, given that dietary components are activators of PXR and CAR (Gao and Xie, 2010).

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Na+-H+ Exchange in Mammalian Digestive Tract

Pawel R. Kiela, Fayez K. Ghishan, in Physiology of the Gastrointestinal Tract (Fourth Edition), 2006

Posttranscriptional Regulation

NHE2 protein has a relatively short half-life (∼3 hours) compared with other NHE isoforms (e.g., 24 hours for NHE1 and 14 hours for NHE3) and is subject to lysosomal degradation, as determined in PS120 fibroblasts and Caco-2 cells (125). This suggests that changes at the level of gene transcription or translation may be more critical for NHE2 regulation than for other isoforms with long half-lives. NHE2 is a residual plasma membrane protein, and unlike NHE3, it does not undergo endosomal recycling (125). Glycosylation of NHE2 may affect its cellular localization, because unglycosylated 75-kDa rabbit NHE2 was found predominantly intracellularly (126), although it is unclear whether this represents a regulatory mechanism or is simply related to the maturational stage of NHE2 protein synthesis. Of the two well-characterized apically expressed NHE isoforms, NHE2 and NHE3, NHE2 activity is considered relatively stable and is not regulated by many factors. Extracellular alkalinization activates NHE2, which is believed to propel increased proton extrusion in gastric parietal cells during secretagogue-stimulated acid secretion (see the Na+-H+ Exchange and Gastric Physiology section later in this chapter). The maximal rate of exchange (Vmax) mediated by NHE2 was shown to be stimulated by serum, fibroblast growth factor (FGF), and protein kinase C (PKC) activator phorbol myristyl acetate in PS120 fibroblasts (127). Intracellular ATP depletion reduced the NHE2 activity by a dramatic decrease in H+ affinity, as well as Vmax, with virtual elimination of the allosteric effect of H+ (127). ATP depletion also eliminated the stimulatory effect of serum, suggesting that growth factor–stimulated NHE2 activity is mediated via its pH-sensing mechanism. Thrombin increased NHE2 Vmax without altering the Hill coefficient (127), although it is unclear whether this could be attributed to increased intracellular Ca2+ ascribed to thrombin-treated fibroblasts. In the same study, thrombin also increased NHE3 activity, whereas it was shown later that increase of intracellular Ca2+ by thapsigargin in Caco-2/bbe cells inhibited

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Gene Regulation in Imaginal Disc and Salivary Gland Development during Drosophila Metamorphosis

CYNTHIA BAYER, ... JAMES W. FRISTROM, in Metamorphosis, 1996

2. rbp Function

The Z1 protein isoform provides rbp function. In the lethal rbp5 mutant, all of the Z1-containing proteins are truncated (Emery et al., 1994). They appear to be prematurely terminated between the conserved core region and the zinc finger domain. Thus, rbp5 animals produce no Z1 proteins with DNA-binding activity. The homozygous viable rbp mutant, rbptn, contains a P element insertion a few kilobases upstream from the Z1 zinc finger domain (Moran and Torkamanzehi, 1990; C. Bayer and J. W. Fristrom, unpublished observations) (Fig. 4). A novel transcript containing both Z1 and Z4 sequences is found in this mutant whereas Z2 and Z3 transcripts are unaffected. The bristle defect in rbptn can be rescued by expression of the Z1 transgene during prepupal development, but not by any of the other BR-C proteins. Consistent with a primary role for Z1 in rbp function, Z1 transgene expression can also rescue the pupal lethality of rbp1 mutants.

A correlation between Z1 and rbp function is also indicated by experiments aimed at rescuing the transcriptional activity of genes dependent on BR-C function. Five salivary gland genes, Sgs4 (Guay and Guild, 1991; Karim et al., 1993; von Kalm et al., 1994) and the late puff genes L71-1,-3,-6, and -9 (Guay and Guild, 1991; Karim et al., 1993), are strongly underexpressed in rbp5 mutants. The transcriptional activity of all of these genes is restored to approximately wild-type levels in rbp5 animals when a Z1 transgene is expressed (K. Crossgrove and G. M. Guild, personal communication; L. von Kalm, C. Bayer, and J. W. Fristrom, unpublished observations). Expression of a Z2 transgene cannot rescue the transcriptional activity of these target genes. It is not yet known if a Z3 or Z4 transgene can also rescue the activity of these genes.

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Epigenetics

William Schierding, ... Wayne S. Cutfield, in Fetal and Neonatal Physiology (Fifth Edition), 2017

Prions

Prions are misfolded isoform proteins that can serve as transmissible agents of disease.64 The role of prions in epigenetics is quite different from that of other mechanisms described in this chapter. Prions propagate by transmitting their misfolded protein state to other proteins.65 The effect of prions on disease state has been shown to be a robust and transmissible epigenetic phenotype (i.e., that self-propagates and is stably heritable), inducing changes in protein conformations that can profoundly alter its mechanistic properties, resulting in a different cellular phenotype.38,66 Prions can be inherited, sporadic, or acquired, and they may be affected by environmental factors.64 There is currently debate as to whether more adverse environmental conditions lead to an increase in protein misfolding and therefore more prions, and whether this change could be beneficial to an individual's fitness.66 In humans, exposure to prions by surgery or blood transfusion can transmit diseases such as variant Creutzfeldt-Jakob disease.65 Kuru, the only known epidemic of human prion disease, was transmitted through ingestion of dead relatives in the Fore tribe of Papua New Guinea.25 Prions have also been linked to many other human neurodegenerative diseases, including Alzheimer, Parkinson, and Lou Gehrig diseases.64,65 Finally, there are also inheritable prion diseases in humans, such as familial Creutzfeldt-Jakob disease and Gerstmann-Sträussler-Scheinker disease.25,66

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The basement membrane and its role in pulmonary disease

Pernille Juhl, ... Jannie M.B. Sand, in Biochemistry of Collagens, Laminins and Elastin (Third Edition), 2024

Localization, remodeling and repair

There are different protein isoforms in the individual basement membranes that are adapted to the physiological function of the tissue. For example, the type IV collagen isoform α3α4α5 is mainly found in the alveolar and glomerular basement membranes, both of which serve a filtration function [7,10]. Interestingly, this isoform is more resistant to proteolysis due to a high degree of disulfide crosslinks, making it suitable for these locations that are subjected to the outside [11]. Different laminin isoforms have also been shown to be differentially expressed and localized in adult human and murine lung, suggesting that laminin α3 and α5 co-localize with both airway and alveolar epithelium, whereas α2 is restricted to airway epithelium and α4 to alveolar epithelium [12–14].

All tissues, including the basement membrane, undergo remodeling, a process that is tightly regulated. Inflammatory cells secrete an excess of proteases responsible for degrading the basement membrane. To maintain tissue homeostasis during these normal processes, basement membrane proteins are degraded to make room for newly synthesized proteins. Remodeling of the basement membrane may release biologically active protein fragments to nearby tissue and cells. Protein fragments can have different functions than the original protein, such as signaling. All the proteins and protein fragments (see Chapter 37) play important roles in maintaining the basement membrane [7].

After an injury, the production of specific proteins is differentially regulated to enhance tissue repair. During development, laminin α1 and α2 are the primary laminin α chains, but a developmental switch ensures that the levels of these subunits are reduced over time. In the adult mouse lung, expression of laminin α1 and α2 chains has been shown to be at least 10 times lower than the most abundant α3 and α5 chains [12,13,15]. Laminin subunit production also undergoes a shift during repair response, where the less abundant α1, α2, and α4 chains are upregulated to a greater degree than the normally abundant α3 and α5 chains [13]. Type XXVIII collagen (see Chapter 28) is normally in the lowest quartile of proteins expressed in a healthy murine lung, but its levels have shown to increase after injury with bleomycin [15]. Additionally, type XXVIII collagen localization changed from the basement membrane to a patchier distribution in fibrotic foci, and the protein became more insoluble, indicating that it is incorporated into the ECM.

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Chemokines: A New Peptide Family of Neuromodulators

PATRICK KITABGI, ... WILLIAM ROSTÈNE, in Handbook of Biologically Active Peptides, 2006

STRUCTURE OF THE PRECURSOR mRNA/GENE

Two SDF-1 protein isoforms, SDF-1 α and SDF-1 β, that arise from alternative mRNA splicing were originally characterized in the process of cloning the chemokine [27]. Their amino acid sequence is shown in Fig. 1. SDF-1 α consists of a 67-residue protein that is preceded by a 21-residue signal peptide at its N-terminus. SDF-1β is identical to SDF-1 α except for a five-residue extension at the C-terminus. Recently, a third mRNA variant, termed SDF-1γ, was described in rat brain [15], but the corresponding protein has yet to be characterized. Should it be translated, SDF-1γ would be identical to SDF-1α with a 31-residue C-terminal extension (Fig. 1). Most functional data on SDF-1 were obtained with the α variant.

FIGURE 1. Amino acid sequences of human, mouse, and rat SDF-1. The 21-aa signal peptide is highlighted in green, and the 67-aa mature SDF-1 a in yellow. Species-variation in the sequences are highlighted in magenta. Note the very high degree of sequence conservation between species. The five-residue extension of mouse and rat SDF-1β is shown highlighted in blue, and the putative 31-residue extension of rat SDF-1γ in italic and gray. The first two Cys residues in the SDF-1 sequence in positions 9 and 11, separated by only one residue, are shown in red. This CXC motif is common to one of the two major subfamilies of chemokines, hence the designation CXCLi for members of this subfamily and CXCRi for their receptors.

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A Survey of Cell Biology

James M. Ervasti, Kevin J. Sonnemann, in International Review of Cytology, 2008

1 INTRODUCTION

Dystrophin is the largest protein isoform expressed from the gene defective in Duchenne muscular dystrophy (Hoffman et al., 1987; Koenig et al., 1988), a lethal muscle-wasting disease that afflicts 1 in 3500 live-born males (Engel, 1986). Based on sequence homology, dystrophin is divided into four distinct domains (Koenig et al., 1988). The amino-terminal 250 residues encode a pair of calponin homology (CH) modules common to several proteins that bind filamentous actin. Adjacent to the amino-terminal domain, more than 2800 amino acids encode 24 homologous triple helical repeats and four hinge domains (Koenig and Kunkel, 1990) that are thought to confer flexibility and elasticity. A third domain of ~400 residues is more complex, encoding a WW module (Bork and Sudol, 1994), two EF hand modules (Koenig et al., 1988), and two ZZ modules in series (Ponting et al., 1996). Finally, the carboxy-terminal ~240 amino acids are unique to dystrophin and its related proteins (Tinsley et al., 1992; Wagner et al., 1993). In total, the four domains of dystrophin are encoded by 3685 amino acids with a molecular weight of 427 kDa.

In skeletal muscle, dystrophin was isolated as part of a large, tightly associated oligomeric complex of proteins synonymously referred to as the dystrophin–glycoprotein complex or dystrophin-associated protein complex (Ervasti et al., 1990; Yoshida and Ozawa, 1990; Ervasti and Campbell, 1991). Investigations into the biological function of the dystrophin–glycoprotein complex suggest it plays an important mechanical function in stabilizing the plasma membrane (the sarcolemma) against stresses imposed during muscle contraction or stretch. The dystrophin–glycoprotein complex has also garnered attention as a putative cellular signaling complex. Here, we review the data supporting current views on the biological function(s) of the dystrophin–glycoprotein complex in striated muscle.

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RET Signaling in Ureteric Bud Formation and Branching

Frank Costantini, in Kidney Development, Disease, Repair and Regeneration, 2016

Effects of Point Mutations That Remove RET Tyrosine Residues Involved in Signal Transduction

Ret encodes two major protein isoforms, RET9 and RET51, which are identical for the first 1063 amino acids but differ in their C-terminal tails. RET9 has a 9-amino acid tail, whereas RET51 has a different 51-amino acid tail (Figure 4.3(A)). Both isoforms are coexpressed in the kidney. Kidneys of mice engineered to express only RET9 were apparently normal [65,108], whereas those expressing only RET51 were normal when a human RET51 sequence was expressed [108] but hypoplastic with reduced UB branching when a chimeric mouse/human form of RET51 was expressed [65]. The reason for this difference is unclear, but the hypomorphic nature of the latter Ret allele provides a useful model of renal hypoplasia caused by reduced/defective RET signaling [65,85].

RET9 and RET51 share three tyrosine (Y) residues that, when phosphorylated, provide docking sites for adapter proteins that activate different signaling pathways (for a review, see Ibanez [27]) (Figure 4.3(A)). Y981 binds Src, activating the MAPK pathway; Y1015 is a docking site for PLCγ; and Y1062 is a multifunctional docking site for several proteins, including SHC, that activate both the MAPK and PI3K pathways. RET51 has an additional docking site at Y1096 that activates the PI3K pathway via GRB2. Thus there is no single RET tyrosine residue that, when mutated, can fully eliminate signaling via either MAPK or PI3K. Nevertheless, mice with mutations of each of these residues have been analyzed. The most severe effects (bilateral renal agenesis or severe hypodysplasia, similar to the Ret-null phenotype) occurred in mice expressing only RET9 with a Y1062 mutation [108,109]. In contrast, the same mutation in mice expressing both RET isoforms [66], or only RET51 [108], caused only reduced branching and milder renal hypoplasia, possibly because Y1096 allowed at least partial activation of PI3K. Mutation of Y981 had no apparent effect in the context of RET51. On the other hand, the Y981 mutation in RET9 caused a partially penetrant unilateral megaureter, even though the kidneys remained relatively normal [108].

Mutation of the PLCγ docking site Y1015 in the context of either RET9 or RET51 caused a spectrum of defects, including bilateral megaureter and multicystic hypodysplasia, which could be traced to the formation of multiple ectopic UBs from the ND, decreased and abnormal UB branching within the kidneys, and failure of the CND to degenerate. However, the mutation did not interfere with the ability of the UBs to invade the MM or initiate early branching [108,110]. The phenotype was similar to that observed in mice lacking Spry1 (discussed in the section GENES THAT ACT DOWNSTREAM OF GDNF/GFRα1/RET SIGNALING) or Slit2, suggesting that Spry1 or Slit2 might normally be upregulated via RET-Y1015 [108]. However, subsequent studies ruled out such a mechanism [110]. Instead, they revealed that in RET-Y1015 mutant mice the more caudal mesonephric mesenchyme, which normally degenerates by E11.5, was initially malpositioned and later failed to degenerate. Furthermore, there was strongly elevated diphospho-Erk in the ND as well as in the adjacent mesenchyme (but no change in phospho-AKT, an indicator of PI3K signaling). How this PLCγ docking site affects MAPK activity in the ND or in the adjacent mesenchyme cells remains unclear; however, the results suggest that RET signaling via Y1015 negatively regulates MAPK and thus balances the activation of this pathway via Y1062 [45,110].

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