Isoforms of proteins

Ouimet, C.C., daCruz, E., Silva, E.F., Greengard, P. The alpha and gamma 1 isoforms of protein phosphatase 1 are highly and specifically concentrated in dendritic spines. Proc. Natl. Acad. Sci. U.S.A. 92 3396, 1995. 

FRET is an extremely useful phenomenon when it comes to the analysis of molecular conformations and interactions. F or the analysis of interactions, in which two separate molecules are labeled with an appropriate pair of fluorophores, an interaction can be shown by observing FRET. Further, FRET can be used as a type of spectroscopic ruler to measure the closeness of interactions. Proteins, lipids, enzymes, DNA, and RNA can all be labeled and interactions documented. This general method can be applied not only to questions of cellular function like kinase dynamics [3] but also to disease pathways, for example, the APP-PS1 interaction that is important in Alzheimer s disease (AD) [4], Alternatively, two parts of a molecule of interest can be labeled with a donor and acceptor fluorophore. Using this technique, changes in protein conformation and differences between isoforms of proteins can be measured, as well as protein cleavage. 

Burns-Hamuro L., Hamuro Y, Kim J., Sigala P., Fayos R., Stranz D., Jennings P., Taylor S., Woods V.L. Jr Distinct interaction modes of an AKAP bound to two regulatory subunit isoforms of protein kinase A revealed by amide hydrogen/ deuterium exchange. Protein Sci. 

Ahmad S, Trepel JB, Ohno S, Suzuki K, Tsuruo T, Glazer R1 (1992) 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. Mol Pharmacol 42 1004-1009... 

Cacace AM, Guadagno SN, Krauss RS, Fabbro D, Weinstein IB (1993) The epsilon isoform of protein kinase C is an oncogene when overexpressed in rat fibroblasts. Oncogene 8 2095-2104... 

Diaz-Meco MT, Municio MM, Frutos S, Sanchez P, Lozano J, Sanz L, Moscat J (1996) The product of par 4, a gene induced during apoptosis, interacts selectively with the atypical isoforms of protein kinase C. Cell 86 777-786 Dietel M, Arps H, Lage H, Niendorf A (1990) Membrane vesicle formation due to acquired mitoxanthrone resistance in human gastric carcinoma cell line EPG85-257. Cancer Res 50 6100-6106... 

PKC signaling is also involved in the plasticity underlying fear learning and memory. Inhibition of PKC in rats impairs acquisition of fear conditioning (Goosens et al. 2000 Li et al. 2002) and mice with a knockout of the isoform of protein kinase C have deficient cued and contextual fear conditioning (Weeber et al. 2000). Inhibitors of PKC also block LTP (MaUnow et al. 1989 Reyman et al. 1988). 

Watanabe Y, Gould E, McEwen BS (1992a) Stress induces atrophy of apical dendrites of hippocampal CA3 pyramidal neurons. Brain Res 588 341-345 Weeber E, Atkins CM, Selcher JC, Varga AW, Mirnikjoo B, Paylor R, Leitges M, Sweatt JD (2000) A role for the p isoform of protein kinase C in fear conditioning. J Neurosci 20 5906-5914... 

The major current working hypothesis for lithium s therapeutic mechanism of action supposes that its effects on phosphoinositol turnover, leading to an early relative reduction of myoinositol in human brain, are part of an initiating cascade of intracellular changes. Effects on specific isoforms of protein kinase C may be most relevant. Alterations of protein kinase C-mediated signaling alter gene expression and the production of proteins implicated in... 

Phosphorylated IRS-1 activates a second signaling pathway by interacting with an 85-kDa SH2-containing protein that is a subunit of phophatidylinositol 3-kinase.384 386 This activates the 110-kDa catalytic subunit of the 3-kinase, which catalyzes formation of phosphatidylinositol 3-phosphate as well as Ptdlns (3,4)P2 and Ptdlns (3,4,5)P3.387/387a These compounds, which remain within membranes, activate other branches of the signaling cascade, some of which may converge with those of the MAP kinase cascade. However, there appears to be specific activation of a ribosomal Ser/Thr kinase that, among other activities, phosphorylates ribosomal protein S6, a component of the small ribosomal subunit.388 It also phosphorylates some isoforms of protein kinase C and other enzymes. Ptdlns 3-kinase may also activate 6-phosphofructo-2-kinase (Fig. 11-2, step ti).384/388... 

Chida, K., Murakami, A., Tagawa, T., Ikuta, T., and Kuroki, T., Cholesterol sulfate, a second messenger for the n isoform of protein kinase C, inhibits promotional phase in mouse skin carcinogenesis, Cancer Res., 55, 4865, 1995. 

A therapeutic strategy could encompass the suppression of protein kinase C activity, thus short-circuiting at least part of the pathway driving VEGF activity in the retina. The initial chemical studied as a protein kinase C inhibitor was known as LY333531 and was found to have very selective inhibition of both [31 and [311 isoforms of protein kinase C. This drug was studied in animal models and was found to reduce VEGF-mediated retinal vascular permeability, increase retinal blood flow, and inhibit retinal neovascularization. 

E.T. Maizels, C.A. Peters, M. Kline, R.E. Cutler and M. Shanmugam, Heat-shock protein-25/27 phosphorylation by the delta isoform of protein kinase C, Biochem. J. 332, 703-712 (1998). 

Luostarinen, R., Siegbahn, A., and Saldeen, T. 1991. Effects of dietary supplementation with vitamin E on human neutrophil chemotaxis and generation of LTB4. Ups. J. Med Sci. 96, 103-111. Madani, S., Hichami, A., Legrand, A., Belleville, J., and Khan, N.A. 2001. Implication of acyl chain of diacylglycerols in activation of different isoforms of protein kinase C. FASEB J. 15, 2595-2601. 

Because of its more complicated signaling cascade, Gq mediated signaling is more complex and cell-type specific. There are many isoforms of protein kinase C that have selective substrates and functions. In addition, different cell types express variable amounts of Ga " -mediated kinases and ion channels and Ca can play a role in events ranging from cell contraction and secretion to gene expression and cell division. 

Another mechanism closely connected to the anti tumoral activity is the inhibition of oncologic signals and enzymes implicated on proliferation, differentiation and transformation processes. Verbascoside and homoplantaginin inhibit EGFR [63] and tirosin-kinase (TK). Calceolarioside A and B, forsythoside, leucosceptoside A, plantainoside, poliumoside, and verbascoside inhibit a-isoform of protein-kinase C (PKC) [69, 100]. The potency of this activity might be related to the number of sugar moieties in an inverse way, but it required more assays with other PPG to find the relationship. It has been found that verbascoside interacts with the catalytic domain of this enzyme, but a structure-activity relationship was not discussed. 

Yet another type of editing has been observed in viral RNA from paramyxovirus. The virally encoded RNA polymerase sometimes "stutters" reading the same template base two or more times, with a resulting insertion of a base. Editing of transcripts usually serves an essential biological function, creating the correct sequence for translation of the mRNA and often generating multiple isoforms of proteins. 

ABBREVIATIONS APS, adaptor protein with PH and SH2 domains CAP, Cbl associated protein Crkll, chicken tumor virus regulator of kinase II GLUT4, glucose transporter 4 Gab-1, Grb-2 associated binder MAP kinase, mitogen-activated protein kinase PDK, phosphoinositide-dependent kinase PI3 kinase, phosphatidylinositol-3-kinase PIP3, phos-phatidylinositol trisphosphate PKB, protein kinase B (also called Akt) aPKC, atypical isoform of protein kinase C Y, tyrosine residue Y-P, phosphorylated tyrosine residue. 

Capillary electrophoresis is primarily limited to small molecules that are water solul le because of their compatibility with the run buffer. Other similar techniques such as isoelectric focusing and capillary zone elec-trophoresis ° have aided in the separation of proteins by allowing for the separation of larger proteins. In addition these techniques can separate isoforms of proteins and peptides by using an extraordinarily low pH range. However, capillary electrophoresis cannot separate neutral compounds, and... 

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