ProMoST (Protein Modification Screening Tool): a web-based tool for mapping protein modifications on two-dimensional gels

Halligan, Brian D.; Ruotti, Victor; Jin, Weihong; Laffoon, Scott; Twigger, Simon; Dratz, Edward A.

doi:10.1093/nar/gkh356

Cited by 107 publications

(93 citation statements)

References 31 publications

(30 reference statements)

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“…Glutamate led to the appearance of at least two more acidic versions of HuR (pI 8.6 and 8.7, respectively) compatible with Ser/Thr monophosphorylation or N-terminal acetylation as indicated by the ProMoST algorithm. 33 Given the known involvement of HuR in mRNA regulation, alterations of its protein should affect the posttranscriptional fates of RNA targets acting to oppose the neurotoxic response. Some of these associations could also be shared with nElavls.…”

Section: Hur Targets and Regulates A Group Of Rnas Involved In Oxidatmentioning

confidence: 99%

Neuroprotection requires the functions of the RNA-binding protein HuR

et al. 2014

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Alterations in the functions of neuronal RNA-binding proteins (RBPs) can contribute to neurodegenerative diseases. However, neurons also express a set of widely distributed RBPs that may have developed specialized functions. Here, we show that the ubiquitous member of the otherwise neuronal Elavl/Hu family of RNA-binding proteins, Elavl1/HuR, has a neuroprotective role. Mice engineered to lack exclusively HuR in the hippocampal neurons of the central nervous system (CNS), maintain physiologic levels of neuronal Elavls and develop a partially diminished seizure response following strong glutamatergic excitation; however, they display an exacerbated neurodegenerative response subsequent to the initial excitotoxic event. This response was phenocopied in hippocampal cells devoid of ionotropic glutamate receptors in which the loss of HuR results in enhanced mitochondrial dysfunction, oxidative damage and programmed necrosis solely after glutamate challenge. The molecular dissection of HuR and nElavl mRNA targets revealed the existence of a HuR-restricted posttranscriptional regulon that failed in HuR-deficient neurons and is involved in cellular energetics and oxidation defense. Thus, HuR acts as a specialized controller of oxidative metabolism in neurons to confer protection from neurodegeneration.

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Section: Hur Targets and Regulates A Group Of Rnas Involved In Oxidatmentioning

confidence: 99%

Neuroprotection requires the functions of the RNA-binding protein HuR

et al. 2014

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“…The increased apparent molecular mass of the D1 Cys protein in yeast fractions compared with the D1 protein in rat liver microsomes might reflect unique posttranslational modifications of D1 protein in yeast. It is known that post-translational modifications such as phosphorylation, glycosylation and acylation of proteins can result in so called 'upshifts' in apparent molecular mass during SDS-PAGE (Halligan et al 2004, Wong et al 2004). The D1 Cys-His-tag protein was detected as several isoforms after affinity-labeling (at least 3) with apparent molecular masses between 27-34 kDa (Fig.…”

Section: Kinetic Analysis Of D1 Activity In Yeast Subcellular Fractionsmentioning

confidence: 99%

Expression of recombinant membrane-bound type I iodothyronine deiodinase in yeast

Kuiper

Klootwijk²,

Visser³

2005

Journal of Molecular Endocrinology

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The bioactivity of thyroid hormone is determined to a large extent by the monodeiodination of the prohormone thyroxine (T4) by the hepatic selenoenzyme type I iodothyronine deiodinase (D1), i.e. by outer ring deiodination (ORD) to the active hormone triiodothyronine (T3) or by inner ring deiodination (IRD) to the inactive metabolite reverse T3 (rT3). Since D1 is a membrane-bound protein with an N-terminal membrane-spanning domain, the enzyme is very difficult to purify in an active state. This study was undertaken in order to develop a heterologous (over)-expression system that would eventually allow the production of large amounts of purified active D1 protein. We have expressed a mutant rat D1 protein, in which the selenocysteine residue in the core catalytic center was replaced by cysteine (D1 Cys) in yeast cells (Saccharomyces cerevisiae). After yeast cell fractionation, kinetic analysis was performed with dithiothreitol as reducing cofactor. ORD activity was associated with membrane fractions, while no activity could be detected in the cytosolic fraction. The D1 Cys protein displayed a tenfold increase in K m (2 µM) for rT3 as compared with native D1 protein in rat liver microsomes. The D1 protein content is about 65 pmol/mg microsomal protein, as compared with about 3 pmol/mg in rat liver microsomal fraction. SDS-PAGE analysis of N-bromoacetyl-[125 I]T3 affinity-labeled D1 protein showed several labeled protein isoforms with apparent molecular masses between 27 and 32 kDa. Immunoblot analysis with a specific D1 antiserum confirmed the observed D1 protein heterogeneity. Site-directed mutagenesis of several potential N-linked glycosylation sites, phosphorylation sites and a unique myristoylation site established that D1 heterogeneity is not caused by N-linked glycosylation, but probably by a combination of O-linked glycosylation and phosphorylation. Deletion of the endoplasmic reticulum (ER)-signal sequence and the membrane-spanning domain (amino acid residue 2-35), did not result in the production of a soluble D1 enzyme. Although this mutated D1 protein is inactive, the fact that it is still membrane bound indicates the existence of additional membrane attachment site(s) or membrane-spanning domains. Overall, our studies indicate that yeast cells provide a useful system for the expression of relatively high levels of D1 protein which could be used for further structure-function analysis.

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“…Based on an average of four 2D gels from each group, Hb␦ and Hb␥ increase from 12% and 0% in normoxic-reared animals to 28% and 22% in hypoxic-reared animals, respectively. Each 2D gel revealed several isoelectric forms of Hb subunits, suggesting a typical phosphorylation train (Halligan et al, 2004).…”

Section: Two-dimensional Gel Electrophoresismentioning

confidence: 97%