Proteomic Profiling of Hsp90 Inhibitors

Voruganti, Sudhakar; Kline, Jake; Balch, Maurie; Rogers, Janet; Matts, Robert L.; Hartson, Steven D.

doi:10.1007/978-1-4939-7477-1_11

Cited by 10 publications

(7 citation statements)

References 32 publications

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“…Specifically, two-dimensional gel electrophoresis has been employed to analyze protein expression profiles and identify novel diagnostic, prognostic or predictive biomarkers in these tumors [38,39,40,41,42,43,44]. Due to the complex interactome of HSP90 and the enormous number of cellular processes in which it is involved, the use of proteomic tools for the study of this chaperone and its clients is a common approach [17,45,46,47]. However, additional information is needed to dissect responses to HSP90 inhibitors in lung cancer.…”

Section: Introductionmentioning

confidence: 99%

Impact of Heat Shock Protein 90 Inhibition on the Proteomic Profile of Lung Adenocarcinoma as Measured by Two-Dimensional Electrophoresis Coupled with Mass Spectrometry

Marrugal

Ferrer

Pastor

et al. 2019

Cells

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Heat shock protein 90 (HSP90) is an important chaperone in lung adenocarcinoma, with relevant protein drivers such as EGFR (epidermal growth factor receptor) and EML4-ALK (echinoderm microtubule-associated protein-like protein4 fused to anaplastic lymphoma kinase) depending on it for their correct function, therefore HSP90 inhibitors show promise as potential treatments for lung adenocarcinoma. To study responses to its inhibition, HSP90 was pharmacologically interrupted by geldanamycin and resorcinol derivatives or with combined inhibition of HSP90 plus HSP70 in lung adenocarcinoma cell lines. Two-dimensional electrophoresis was performed to identify proteomic profiles associated with inhibition which will help to understand the biological basis for the responses. HSP90 inhibition resulted in altered protein profiles that differed according the treatment condition studied. Results revealed 254 differentially expressed proteins after treatments, among which, eukaryotic translation initiation factor3 subunit I (eIF3i) and citrate synthase demonstrated their potential role as response biomarkers. The differentially expressed proteins also enabled signalling pathways involved in responses to be identified; these included apoptosis, serine-glycine biosynthesis and tricarboxylic acid cycle. The proteomic profiles identified here contribute to an improved understanding of HSP90 inhibition and open possibilities for the detection of potential response biomarkers which will be essential to maximize treatment efficacy in lung adenocarcinoma.

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Section: Introductionmentioning

confidence: 99%

Impact of Heat Shock Protein 90 Inhibition on the Proteomic Profile of Lung Adenocarcinoma as Measured by Two-Dimensional Electrophoresis Coupled with Mass Spectrometry

Marrugal

Ferrer

Pastor

et al. 2019

Cells

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“…g ., Voruganti et al). 40 Trypsinolytic peptides were injected onto a 75 μm × 50 cm nanocolumn (Acclaim PepMap, Thermo PN 164942) and separated using a water–acetonitrile gradient (3–30% acetonitrile in 120 min) containing 0.1% formic acid. Peptides were eluted through a stainless steel emitter and ionized within a NanoSpray Flex ion source (Thermo).…”

Section: Methodsmentioning

confidence: 99%

“…From these coomassie-stained gels, the γ-sarcoglycan protein bands were excised and digested with trypsin using standard methodologies (e.g., Voruganti et al). 40 Trypsinolytic peptides were injected onto a 75 μm × 50 cm nanocolumn (Acclaim PepMap, Thermo PN 164942) and separated using a water− acetonitrile gradient (3−30% acetonitrile in 120 min) containing 0.1% formic acid. Peptides were eluted through a stainless steel emitter and ionized within a NanoSpray Flex ion source (Thermo).…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

In Vitro Glycosylation of Membrane Proteins Using N-Glycosyltransferase

2021

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Glycoproteins are post-translationally modified proteins that take part in nearly every biological process and make up a large percent of the proteome. N-Linked glycosylation can be performed by N -glycosyltransferase (NGT), which recognizes the consensus amino acid sequence, -Asn-X-Ser/Thr- (NXT), within the protein. The enzyme catalyzes glycosidic bond formation between the oligosaccharide donor, containing nucleoside phosphatase, and the amide nitrogen of the asparagine residue. The attachment of the sugar moiety can influence physiological and biological properties of the protein by affecting their folding, modulating interactions with other biomolecules, and modifying their functions at the cellular level. We are specifically interested in the properties of membrane glycoproteins, which are key components in a number of different disease states. Therefore, the use of in vitro protein glycosylation can help further evaluate the effects of the properties for these important macromolecules. In vitro studies of N-linked glycosylation were done in a stepwise fashion in a membrane-mimetic environment to confirm that the methods for glycosylating soluble proteins could be applicable to membrane proteins. Detergent and lipid systems were used since hydrophobic peptides and membrane proteins are insoluble in aqueous solvents. The stepwise method consisted of the glycosylation of a soluble 7-residue peptide, a hydrophobic WALP-NVT peptide, and a γ-sarcoglycan membrane protein, all of which contained the glycosylation site Asn-Val-Thr (NVT). Glycosylation of the samples was performed using Escherichia coli -expressed NGT from the Actinobacillus pleuropneumoniae genome, and a single sugar moiety of glucose, provided from a nucleotide-linked donor, was added to the glycosylation site. Gel electrophoresis, mass spectrometry, and NMR studies were used for the detection of glycosyltransferase activity and to show the attachment of a single glucose molecule. Our experiments demonstrated that small or large membrane proteins that contain an N-glycosylation consensus sequence can be glycosylated by NGT in membrane-mimetic environments.

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“…Adsorption reactions were eluted with SDS-PAGE sample buffer and separated on 12% gels. Gels were fractionated and trypsinolyzed as described [40]. For solution digests, resins were eluted with 8M buffered urea, and then the eluates were reduced and alkylated, diluted to 2M urea, and digested with trypsin using standard methods.…”

Section: Lc-ms/msmentioning

confidence: 99%

Complex protein interactions mediate Drosophila Lar function in muscle tissue

et al. 2022

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The type IIa family of receptor protein tyrosine phosphatases (RPTPs), including Lar, RPTPσ and RPTPδ, are well-studied in coordinating actin cytoskeletal rearrangements during axon guidance and synaptogenesis. To determine whether this regulation is conserved in other tissues, interdisciplinary approaches were utilized to study Lar-RPTPs in the Drosophila musculature. Here we find that the single fly ortholog, Drosophila Lar (Dlar), is localized to the muscle costamere and that a decrease in Dlar causes aberrant sarcomeric patterning, deficits in larval locomotion, and integrin mislocalization. Sequence analysis uncovered an evolutionarily conserved Lys-Gly-Asp (KGD) signature in the extracellular region of Dlar. Since this tripeptide sequence is similar to the integrin-binding Arg-Gly-Asp (RGD) motif, we tested the hypothesis that Dlar directly interacts with integrin proteins. However, structural analyses of the fibronectin type III domains of Dlar and two vertebrate orthologs that include this conserved motif indicate that this KGD tripeptide is not accessible and thus unlikely to mediate physical interactions with integrins. These results, together with the proteomics identification of basement membrane (BM) proteins as potential ligands for type IIa RPTPs, suggest a complex network of protein interactions in the extracellular space that may mediate Lar function and/or signaling in muscle tissue.

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Proteomic Profiling of Hsp90 Inhibitors

Cited by 10 publications

References 32 publications

Impact of Heat Shock Protein 90 Inhibition on the Proteomic Profile of Lung Adenocarcinoma as Measured by Two-Dimensional Electrophoresis Coupled with Mass Spectrometry

Impact of Heat Shock Protein 90 Inhibition on the Proteomic Profile of Lung Adenocarcinoma as Measured by Two-Dimensional Electrophoresis Coupled with Mass Spectrometry

In Vitro Glycosylation of Membrane Proteins Using N-Glycosyltransferase

Complex protein interactions mediate Drosophila Lar function in muscle tissue

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