Regulation of microtubule dynamics by DIAPH3 influences amoeboid tumor cell mechanics and sensitivity to taxanes

Morley, Samantha; You, Sungyong; Pollan, Sara; Choi, Ji-Young; Zhou, Bo; Hager, Martin H.; Steadman, Kenneth; Spinelli, Cristiana; Rajendran, Kavitha; Gertych, Arkadiusz; Kim, Jayoung; Adam, Rosalyn M.; Yang, Wei; Krishnan, Ramaswamy; Knudsen, Beatrice S.; Vizio, Dolores Di; Freeman, Michael R.

doi:10.1038/srep12136

Cited by 42 publications

(41 citation statements)

References 59 publications

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“…18 The resulting peptides were separated by EASY-Spray columns and analyzed by an LTQ Velos, an LTQ Orbitrap Elite, or an Orbitrap Fusion Lumos mass spectrometer essentially as described. 19,20 Database searching analyses were conducted using MaxQuant 21 and Andromeda 22 . Data analysis was performed using Perseus 23 and R (www.R-project.org).…”

Section: Methodsmentioning

confidence: 99%

Low-background Acyl-biotinyl Exchange Largely Eliminates the Co-isolation of Non-S-acylated Proteins and Enables Deep S-acylproteomic Analysis

Zhou

Wang

Yan

et al. 2019

Preprint

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Protein S-acylation (also called palmitoylation) is a common post-translational modification whose deregulation plays a key role in the pathogenesis of many diseases. Acyl-biotinyl exchange (ABE), a widely used method for the enrichment of S-acylated proteins, has the potential of capturing the entire S-acylproteome in any types of biological samples. Here, we showed that current ABE methods suffer from high background arising from the co-isolation of non-S-acylated proteins. The background can be substantially reduced by an additional blockage of residual free cysteine residues with 2,2'-dithiodipyridine prior to biotin-HPDP reaction. Coupling the low-background ABE (LB-ABE) method with label-free quantitative proteomics, 2,895 highconfidence candidate S-acylated proteins (including 1,591 known S-acylated proteins) were identified from human prostate cancer LNCaP cells, representing so-far the largest S-acylproteome dataset identified in a single study. Immunoblotting analysis confirmed the S-acylation of five known and five novel prostate cancer-related S-acylated proteins in LNCaP cells and suggested that their Sacylation levels were about 0.6-1.8%. In summary, the LB-ABE method largely eliminates the co-isolation of non-S-acylated proteins and enables deep S-acylproteomic analysis. It is expected to facilitate much more comprehensive and accurate quantification of S-acylproteomes than previous ABE methods.

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Section: Methodsmentioning

confidence: 99%

Low-background Acyl-biotinyl Exchange Largely Eliminates the Co-isolation of Non-S-acylated Proteins and Enables Deep S-acylproteomic Analysis

Zhou

Wang

Yan

et al. 2019

Preprint

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“…S6A and Dataset S8). Several of these genes were previously associated with PCa, such as AOX1, ID4, RXRA, BUB1B, CITED1, CDC25C, SIX1, MCM10, and DIAPH3 (39,40,50,(55)(56)(57)(58)(59)(60) and correlated with poor prognosis (LENG9 and GLB1L2) (61).…”

Section: Modeling Of Pca With Organoids Identifies a Gene Signature Omentioning

confidence: 98%

TIP5 primes prostate luminal cells for the oncogenic transformation mediated by PTEN -loss

Pietrzak

Kuzyakiv

Simon

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Prostate cancer (PCa) is the second leading cause of cancer death in men. Its clinical and molecular heterogeneities and the lack of in vitro models outline the complexity of PCa in the clinical and research settings. We established an in vitro mouse PCa model based on organoid technology that takes into account the cell of origin and the order of events. Primary PCa with deletion of the tumor suppressor gene PTEN (PTEN-del) can be modeled through Pten-down-regulation in mouse organoids. We used this system to elucidate the contribution of TIP5 in PCa initiation, a chromatin regulator that is implicated in aggressive PCa. High TIP5 expression correlates with primary PTEN-del PCa and this combination strongly associates with reduced prostate-specific antigen (PSA) recurrence-free survival. TIP5 is critical for the initiation of PCa of luminal origin mediated by Pten-loss whereas it is dispensable once Pten-loss mediated transformation is established. Cross-species analyses revealed a PTEN gene signature that identified a group of aggressive primary PCas characterized by PTEN-del, high-TIP5 expression, and a TIP5-regulated gene expression profile. The results highlight the modeling of PCa with organoids as a powerful tool to elucidate the role of genetic alterations found in recent studies in their time orders and cells of origin, thereby providing further optimization for tumor stratification to improve the clinical management of PCa.

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“…Tryptic peptides were then recovered, dried down in a SpeedVac concentrator (Thermo Scientific), and redissolved in 0.2% formic acid (Sigma) up to a concentration of 0.15 µg/mL. Label-free proteomic analysis was performed using an EASY-nLC 1000 connected to an LTQ Orbitrap Elite hybrid mass spectrometer essentially as we previously described 29,30 . Briefly, 7 μL of peptide solution was loaded onto a 2-cm trap column (75 μm × 2 cm, C18) and separated on a 50-cm EASY-Spray analytical column (PepMap RSLC C18, 2 μm, 100Å, 50 μm × 15 cm) heated to 55°C, using a 2-h gradient consisting of 2-40% B in 150 min, 40-100% B in 20 min, and 100% B in 10 min at the flow rate of 150 nL/min.…”

Section: Lc-ms/ms Analysis and Data Processingmentioning

confidence: 99%

Comprehensive palmitoyl-proteomic analysis identifies distinct protein signatures for large and small cancer-derived extracellular vesicles

Mariscal

Vagner

Kim

et al. 2019

Preprint

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Extracellular vesicles (EVs) are membrane-enclosed particles that play an important role in cancer progression and have emerged as a promising source of circulating biomarkers. Protein S-acylation, also known as palmitoylation, has been proposed as a post-translational mechanism that modulates the dynamics of EV biogenesis and protein cargo sorting. However, technical challenges have limited large-scale profiling of the whole palmitoyl-proteins of EVs. We successfully employed a novel approach that combines low-background acyl-biotinyl exchange (LB-ABE) with label-free proteomics to analyze the palmitoyl proteome of large EVs (L-EVs) and small EVs (S-EVs) from prostate cancer cells. Here we report the first palmitoyl-protein signature of EVs, and demonstrate that L-and S-EVs harbor proteins associated with distinct biological processes and subcellular origin. We identified STEAP1, STEAP2, and ABBC4 as prostate cancer-specific palmitoyl proteins enriched in both EV populations in comparison with the originating cell lines. Importantly, the presence of the above proteins in EVs was significantly reduced upon inhibition of palmitoylation in the producing cells. These results suggest that palmitoylation may be involved in the differential sorting of proteins to distinct EV populations and allow for better detection of disease biomarkers.

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Regulation of microtubule dynamics by DIAPH3 influences amoeboid tumor cell mechanics and sensitivity to taxanes

Cited by 42 publications

References 59 publications

Low-background Acyl-biotinyl Exchange Largely Eliminates the Co-isolation of Non-S-acylated Proteins and Enables Deep S-acylproteomic Analysis

Low-background Acyl-biotinyl Exchange Largely Eliminates the Co-isolation of Non-S-acylated Proteins and Enables Deep S-acylproteomic Analysis

TIP5 primes prostate luminal cells for the oncogenic transformation mediated by PTEN -loss

Comprehensive palmitoyl-proteomic analysis identifies distinct protein signatures for large and small cancer-derived extracellular vesicles

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