scholarly journals Identification of phosphosites that alter protein thermal stability

Author(s):  
Ian R. Smith ◽  
Kyle N. Hess ◽  
Anna A. Bakhtina ◽  
Anthony S. Valente ◽  
Ricard A. Rodríguez-Mias ◽  
...  

ABSTRACTProteomics has enabled the cataloguing of 100,000s of protein phosphorylation sites 1, however we lack methods to systematically annotate their function. Phosphorylation has numerous biological functions, yet biochemically all involve changes in protein structure and interactions. These biochemical changes can be recapitulated by measuring the difference in stability between the protein and the phosphoprotein. Building on recent work, we present a method to infer phosphosite functionality by reliably measuring such differences at the proteomic scale.

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Niraj Thapa ◽  
Meenal Chaudhari ◽  
Anthony A. Iannetta ◽  
Clarence White ◽  
Kaushik Roy ◽  
...  

AbstractProtein phosphorylation, which is one of the most important post-translational modifications (PTMs), is involved in regulating myriad cellular processes. Herein, we present a novel deep learning based approach for organism-specific protein phosphorylation site prediction in Chlamydomonas reinhardtii, a model algal phototroph. An ensemble model combining convolutional neural networks and long short-term memory (LSTM) achieves the best performance in predicting phosphorylation sites in C. reinhardtii. Deemed Chlamy-EnPhosSite, the measured best AUC and MCC are 0.90 and 0.64 respectively for a combined dataset of serine (S) and threonine (T) in independent testing higher than those measures for other predictors. When applied to the entire C. reinhardtii proteome (totaling 1,809,304 S and T sites), Chlamy-EnPhosSite yielded 499,411 phosphorylated sites with a cut-off value of 0.5 and 237,949 phosphorylated sites with a cut-off value of 0.7. These predictions were compared to an experimental dataset of phosphosites identified by liquid chromatography-tandem mass spectrometry (LC–MS/MS) in a blinded study and approximately 89.69% of 2,663 C. reinhardtii S and T phosphorylation sites were successfully predicted by Chlamy-EnPhosSite at a probability cut-off of 0.5 and 76.83% of sites were successfully identified at a more stringent 0.7 cut-off. Interestingly, Chlamy-EnPhosSite also successfully predicted experimentally confirmed phosphorylation sites in a protein sequence (e.g., RPS6 S245) which did not appear in the training dataset, highlighting prediction accuracy and the power of leveraging predictions to identify biologically relevant PTM sites. These results demonstrate that our method represents a robust and complementary technique for high-throughput phosphorylation site prediction in C. reinhardtii. It has potential to serve as a useful tool to the community. Chlamy-EnPhosSite will contribute to the understanding of how protein phosphorylation influences various biological processes in this important model microalga.


2021 ◽  
Author(s):  
Ian R. Smith ◽  
Kyle N. Hess ◽  
Anna A. Bakhtina ◽  
Anthony S. Valente ◽  
Ricard A. Rodríguez-Mias ◽  
...  

Epigenomes ◽  
2021 ◽  
Vol 5 (1) ◽  
pp. 5 ◽  
Author(s):  
Ryohei Wada ◽  
Wataru Yoshida

N6-methyladenine modification (m6dA) has recently been identified in eukaryote genomic DNA. The methylation destabilizes the duplex structure when the adenine forms a Watson–Crick base pair, whereas the methylation on a terminal unpaired adenine stabilizes the duplex structure by increasing the stacking interaction. In this study, the effects of m6dA modification on the thermal stability of four distinct telomeric G-quadruplex (G4) structures were investigated. The m6dA-modified telomeric oligonucleotide d[AGGG(TTAGGG)3] that forms a basket-type G4 in Na+, d[(TTAGGG)4TT] that forms a hybrid-type G4 in K+ (Form-2), d[AAAGGG(TTAGGG)3AA] that forms a hybrid-type G4 in K+ (Form-1), and d[GGG(TTAGGG)3T] that forms a basket-type G4 with two G-tetrads in K+ (Form-3) were analyzed. Circular dichroism melting analysis demonstrated that (1) A7- and A19-methylation destabilized the basket-type G4 structure that formed in Na+, whereas A13-methylation stabilized the structure; (2) A15-methylation stabilized the Form-2 G4 structure; (3) A15- and A21-methylations stabilized the Form-1 G4 structure; and (4) A12-methylation stabilized the Form-3 G4 structure. These results suggest that m6dA modifications may affect the thermal stability of human telomeric G4 structures in regulating the biological functions.


2009 ◽  
Vol 8 (7) ◽  
pp. 922-932 ◽  
Author(s):  
Jens Boesger ◽  
Volker Wagner ◽  
Wolfram Weisheit ◽  
Maria Mittag

ABSTRACT Cilia and flagella are cell organelles that are highly conserved throughout evolution. For many years, the green biflagellate alga Chlamydomonas reinhardtii has served as a model for examination of the structure and function of its flagella, which are similar to certain mammalian cilia. Proteome analysis revealed the presence of several kinases and protein phosphatases in these organelles. Reversible protein phosphorylation can control ciliary beating, motility, signaling, length, and assembly. Despite the importance of this posttranslational modification, the identities of many ciliary phosphoproteins and knowledge about their in vivo phosphorylation sites are still missing. Here we used immobilized metal affinity chromatography to enrich phosphopeptides from purified flagella and analyzed them by mass spectrometry. One hundred forty-one phosphorylated peptides were identified, belonging to 32 flagellar proteins. Thereby, 126 in vivo phosphorylation sites were determined. The flagellar phosphoproteome includes different structural and motor proteins, kinases, proteins with protein interaction domains, and many proteins whose functions are still unknown. In several cases, a dynamic phosphorylation pattern and clustering of phosphorylation sites were found, indicating a complex physiological status and specific control by reversible protein phosphorylation in the flagellum.


Blood ◽  
1987 ◽  
Vol 70 (2) ◽  
pp. 511-515
Author(s):  
DJ Carty ◽  
DL Freas ◽  
AR Gear

We developed a general quenched-flow approach to study platelet function as early as 0.3 seconds after stimulation. Phosphorylation of 20- and 47-kiloDalton (kD) proteins was analyzed during the first 5 seconds of platelet response to ADP from 0.5 to 10.0 mumol/L and compared with the progress of aggregation. The onset time for aggregation and phosphorylation of both proteins was less than 1 second; 20-K phosphorylation was increased greater than 200% and 47-K phosphorylation was increased 50%. The ADP sensitivity of 20-K phosphorylation was greater than that of 47-K phosphorylation (P less than .025), and of that of aggregation (P less than .01), with Ka values of 0.7, 1.0, and 1.2 mumol/L of ADP, respectively. The cyclooxygenase inhibitor indomethacin had no effect on aggregation, but inhibited both phosphorylations. Its inhibition of 20-K phosphorylation was greater than that of 47-K phosphorylation. Platelet activation by ADP thus induced biochemical changes well before 1 second. The quenched- flow approach may help to reveal relationships between phospholipase activation, calcium fluxes, and protein phosphorylation during these early periods of platelet activation.


Konturen ◽  
2015 ◽  
Vol 8 ◽  
pp. 8
Author(s):  
Jonathan Monroe

Opening questions about “things” onto the bureaucratically-maintained, compartmentalized discursive, disciplinary claims of “philosophy,” “theory,” and “poetry,” “Urgent Matter” explores these three terms in relation to one another through attention to recent work by Giorgio Agamben, Jacques Rancière, the German-American poet Rosmarie Waldrop, and the German poet Ulf Stolterfoht, whose fachsprachen. Gedichte. I-IX (Lingos I-IX. Poems) Waldrop rendered into English in an award-winning translation. The difference between the "things" called "poetry" and "philosophy," as now institutionalized within the academy, is not epistemological, ontological, ahistorical, but a matter of linguistic domains, of so-called concrete "images" as the policed domain of the former and of "abstraction" as the policed domain of the latter. Challenging the binary logics that dominate language use in diverse discursive/disciplinary cultures, Waldrop’s linguistically self-referential, appositional procedures develop ways to use language that are neither linear, nor so much without direction, as multi-directional, offering complexes of adjacency, of asides, of digression, of errancy, of being “alongside,” in lieu of being “opposed to,” that constitute at once a poetics, an aesthetics, an ethics, and a politics. Elaborating a complementary understanding of poetry as “the most philosophic of all writing,” a medium of being “contemporary,” Waldrop and Stolterfoht question poetry’s purposes as one kind of language apparatus among others in the general economy. Whatever poetry might be, it aspires to be in their hands not a thing in itself but a form of self-questioning, of all discourses, all disciplines, that “thing” that binds “poetry” and “philosophy” together, as urgent matter, in continuing.


2021 ◽  
Vol 35 (S1) ◽  
Author(s):  
Jacob Canepa ◽  
Julie Torgerson ◽  
Da Eun Kim ◽  
Elizabeth Lindahl ◽  
Rei Takahashi ◽  
...  

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