scholarly journals Development and validation of inducible protein degradation and quantitative phosphoproteomics to identify kinase-substrate relationships.

2021 ◽  
Author(s):  
Rufus Hards ◽  
Charles L. Howarth ◽  
Kwame Wiredu ◽  
Ian LaCroix ◽  
Juan Carlos Mercado del Valle ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Hela Cells ◽  
Kinase Activity ◽  
General Strategy ◽  
Chemical Proteomics ◽  
Kinase Substrate ◽  
Protein Substrates ◽  
Degradation Rates ◽  
The Impact ◽  
Quantitative Phosphoproteomics

Phosphorylation signaling is an essential post-translational regulatory mechanism that governs almost all eukaryotic biological processes and is controlled by an interplay between protein kinases and phosphatases. Knowledge of direct substrates of kinases provides evidence of mechanisms that relate activity to biological function. Linking kinases to their protein substrates can be achieved by inhibiting or reducing kinase activity and quantitative comparisons of phosphoproteomes in the presence and absence of kinase activity. Unfortunately, most of the human kinases lack chemical inhibitors with selectivity required to unambiguously assign protein substrates to their respective kinases. Here, we develop and validate a chemical proteomics strategy for linking kinase activities to protein substrates via targeted protein degradation and quantitative phosphoproteomics and apply it to the well-studied, essential mitotic regulator polo-like kinase 1 (Plk1). We leveraged the Tir1/auxin system to engineer HeLa cells with endogenously homozygous auxin-inducible degron (AID)-Plk1). We used HeLa cells and determined the impact of AID-tagging on Plk1 activity, localization, protein interactors, and substrate motifs. Using quantitative proteomics, we show that of over 8,000 proteins quantified, auxin addition is highly selective for degrading AID-Plk1 in mitotic cells. Comparison of phosphoproteome changes in response to chemical Plk1 inhibition to auxin-induced degradation revealed a striking degree of correlation. Finally, we explored basal protein turnover as a potential basis for clonal differences in auxin-induced degradation rates for AID-Plk1 cells. Taken together, our work provides a roadmap for the application of AID technology as a general strategy for the kinome-wide discovery of kinase-substrate relationships.

scholarly journals TTBK2 kinase substrate specificity and the impact of spinocerebellar-ataxia-causing mutations on expression, activity, localization and development

2011 ◽  
Vol 437 (1) ◽  
pp. 157-167 ◽  
Author(s):  
Michale Bouskila ◽  
Noor Esoof ◽  
Laurie Gay ◽  
Emily H. Fang ◽  
Maria Deak ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Protein Expression ◽  
Spinocerebellar Ataxia ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Kinase Domain ◽  
Spinocerebellar Ataxia Type ◽  
Protein Kinase Activity ◽  
Kinase Substrate ◽  
The Impact

Mutations that truncate the C-terminal non-catalytic moiety of TTBK2 (tau tubulin kinase 2) cause the inherited, autosomal dominant, SCA11 (spinocerebellar ataxia type 11) movement disorder. In the present study we first assess the substrate specificity of TTBK2 and demonstrate that it has an unusual preference for a phosphotyrosine residue at the +2 position relative to the phosphorylation site. We elaborate a peptide substrate (TTBKtide, RRKDLHDDEEDEAMSIYpA) that can be employed to quantify TTBK2 kinase activity. Through modelling and mutagenesis we identify a putative phosphate-priming groove within the TTBK2 kinase domain. We demonstrate that SCA11 truncating mutations promote TTBK2 protein expression, suppress kinase activity and lead to enhanced nuclear localization. We generate an SCA11-mutation-carrying knockin mouse and show that this leads to inhibition of endogenous TTBK2 protein kinase activity. Finally, we find that, in homozygosity, the SCA11 mutation causes embryonic lethality at embryonic day 10. These findings provide the first insights into some of the intrinsic properties of TTBK2 and reveal how SCA11-causing mutations affect protein expression, catalytic activity, localization and development. We hope that these findings will be helpful for future investigation of the regulation and function of TTBK2 and its role in SCA11.

Effect of Thrombin on Phosphorylation of Platelet Membrane Proteins

1976 ◽  
Vol 35 (03) ◽  
pp. 635-642 ◽  
Author(s):  
M Steiner
Keyword(s):  
Protein Kinase ◽  
Kinase Activity ◽  
Qualitative Change ◽  
Protein Kinase Activity ◽  
Protein Substrates ◽  
Phosphoprotein Phosphatase ◽  
Progressive Loss ◽  
Platelet Membranes ◽  
Endogenous Protein ◽  
Considerable Loss

SummaryThe effect of thrombin on the phosphorylating activity of platelet membranes was compared to that of trypsin. Preincubation of non-32P phosphorylated platelet membranes with or without either of these two enzymes resulted in a considerable loss of membrane protein kinase activity which was most severe when trypsin was used. Protein kinase activity and endogenous protein acceptors decreased in parallel. 32P-phosphorylated membranes showed a slow but progressive loss of label which was accelerated by trypsin. Thrombin under these conditions prevented the loss of 32P-phosphate. These results are interpreted to indicate a thrombin-induced destruction of a phosphoprotein phosphatase. The protein kinase activity of phosphorylated platelet membranes using endogenous or exogenous protein substrates showed a significant reduction compared to non-phosphorylated membranes suggesting a deactivation of protein kinase by phosphorylation of platelet membranes. Neither thrombin nor trypsin caused a qualitative change in the membrane polypeptides accepting 32P-phosphate but resulted in quantitative alterations of their ability to become phosphorylated.

scholarly journals Inhibition of p38 Mitogen-Activated Protein Kinase Impairs Mayaro Virus Replication in Human Dermal Fibroblasts and HeLa Cells

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1156
Author(s):  
Madelaine Sugasti-Salazar ◽  
Yessica Y. Llamas-González ◽  
Dalkiria Campos ◽  
José González-Santamaría
Keyword(s):  
Protein Expression ◽  
Hela Cells ◽  
Plaque Assay ◽  
Dermal Fibroblasts ◽  
Dependent Manner ◽  
Human Dermal Fibroblasts ◽  
Mitogen Activated Protein ◽  
Mayaro Virus ◽  
The Impact ◽  
Dose Dependent

Mayaro virus (MAYV) hijacks the host’s cell machinery to effectively replicate. The mitogen-activated protein kinases (MAPKs) p38, JNK, and ERK1/2 have emerged as crucial cellular factors implicated in different stages of the viral cycle. However, whether MAYV uses these MAPKs to competently replicate has not yet been determined. The aim of this study was to evaluate the impact of MAPK inhibition on MAYV replication using primary human dermal fibroblasts (HDFs) and HeLa cells. Viral yields in supernatants from MAYV-infected cells treated or untreated with inhibitors SB203580, SP600125, U0126, or Losmapimod were quantified using plaque assay. Additionally, viral protein expression was analyzed using immunoblot and immunofluorescence. Knockdown of p38⍺/p38β isoforms was performed in HDFs using the PROTACs molecule NR-7h. Our data demonstrated that HDFs are highly susceptible to MAYV infection. SB203580, a p38 inhibitor, reduced MAYV replication in a dose-dependent manner in both HDFs and HeLa cells. Additionally, SB203580 significantly decreased viral E1 protein expression. Similarly, knockdown or inhibition of p38⍺/p38β isoforms with NR-7h or Losmapimod, respectively, affected MAYV replication in a dose-dependent manner. Collectively, these findings suggest that p38 could play an important role in MAYV replication and could serve as a therapeutic target to control MAYV infection.

scholarly journals Photocatalytic Degradation of Sulfolane Using a LED-Based Photocatalytic Treatment System

Catalysts ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 624
Author(s):  
Sripriya Dharwadkar ◽  
Linlong Yu ◽  
Gopal Achari
Keyword(s):  
Photocatalytic Degradation ◽  
Oil And Gas ◽  
Light Emitting Diode ◽  
Tap Water ◽  
Aqueous Media ◽  
Ultrapure Water ◽  
Light Emitting ◽  
Degradation Rates ◽  
Reduced Graphene ◽  
The Impact

Sulfolane is an emerging industrial pollutant detected in the environments near many oil and gas plants in North America. So far, numerous advanced oxidation processes have been investigated to treat sulfolane in aqueous media. However, there is only a few papers that discuss the degradation of sulfolane using photocatalysis. In this study, photocatalytic degradation of sulfolane using titanium dioxide (TiO2) and reduced graphene oxide TiO2 composite (RGO-TiO2) in a light-emitting diode (LED) photoreactor was investigated. The impact of different waters (ultrapure water, tap water, and groundwater) and type of irradiation (UVA-LED and mercury lamp) on photocatalytic degradation of sulfolane were also studied. In addition, a reusability test was conducted for the photocatalyst to examine the degradation of sulfolane in three consecutive cycles with new batches of sulfolane-contaminated water. The results show that LED-based photocatalysis was effective in degrading sulfolane in waters even after three photocatalytic cycles. UVA-LEDs displayed more efficient use of photon energy when compared with the mercury lamps as they have a narrow emission spectrum coinciding with the absorption of TiO2. The combination of UVA-LED and TiO2 yielded better performance than UVA-LED and RGO-TiO2 for the degradation of sulfolane. Much lower sulfolane degradation rates were observed in tap water and groundwater than ultrapure water.

scholarly journals High-Throughput Quantitative Assay Technologies for Accelerating the Discovery and Optimization of Targeted Protein Degradation Therapeutics

2021 ◽  
pp. 247255522098504
Author(s):  
Jeffrey R. Simard ◽  
Linda Lee ◽  
Ellen Vieux ◽  
Reina Improgo ◽  
Trang Tieu ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Protein Degradation ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
De Novo ◽  
Rapid Development ◽  
Dependent Manner ◽  
Western Blots ◽  
Advantages And Disadvantages ◽  
Degradation Rates

The aberrant regulation of protein expression and function can drastically alter cellular physiology and lead to numerous pathophysiological conditions such as cancer, inflammatory diseases, and neurodegeneration. The steady-state expression levels of endogenous proteins are controlled by a balance of de novo synthesis rates and degradation rates. Moreover, the levels of activated proteins in signaling cascades can be further modulated by a variety of posttranslational modifications and protein–protein interactions. The field of targeted protein degradation is an emerging area for drug discovery in which small molecules are used to recruit E3 ubiquitin ligases to catalyze the ubiquitination and subsequent degradation of disease-causing target proteins by the proteasome in both a dose- and time-dependent manner. Traditional approaches for quantifying protein level changes in cells, such as Western blots, are typically low throughput with limited quantification, making it hard to drive the rapid development of therapeutics that induce selective, rapid, and sustained protein degradation. In the last decade, a number of techniques and technologies have emerged that have helped to accelerate targeted protein degradation drug discovery efforts, including the use of fluorescent protein fusions and reporter tags, flow cytometry, time-resolved fluorescence energy transfer (TR-FRET), and split luciferase systems. Here we discuss the advantages and disadvantages associated with these technologies and their application to the development and optimization of degraders as therapeutics.

Energy-Saving Methods for Urban Travel and Public Transportation in Smart Cities

2022 ◽  
Author(s):  
Shan Li ◽  
Ying Gao ◽  
Tao Ba ◽  
Wei Zhao
Keyword(s):  
Energy Saving ◽  
Public Transportation ◽  
Traffic Control ◽  
Smart Cities ◽  
Learning Technologies ◽  
General Strategy ◽  
Smart Transportation ◽  
Urban Travel ◽  
Emissions Mitigation ◽  
The Impact

In many countries, energy-saving and emissions mitigation for urban travel and public transportation are important for smart city developments. It is essential to understand the impact of smart transportation (ST) in public transportation in the context of energy savings in smart cities. The general strategy and significant ideas in developing ST for smart cities, focusing on deep learning technologies, simulation experiments, and simultaneous formulation, are in progress. This study hence presents simultaneous transportation monitoring and management frameworks (STMF ). STMF has the potential to be extended to the next generation of smart transportation infrastructure. The proposed framework consists of community signal and community traffic, ST platforms and applications, agent-based traffic control, and transportation expertise augmentation. Experimental outcomes exhibit better quality metrics of the proposed STMF technique in energy saving and emissions mitigation for urban travel and public transportation than other conventional approaches. The deployed system improves the accuracy, consistency, and F-1 measure by 27.50%, 28.81%, and 31.12%. It minimizes the error rate by 75.35%.

scholarly journals Combination of in vivo phage therapy data with in silico model highlights key parameters for treatment efficacy

2021 ◽  
Author(s):  
Raphaelle Delattre ◽  
Jeremy Seurat ◽  
Feyrouz Haddad ◽  
Thu-Thuy Nguyen ◽  
Baptiste Gaborieau ◽  
...  
Keyword(s):  
Phage Therapy ◽  
Biological Properties ◽  
General Strategy ◽  
In Vivo Experiments ◽  
Bacterial Dynamics ◽  
Optimal Dosing ◽  
Dosing Regimens ◽  
The Impact

The clinical (re)development of phage therapy to treat antibiotic resistant infections requires grasping specific biological properties of bacteriophages (phages) as antibacterial. However, identification of optimal dosing regimens is hampered by the poor understanding of phage-bacteria interactions in vivo. Here we developed a general strategy coupling in vitro and in vivo experiments with a mathematical model to characterize the interplay between phage and bacterial dynamics during pneumonia induced by a pathogenic strain of Escherichia coli. The model estimates some key parameters for phage therapeutic efficacy, in particular the impact of dose and route of administration on phage dynamics and the synergism of phage and the innate immune response on the bacterial clearance rate. Simulations predict a low impact of the intrinsic phage characteristics in agreement with the current semi-empirical choices of phages for compassionate treatments. Model-based approaches will foster the deployment of future phage therapy clinical trials.

scholarly journals The activities of LRRK2 and GCase are positively correlated in clinical biospecimens and experimental models of Parkinson’s disease

2021 ◽  
Author(s):  
Maria Kedariti ◽  
Emanuele Frattini ◽  
Pascale Baden ◽  
Susanna Cogo ◽  
Laura Civiero ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Kinase Activity ◽  
Experimental Models ◽  
Cellular Functions ◽  
Gene Encoding ◽  
Cellular Models ◽  
Lrrk2 Kinase ◽  
The Impact ◽  
Lrrk2 Kinase Activity

AbstractLRRK2 is a kinase involved in different cellular functions, including autophagy, endolysosomal pathways and vesicle trafficking. Mutations in LRRK2 cause autosomal dominant forms of Parkinson’s disease (PD). Heterozygous mutations in GBA1, the gene encoding the lysosomal enzyme glucocerebrosidase (GCase), are the most common genetic risk factors for PD. Moreover, GCase function is altered in idiopathic PD and in other genetic forms of the disease. Recent work suggests that LRRK2 kinase activity can regulate GCase function. However, both a positive and a negative correlation have been described. To gain insights into the impact of LRRK2 on GCase, we investigated GCase levels and activity in LRRK2 G2019S knockin mice, in clinical biospecimens from PD patients carrying this mutation and in patient-derived cellular models. In these models we found a positive correlation between the activities of LRRK2 and GCase, which was further confirmed in cell lines with genetic and pharmacological manipulation of LRRK2 kinase activity. Overall, our study indicates that LRRK2 kinase activity affects both the levels and the catalytic activity of GCase.

Analysis of cell signaling during floral abscission in arabidopsis

2017 ◽  
Author(s):  
◽  
Isaiah Taylor
Keyword(s):  
Protein Degradation ◽  
Future Research ◽  
Loss Of Function ◽  
Kinase Gene ◽  
University Of Missouri ◽  
Gene Map ◽  
Map Kinase Phosphatase ◽  
Floral Abscission ◽  
The University ◽  
The Impact

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] The shedding of plant organs is known as abscission. Floral abscission in Arabidopsis is regulated by two related receptor[negation symbol]-like protein kinases (RLKs), HAESA and HAESA[negation symbol-like 2 (HAE/HSL2). Double mutants of HAE/HSL2 are completely defective in abscission and retain sepals, petals, and stamen indefinitely. We have utilized genetic suppressor screens of hae hsl2 mutant to identify additional regulatory mechanisms of floral abscission. We have uncovered a series of gain-of-function alleles of the receptor-like protein kinase gene SERK1, as well as loss of function alleles of the gene MAP-KINASE-PHOSPHATASE-1/MKP1. We further show that mutation of two components of the endoplasmic reticulum-associated protein degradation system can suppress a weak hae hsl2 mutant, suggesting that the weak hae hsl2 mutant receptor proteins undergo ER-associated protein degradation. We further perform a number of experiments to examine the impact of phosphorylation on the activity of HAE. These results provide a number of important mechanistic details to our understanding of floral abscission, and suggest many lines of inquiry for future research.

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