Diverse Hotspot Thermal Profiling Methods Detect Phosphorylation-Dependent Changes in Protein Stability

Hotspot thermal profiling (HTP) methods utilize modified-peptide level information in order to interrogate proteoform-specific stability inside of live cells. The first demonstration of HTP involved the integration of phosphopeptide enrichment into a TMT-based, single-LC separation thermal profiling workflow1. Here we present a new "label-fractionate-enrich" (LFE)-HTP method that involves high-pH reverse phase fractionation of TMT-labeled peptides prior to phosphopeptide enrichment, followed by peptide detection and quantitation using multi-notch LC-MS3. We find that LFE-HTP, while more resource intensive, improves the depth and precision of (phospho)proteoform coverage relative to the initial published HTP workflow. The fraction of detected phosphorylation sites that are significantly perturbed in this new dataset are consistent with those seen in our previous study, as well as those published by others, when compared head-to-head with the same analysis pipelines. Likewise, many hotspot phosphorylation sites identified in our paper are consistently reproduced by LFE-HTP as well as other modified HTP methods. The LFE-HTP dataset contains many novel hotspot phosphorylation sites that regulate the stability of diverse proteins, including phosphosites in the central glycolytic enzyme Aldolase A that are associated with monomer-to-oligomer formation, enzymatic activity and metabolic regulation in cancer cells. Our comparative analyses confirm that several variants of the HTP method can track modified proteoforms in live cells to detect and prioritize PTM-dependent changes in protein stability that may be associated with function.

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Applicability of Instability Index for In vitro Protein Stability Prediction

Protein and Peptide Letters ◽

10.2174/0929866526666190228144219 ◽

2019 ◽

Vol 26 (5) ◽

pp. 339-347 ◽

Cited By ~ 4

Author(s):

Dilani G. Gamage ◽

Ajith Gunaratne ◽

Gopal R. Periyannan ◽

Timothy G. Russell

Keyword(s):

Protein Stability ◽

Caulobacter Crescentus ◽

Effect Of Temperature ◽

Instability Index ◽

Dipeptide Composition ◽

Experimental Conditions ◽

The Stability ◽

Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

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PAROXYSMAL NOCTURNAL HEMOGLOBINURIA IN CHILDHOOD AND ADOLESCENCE

PEDIATRICS ◽

10.1542/peds.39.5.675 ◽

1967 ◽

Vol 39 (5) ◽

pp. 675-688

Author(s):

Denis R. Miller ◽

Robert L. Baehner ◽

Louis K. Diamond

Keyword(s):

Fetal Hemoglobin ◽

Normal Activity ◽

Glycolytic Enzyme ◽

Clinical Severity ◽

Childhood And Adolescence ◽

Red Cell ◽

Red Cell Membrane ◽

Severity Of The Disease ◽

Increased Activity ◽

Aldolase A

Two cases of PNH in adolescence and childhood are reported. The first presented at age 7½ years with aplastic anemia and improved after splenectomy performed at age 14. The second, a 15-year-old girl, presented with a Coombs-positive hemolytic anemia and has had a course complicated by multiple peripheral thromboses. The clinical and laboratory manifestations, complications, and certain therapeutic aspects of PNH are discussed. Anticoagulant therapy appears indicated in the presence of multiple thrombotic episodes. Erythrocyte metabolic studies revealed normal glycolysis, ATP stability, and GSH content in the cells of a child with a normal reticulocyte count. Mild elevations of glycolysis, noted in the child with a reticulocytosis, was ascribed to a younger mean red cell population since further elevations found in the "top" reticulocyte-rich layer after centrifugation. Heparin, the anticoagulant used in these studies, had no adverse effect on glycolysis but did inhibit hemolysis and minimize ATP instability when compared to cells suspended in defibrinated serum. Erythrocytes fractionated by centrifugation revealed increased glycolytic enzyme activities of hexokinase, G3PD, PGK, TPI, PK, LDH, G6PD, and 6PGD in the reticulocyte-rich layer. Normal, rather than increased activity of aldolase, a membrane enzyme, may reflect damage to the red cell membrane. PFK, known to be decreased in the erythrocyte of neonates, showed normal activity, but it was lowest in the reticulocyte-rich layer. Fetal hemoglobin was elevated in this layer. AChE deficiency and increased suceptibility to hydrogen perioxide and acid hemolysis confirmed previous reports and were most marked in the young cell layer. The level of increased glycolytic rates and enzyme activity, AChE deficiency, acid hemolysis and peroxide hemolysis were related to the clinical severity of the disease.

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A glycine-specific N-degron pathway mediates the quality control of protein N-myristoylation

Science ◽

10.1126/science.aaw4912 ◽

2019 ◽

Vol 365 (6448) ◽

pp. eaaw4912 ◽

Cited By ~ 27

Author(s):

Richard T. Timms ◽

Zhiqian Zhang ◽

David Y. Rhee ◽

J. Wade Harper ◽

Itay Koren ◽

...

Keyword(s):

Quality Control ◽

Protein Stability ◽

Protein Degradation ◽

E3 Ligase ◽

Cleavage Sites ◽

Caspase Cleavage ◽

E3 Ligases ◽

Ring E3 Ligase ◽

The Stability ◽

Ring E3

The N-terminal residue influences protein stability through N-degron pathways. We used stability profiling of the human N-terminome to uncover multiple additional features of N-degron pathways. In addition to uncovering extended specificities of UBR E3 ligases, we characterized two related Cullin-RING E3 ligase complexes, Cul2ZYG11B and Cul2ZER1, that act redundantly to target N-terminal glycine. N-terminal glycine degrons are depleted at native N-termini but strongly enriched at caspase cleavage sites, suggesting roles for the substrate adaptors ZYG11B and ZER1 in protein degradation during apoptosis. Furthermore, ZYG11B and ZER1 were found to participate in the quality control of N-myristoylated proteins, in which N-terminal glycine degrons are conditionally exposed after a failure of N-myristoylation. Thus, an additional N-degron pathway specific for glycine regulates the stability of metazoan proteomes.

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Engineering a bioluminescent indicator for cyclic AMP-dependent protein kinase

Biochemical Journal ◽

10.1042/bj2790727 ◽

1991 ◽

Vol 279 (3) ◽

pp. 727-732 ◽

Cited By ~ 20

Author(s):

G B Sala-Newby ◽

A K Campbell

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Specific Activity ◽

Phosphorylation Site ◽

Live Cells ◽

Phosphorylation Sites ◽

Dependent Protein Kinase ◽

Ph Optimum ◽

Dependent Protein

cDNA coding for the luciferase in the firefly Photinus pyralis was amplified in vitro to generate cyclic AMP-dependent protein kinase phosphorylation sites. The DNA was transcribed and translated to generate light-emitting protein. A valine at position 217 was mutated to arginine to generate a site RRFS and the heptapeptide kemptide, the phosphorylation site of the porcine pyruvate kinase, was added at the N- or C-terminus of the luciferase. The proteins carrying phosphorylation sites were characterized for their specific activity, pI, effect of pH on the colour of the light emitted and effect of the catalytic subunit of protein kinase A in the presence of ATP. Only one of the recombinant proteins (RRFS) was significantly different from wild-type luciferase. The RRFS mutant had a lower specific activity, lower pH optimum, emitted greener light at low pH and when phosphorylated it decreased its activity by up to 80%. This latter effect was reversed by phosphatase. This recombinant protein is a good candidate to measure for the first time cyclic AMP-dependent phosphorylation in live cells.

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Cellular ATP Levels Determine the Stability of a Nucleotide Kinase

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.790304 ◽

2021 ◽

Vol 8 ◽

Author(s):

Oliver Brylski ◽

Puja Shrestha ◽

Patricia Gnutt ◽

David Gnutt ◽

Jonathan Wolf Mueller ◽

...

Keyword(s):

Protein Stability ◽

Bound States ◽

Atp Depletion ◽

Aps Kinase ◽

Cellular Processes ◽

Atp Levels ◽

Stability Change ◽

Cell Stability ◽

The Stability ◽

Nucleotide Kinase

The energy currency of the cell ATP, is used by kinases to drive key cellular processes. However, the connection of cellular ATP abundance and protein stability is still under investigation. Using Fast Relaxation Imaging paired with alanine scanning and ATP depletion experiments, we study the nucleotide kinase (APSK) domain of 3′-phosphoadenosine-5′-phosphosulfate (PAPS) synthase, a marginally stable protein. Here, we show that the in-cell stability of the APSK is determined by ligand binding and directly connected to cellular ATP levels. The observed protein stability change for different ligand-bound states or under ATP-depleted conditions ranges from ΔGf0 = -10.7 to +13.8 kJ/mol, which is remarkable since it exceeds changes measured previously, for example upon osmotic pressure, cellular stress or differentiation. The results have implications for protein stability during the catalytic cycle of APS kinase and suggest that the cellular ATP level functions as a global regulator of kinase activity.

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Poly(catecholamine) Coating and Biofunctionalization of CsPbBr3 Microcrystals

10.26434/chemrxiv.14049710.v2 ◽

2021 ◽

Author(s):

Sangyeon Cho ◽

Seok-Hyun Yun

Keyword(s):

Lipid Bilayers ◽

Environmental Stability ◽

Live Cells ◽

Functional Coatings ◽

Common Strategy ◽

Novel Method ◽

Halide Perovskites ◽

The Stability ◽

New Applications

Lead halide perovskites (LHP) microcrystals are promising materials for various optoelectronic applications. Surface coating on particles is a common strategy to improve their functionality and environmental stability, but LHP is not amenable to most coating chemistries because of its intrinsic weakness against polar solvents. Here, we describe a novel method of synthesizing LHP microcrystals in a super-saturated polar solvent using sonochemistry and applying various functional coatings on individual microcrystals in situ. We synthesize cesium lead bromine perovskite (CsPbBr3) microparticles capped with organic poly-norepinephrine (pNE) layers. The catechol group of pNE coordinates to bromine-deficient lead atoms, forming a defect-passivating and diffusion-blocking shell. The pNE layer enhances the stability of CsPbBr3 in water by 2,000-folds, enabling bright luminescence and lasing from single microcrystals in water. Furthermore, the pNE shell permits biofunctionalization with proteins, small molecules, and lipid bilayers. Luminescence from CsPbBr3 microcrystals is sustained in water over 1 hour and observed in live cells. The functionalization method may enable new applications of LHP particles in water-rich environments.

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Distinct phosphorylation sites in a prototypical GPCR differently orchestrate β-arrestin interaction, trafficking, and signaling

Science Advances ◽

10.1126/sciadv.abb8368 ◽

2020 ◽

Vol 6 (37) ◽

pp. eabb8368 ◽

Cited By ~ 1

Author(s):

Hemlata Dwivedi-Agnihotri ◽

Madhu Chaturvedi ◽

Mithu Baidya ◽

Tomasz Maciej Stepniewski ◽

Shubhi Pandey ◽

...

Keyword(s):

G Protein ◽

Phosphorylation Site ◽

Dynamics Simulation ◽

Phosphorylation Sites ◽

Functional Responses ◽

Biased Signaling ◽

The Stability ◽

G Protein Coupled ◽

Structural Aspects

Agonist-induced phosphorylation of G protein–coupled receptors (GPCRs) is a key determinant for their interaction with β-arrestins (βarrs) and subsequent functional responses. Therefore, it is important to decipher the contribution and interplay of different receptor phosphorylation sites in governing βarr interaction and functional outcomes. Here, we find that several phosphorylation sites in the human vasopressin receptor (V2R), positioned either individually or in clusters, differentially contribute to βarr recruitment, trafficking, and ERK1/2 activation. Even a single phosphorylation site in V2R, suitably positioned to cross-talk with a key residue in βarrs, has a decisive contribution in βarr recruitment, and its mutation results in strong G-protein bias. Molecular dynamics simulation provides mechanistic insights into the pivotal role of this key phosphorylation site in governing the stability of βarr interaction and regulating the interdomain rotation in βarrs. Our findings uncover important structural aspects to better understand the framework of GPCR-βarr interaction and biased signaling.

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An Intelligent Multi-View Active Learning Method Based on a Double-Branch Network

Entropy ◽

10.3390/e22080901 ◽

2020 ◽

Vol 22 (8) ◽

pp. 901

Author(s):

Fucong Liu ◽

Tongzhou Zhang ◽

Caixia Zheng ◽

Yuanyuan Cheng ◽

Xiaoli Liu ◽

...

Keyword(s):

Artificial Intelligence ◽

Deep Learning ◽

Active Learning ◽

Sample Selection ◽

Training Dataset ◽

Learning Method ◽

Active Learning Method ◽

Level Information ◽

The Stability ◽

Different Characteristics

Artificial intelligence is one of the most popular topics in computer science. Convolutional neural network (CNN), which is an important artificial intelligence deep learning model, has been widely used in many fields. However, training a CNN requires a large amount of labeled data to achieve a good performance but labeling data is a time-consuming and laborious work. Since active learning can effectively reduce the labeling effort, we propose a new intelligent active learning method for deep learning, which is called multi-view active learning based on double-branch network (MALDB). Different from most existing active learning methods, our proposed MALDB first integrates two Bayesian convolutional neural networks (BCNNs) with different structures as two branches of a classifier to learn the effective features for each sample. Then, MALDB performs data analysis on unlabeled dataset and queries the useful unlabeled samples based on different characteristics of two branches to iteratively expand the training dataset and improve the performance of classifier. Finally, MALDB combines multiple level information from multiple hidden layers of BCNNs to further improve the stability of sample selection. The experiments are conducted on five extensively used datasets, Fashion-MNIST, Cifar-10, SVHN, Scene-15 and UIUC-Sports, the experimental results demonstrate the validity of our proposed MALDB.

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VBP1 modulates Wnt/β-catenin signaling by mediating the stability of the transcription factors TCF/LEFs

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.015282 ◽

2020 ◽

Vol 295 (49) ◽

pp. 16826-16839

Author(s):

Haifeng Zhang ◽

Xiaozhi Rong ◽

Caixia Wang ◽

Yunzhang Liu ◽

Ling Lu ◽

...

Keyword(s):

Transcription Factors ◽

Protein Stability ◽

Signaling Pathway ◽

Cultured Cells ◽

Critical Role ◽

Family Members ◽

Von Hippel Lindau ◽

T Cell Factor ◽

Protein Levels ◽

The Stability

The Wnt/β-catenin pathway is one of the major pathways that regulates embryonic development, adult homeostasis, and stem cell self-renewal. In this pathway, transcription factors T-cell factor and lymphoid enhancer factor (TCF/LEF) serve as a key switch to repress or activate Wnt target gene transcription by recruiting repressor molecules or interacting with the β-catenin effector, respectively. It has become evident that the protein stability of the TCF/LEF family members may play a critical role in controlling the activity of the Wnt/β-catenin signaling pathway. However, factors that regulate the stability of TCF/LEFs remain largely unknown. Here, we report that pVHL binding protein 1 (VBP1) regulates the Wnt/β-catenin signaling pathway by controlling the stability of TCF/LEFs. Surprisingly, we found that either overexpression or knockdown of VBP1 decreased Wnt/β-catenin signaling activity in both cultured cells and zebrafish embryos. Mechanistically, VBP1 directly binds to all four TCF/LEF family members and von Hippel-Lindau tumor-suppressor protein (pVHL). Either overexpression or knockdown of VBP1 increases the association between TCF/LEFs and pVHL and then decreases the protein levels of TCF/LEFs via proteasomal degradation. Together, our results provide mechanistic insights into the roles of VBP1 in controlling TCF/LEFs protein stability and regulating Wnt/β-catenin signaling pathway activity.

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Assembly and Dynamics of the Bacterial Flagellum

Annual Review of Microbiology ◽

10.1146/annurev-micro-090816-093411 ◽

2020 ◽

Vol 74 (1) ◽

pp. 181-200 ◽

Cited By ~ 1

Author(s):

Judith P. Armitage ◽

Richard M. Berry

Keyword(s):

Single Molecule ◽

Protein Complexes ◽

Complex Structure ◽

Live Cells ◽

Flagellar Motor ◽

Interacting Protein ◽

Bacterial Flagellum ◽

Bacterial Flagellar Motor ◽

The Stability

The bacterial flagellar motor is the most complex structure in the bacterial cell, driving the ion-driven rotation of the helical flagellum. The ordered expression of the regulon and the assembly of the series of interacting protein rings, spanning the inner and outer membranes to form the ∼45–50-nm protein complex, have made investigation of the structure and mechanism a major challenge since its recognition as a rotating nanomachine about 40 years ago. Painstaking molecular genetics, biochemistry, and electron microscopy revealed a tiny electric motor spinning in the bacterial membrane. Over the last decade, new single-molecule and in vivo biophysical methods have allowed investigation of the stability of this and other large protein complexes, working in their natural environment inside live cells. This has revealed that in the bacterial flagellar motor, protein molecules in both the rotor and stator exchange with freely circulating pools of spares on a timescale of minutes, even while motors are continuously rotating. This constant exchange has allowed the evolution of modified components allowing bacteria to keep swimming as the viscosity or the ion composition of the outside environment changes.

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