bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantifying Protein Dephosphoylation as a Function of Mechanical Sample Disruption Techniques Towards an Optimized Sample Prep Protocol for Phosphoproteomics
Authors: Garrett Shari , Atwood James , Easparro Brandon 
Published: November 2023

Phosphorylation is one of the most commonly studied post translational modifications. Phosphorylation plays a critical role in many cellular processes and is extensively seen in eukaryotic cells. Quantitative phosphoproteomic studies aim to measure the degree of protein phosphorylation as a function of biological state. These studies are routinely performed using tissues and cells. In all cases the proteins must be first liberated from the target sample. Especially for tougher tissue types, mechanical methods of sample disruption are routinely employed; including bead milling, sonication or rotor‐stator homogenization.However, with mechanical sample disruption, there is always a concern that excessive processing will generate heat or shear forces that may dephosphorylate the target proteins. Maintaining the native state of phosphoproteins is imperative when detecting the removal or addition of a phosphate moiety through a quantitative phosphoproteomic study.In this study, we quantify protein dephosphorylation as a function of mechanical disruption methods, processing time, and power to develop an optimized method that both provides adequate sample disruption while maintaining phosphoprotein integrity. Beta‐caesin and protein lysates from eukaryotic cells were processed via manual disruption methods and via bead milling using multiple different parameters. Following homogenization, the effect of desphosphorylation was quantified via western blotting and quantitative LC‐MS/MS.
High resolution mass spectrometry coupled with multivariate data analysis revealing plasma lipidomic alteration in patients with colorectal cancer
Authors: De Oliveira Carvalho Patricia , Messias Marcia Cristina Fernandes , Mecatti Giovana Colozza , Angolini Célio Fernando Figueiredo 
Published: November 2023

Colorectal cancer (CRC) is one of the most frequent neoplasias in the adult population worldwide and presents a high rate of morbidity and lethality. In this study, a lipidomic approach based on gas chromatography and liquid chromatography‐electrospray ionization‐qToF‐mass spectrometry (LC‐ESI‐qToF‐MS) combined with multivariate data analysis, including principal component analysis (PCA) and orthogonal partial least squared discriminant analysis (OPLS‐DA) was adopted for the investigation of potential diagnostic biomarkers in the plasma of CRC patients. Variable Importance in Projection (VIP) was used to identify the ions that had the greatest discriminatory effect among the groups (cancer patients and healthy volunteers). It was possible to identify 15 differential lipids belonging to the glycerophospholipids class. The most abundant ions identified in the group of cancer patients were the phosphatidylcholines and phosphatidylethanolamines whereas the lysophosphatidylcholine (LPC) metabolite identified as LPC (P‐16:1), which presented a major VIP score through the OPLS‐DA model, was down regulated in patients with CRC. Also, a relative reduction of palmitoleic (C16:1), arachidonic (C20:4) and n‐3 polyunsaturated fatty acids was observed in these patients. This study may provide a new insight into underlying mechanisms for CRC and proves that MS‐based lipidomics is a powerful method for discovering new potential clinical biomarkersSupport or Funding InformationFAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo)
High‐Throughput, Fluorescent Analysis of Reactive Oxygen Species in C. elegans after Knockdown of mrck‐1
Authors: Brown Callista , Heying Ruth , Russo Matthew , Rigoulot Stephen , Erickson Patti 
Published: November 2023

Reactive oxygen species (ROS) produced in living organisms can generate oxidative stress, which contributes to cell damage associated with neurodegenerative diseases, such as Alzheimer's and Parkinson's. Studying responses to oxidative stress in model organisms, like the nematode worm Caenorhabditis elegans, may provide insight into basic underlying cellular mechanisms shared with humans. MRCK‐1 (myotonic dystrophy kinase‐related Cdc42 binding kinase), a highly conserved intracellular kinase, is activated by the G‐protein, Cdc42, which contains a redox sensitive motif. Previous studies have shown that MRCK‐1 regulates cell polarity and cytoskeleton reorganization, particularly during cell division and in response to cellular damage. To determine if MRCK‐1 plays a role in other cellular defense responses, we are testing if RNA interference (RNAi) of mrck‐1 results in the production of more ROS under stressed conditions. The cell permeable fluorescent ROS indicator, carboxymethyl dichlorodihydrofluorescein derivative, CM‐H2DCFDA, shows significantly increased fluorescence in hydrogen peroxide‐treated worms when quantified using the SpectraMax microplate reader. Oxidative stress conditions were induced with hydrogen peroxide (H2O2) in the presence of CM‐H2DCFDA, and fluorescence was measured over a period of 20 hours. Measuring the fluorescence of worms exposed to dsRNA from two independent mrck‐1 RNAi constructs, mrck‐m and mrck‐s, enables the quantification of ROS levels when MRCK‐1 function is decreased. Exploring the levels of ROS in RNAi‐treated C. elegans may give further insight into the cellular role of MRCK‐1 both in worms and in humans.Support or Funding InformationSpecial acknowledgments to the Salisbury University Department of Biological Sciences, the Henson Undergraduate Research Award, and the National Science Foundation MRI Acquisition Award DBI‐1337534 for support.
Biochemical Study of A Cancer Driver Fusion Protein, DnaJB1‐PKA
Authors: Lu Tsan‐Wen , Zhang Ping , Cianfrocco Michael , Simon Sanford , Leschziner Andres , Taylor Susan 
Published: November 2023

Fibrolamellar Hepatocellular Carcinoma (FL‐HCC), a rare liver cancer disease, usually occurs in young adults without any liver disease history. Until now, surgery is still the main treatment because FLHCC does not respond well to chemotherapy. Given the low survival rate and lack of available treatment options, there is a pressing need for new diagnostic and therapeutic approaches to FLHCC.Whole genome sequencing and transcriptome sequencing of FLHCC tumors show a single, consistent genetic alteration in the FLHCC tumor cells: a deletion of ~400 kB between the first exon of the heat shock protein, DNAJB1, and the first exon of the catalytic subunit of PKA, PRKACA, on one copy of chromosome 19. This deletion produces a chimeric gene, which leads to a chimeric protein. This chimeric DNAJB1‐PRKACA protein is now recognized as the driver of FLHCC. Inhibition of the tumor driver DNAJB1‐PRKACA chimera offers tremendous potential to cure FLHCC. Full structural and biochemical characterization of DNAJB1‐PRKACA and its comparison to native protein PKA will provide valuable insights toward developing a drug that selectively blocks the FLHCC chimera. Here, we show DnaJB1‐PKA has similar kinase activity as wild type PRKACA. It can also form an R2 DnaJB1‐PRKACA2 holoenzyme with PKA regulatory subunits similar to wild type R2‐PRKACA2 holoenzymes even though they elute at different positions from gel filtration, indicating they may have different cell dimensions. Both cryo‐EM and X‐ray crystallography are being used to elucidate differences between wild type and mutant holoenzymes. Differences in holoenzyme functional properties are also being explored.Support or Funding InformationFunding for this research was provided by the National Institutes of Health Grants GM034921
The secret life of kinases: insights into non‐catalytic functions from pseudokinases
Authors: Murphy James M , Petrie Emma J , Davies Katherine , Tanzer Maria C , Jacobsen Annette V , Hildebrand Joanne M , Lucet Isabelle S , Silke John , Czabotar Peter E 
Published: November 2023

While protein kinases are best understood for their phosphoryl transfer enzymatic activities, recent mechanistic studies have led to recognition of non‐catalytic functions in cell signaling. Studies of pseudokinases ‐ the catalytically‐impaired or dead cousins of conventional protein kinases ‐ have been instrumental in shining light on the vast, varied and ever‐expanding repertoire of mechanisms by which kinase‐like domains can mediate protein interactions to modulate cellular signaling.Although best characterized as allosteric regulators of conventional, active protein kinases, our work and that of others suggests more broadly that pseudokinase domains are important protein interaction modules that can function as molecular switches, scaffolds and adaptors in cell signaling pathways. Here, I will describe how our work has attributed these functions to the pseudokinase, Mixed lineage kinase domain‐like (MLKL). Overall, these studies suggest how conventional protein kinases could moonlight as protein interaction modules, scaffolds or molecular switches to complement their better‐understood catalytic roles.Support or Funding InformationNational Health and Medical Research Council of Australia (fellowship 1105754, grants 1057905, 1067289, infrastructure 9000220) and Victorian Government Operational Infrastructure Support.
Calcium Signals Alter the Mobility and Localization of CaMKIIδ in Cardiomyocytes
Authors: Wood Brent , Galice Samuel , Bers Donald , Bossuyt Julie 
Published: November 2023

Ca2+‐calmodulin dependent protein kinase II δ (CaMKIIδ) is an important regulator of cardiac myocyte ion channels, Ca2+ regulation, contraction and transcriptional control; and its activity is upregulated in pathological states. CaMKIIδ has two main splice variants (δB and δC), which differ only by a nuclear localization sequence, so δB is more concentrated in the nucleus. While activity‐triggered translocation of neuronal CaMKIIα to dendritic spines is established as part of memory formation, very little is known about the spatiotemporal dynamics of CaMKIIδ activation. Prevailing views have been that cardiac CaMKII forms extremely stable dodecamers anchored at target‐rich loci and displays very little mobility (partially due to its size). We tested this using adenovirally‐expressed δC‐GFP and δB‐GFP and fluorescence recovery after photobleaching (FRAP). We found that δC‐GFP is remarkably mobile in the cytoplasm (Tau 11.09±0.27s) in resting rabbit cardiac myocytes. Moreover calcium chelation with BAPTA slowed, but 0.5 Hz pacing increased recovery kinetics (Tau 17.08±1.7 and 8.76±0.42s, respectively). δB‐GFP had similar differences as δC‐GFP in mobility under resting, BAPTA and paced conditions (Tau of 11.41±0.61, 19.17±2.5, and 10.04±0.59s). Autonomous δCT287D‐GFP moved rapidly even without pacing (Tau 9.90±0.41s). Calcium effects on nuclear FRAP followed a similar pattern, but recovery was significantly slower (Tau at rest 70.2±4.5s and 56.68±2.2s for δC‐GFP and δB‐GFP). The recovery plateau in the nucleus was also only 0.394±0.02 and 0.105±0.01 for δC‐GFP and δB‐GFP respectively (vs. 0.865±0.01 and 0.824±0.01 in cytosol), indicating a significant non‐mobile fraction of CaMKIIδ in the nucleus. Immunocytochemistry revealed pacing drives endogenous CaMKIIδ out of the nucleus and away from the Z‐lines. Nuclear localization of endogenous CaMKII was even further decreased in resting heart failure rabbit myocytes compared to age matched controls. These results indicate that CaMKIIδ activation promotes its mobility and suggest that CaMKII translocation may be an essential component of signal spread in cardiomyocytes.Support or Funding InformationPO1‐HL80101 to DB and RO1 HL103933 to JB
Structural Elucidation of the Binding of the Vav‐SH2 Domain to the EphA2 Cytoplasmic Region
Authors: Zhang Kaibo , Ma Jia , Post Carol B , Stauffacher Cynthia V 
Published: November 2023

The Vav guanine nucleotide exchange factors (GEFs) facilitate the GDP/GTP exchange of the Rac/Rho family of small GTPases to modulate cytoskeletal organization, cell morphology and mobility. In endothelial cells, Vav interacts with the EphA2 receptor tyrosine kinase (RTK) with its C‐terminal SH2 domain and plays a crucial role in EphA2‐mediated angiogenesis in cancers. Two closely‐spaced tyrosines in the juxtamembrane segment (JM) of the EphA2 cytoplasmic region, Y588 and Y594, were the postulated Vav‐SH2 recognition sites. Quantitative mass spectrometry data suggests that both JM tyrosines are phosphorylated with high stoichiometry in mammalian cells. However, they exhibit different rates of dephosphorylation by human cytoplasmic protein tyrosine phosphatases (HCPTPs). To characterize the role of each JM phosphotyrosine in Vav‐SH2 binding, isothermal titration calorimetry (ITC) and 15N‐HSQC titration experiments were performed using bacterially expressed Vav1‐SH2 and synthetic JM peptides with different tyrosine phosphorylation patterns. The ITC data indicates Vav1‐SH2 binds preferentially to the doubly phosphorylated and the Y594‐phosphorylated version of JM peptide (JMpYpY and JMYpY), with a KD 10‐fold smaller than the Y588‐phosphorylated version (JMpYY). The 15N‐HSQC spectra suggests all three versions of synthetic JM phosphopeptides bind Vav‐SH2 in similar fashions. The chemical shift perturbation (CSP) analysis of the titration spectra reveals both the primary and secondary phosphotyrosine binding pockets of Vav1‐SH2 are involved in JM phosphopeptide recognition, suggesting each phosphotyrosine has its designated binding pocket. Subsequent 3D NOESY‐HSQC experiments and crystallization trials are undergoing in order to resolve the peptide configuration in the complex of the Vav‐SH2 domain and the JM phosphopeptides.Support or Funding InformationPurdue University Center for Cancer Research
AT THE CROSSROADS BETWEEN TYR AND SER/THR SIGNALING: A NEW PARADIGM IN THE REGULATION OF PP2A BY SRC KINASE
Authors: Sontag Estelle , Sontag Jean‐Marie , Gomez Rey J , Hoffman Alexander , Taleski Goce , Mazalouskas Matthew D , Hanks Steven K , Frohner Ingrid , Ogris Egon , Wadzinski Brian E 
Published: November 2023

The major enzyme, Ser/Thr protein phosphatase 2A (PP2A), is a key regulator of cellular signal transduction pathways. While PP2A dysfunction has been linked to human cancer and neurodegenerative disorders such as Alzheimer disease (AD), PP2A regulation remains relatively poorly understood, precluding the development of effective PP2A‐centric therapies. It has been reported more than two decades ago that the PP2A catalytic subunit (PP2Ac) is inactivated in vitro by phosphorylation at Tyr307 by tyrosine kinases such as v‐Src. Numerous studies relying on the use of commercial “PP2Ac pY307 antibodies” have subsequently linked enhanced phosphorylation of PP2A at Tyr307 with pathological cascades in cancer and AD. In contrast, we present here novel evidence in multiple cell types showing that PP2Ac is not phosphorylated at Tyr307. Rather, we uncover a novel mechanism of regulation of PP2A by Src that affects PP2A substrate specificity. Using specific PP2A mutants, we show that Src‐dependent PP2A deregulation can affect the phosphorylation of tau at Ser/Thr epitopes, a pathological hallmark of AD. It also influences the activation of ERK, a major growth regulatory kinase upregulated in many cancers. Our findings unveil a novel functional Src kinase/PP2A signalling module that positions these enzymes at the interface between Tyr and Ser/Thr signaling pathways. We propose that dysregulation of this Src‐PP2A signaling node may contribute to cancer and AD.Support or Funding InformationSupported by NHMRC grant APP1002744 and a grant from the Hunter Medical Research Institute, Australia (ES and JMS), and NIH grants GM051366 and DK070787 (BEW), and T32 GM08554 (MDM).
NADPH Oxidase 4 Expression Is Increased through TRPV4 Channel in D. farinae‐Induced Airway Remodeling and TGF‐β1‐Mediated Fibroblast Differentiation into Myofibroblasts
Authors: Al‐Azzam Nosayba Zakariya , Gombedza Farai , Snyderman Mathew , Thodeti Charles , Paruchuri Sailaja 
Published: November 2023

NADPH oxidase 4 (NOX4) is a member of the NOX family of NADPH oxidases, encoded by NOX4 gene and has been implicated in TGF‐β1‐induced human cardiac fibroblast to myofibroblast differentiation. Further, NOX4 has been shown to potentiate lung fibroblast differentiation in vitro and its transcript level is enhanced in fibroblasts isolated from idiopathic pulmonary fibrosis (IPF) patients compared to normal ones. Our lab has previously identified that Transient Receptor Potential Vanilloid 4 (TRPV4) TRPV4, a mechano‐sensitive ion channel is involved in D. farinae‐induced airway remodeling during asthma in vivo and myofibroblast differentiation in vitro. However, the role of NOX4 in asthma, its interaction with TRPV4 and the mechanism of action still remains elusive. In the present study, we show that NOX4 is up‐regulated during lung remodeling in asthma and fibroblast differentiation in vitro in a TRPV4‐dependent pathway.Using NOX4 antagonist and NOX4 siRNA, we demonstrate that NOX4 is involved in TGFβ‐1 induced α smooth muscle actin (α SMA) and fibronectin (FN) expression in human lung fibroblasts (HLF). Further, TGFβ‐1 treatment up‐regulated NOX4 gene expression as determined by real time PCR.. Further, to investigate the effect of TRPV4 on NOX4, we inhibited TRPV4 channel using a pharmacological antagonist RN 1734, and this led to a reduction in TGFβ‐1‐mediated NOX4 expression and differentiation. Interestingly, NOX4 transcripts were up‐regulated in lung tissues in response to D. farinae in an asthma model. More importantly, TRPV4 KO mice which were protected from D. farinae‐induced asthma also exhibited lower levels of NOX4 gene expression, suggesting that TRPV4 promotes airway remodeling through NOX4. Our results unravel a unique role for NOX4 in TRPV4 mediated fibroblast differentiation and lung remodeling and can be used as a new therapeutic target in asthma treatment.
Up‐regulation of atrial and neuronal Kir3 activity by cholesterol
Authors: Rosenhouse‐Dantsker Avia , Bukiya Anna N 
Published: November 2023

Cholesterol is one of the major lipid components of the plasma membrane in mammalian cells, and its excess is associated with multiple pathological conditions including cardiovascular and neurodegenerative disease. In recent years, cholesterol has been emerging as a major regulator of ion channel function. Here we focus on G protein‐gated inwardly rectifying K+ (GIRK or Kir3) channels that play a key role in the regulation of membrane excitability in neuronal and cardiac cells.The most common effect of cholesterol on ion channels is a decrease in channel activity. In contrast, we have recently shown that unexpectedly cholesterol enrichment up‐regulates Kir3 activity in atrial myocytes. Notably, we show here that also in freshly isolated CA1 pyramidal neurons from hippocampus, in‐vitro cholesterol enrichment results in a strong increase in physiological Kir3 currents.To obtain mechanistic insights into the up‐regulation of cardiac and neuronal Kir3 channels by cholesterol, we employed planar lipid bilayers, mutagenesis studies using the Xenopus oocytes heterologous expression system, and molecular modeling. We first investigated the biophysical properties of the channel. Our data showed that the increase in channel activity following an increase in cholesterol levels is a result of an increase in the open probability of the channel and not in the unitary current amplitude and channel conductance. Next, we investigated why and how cholesterol up‐regulates Kir3 channels. Our studies traced the unique impact of cholesterol on cardiac Kir3 channels (up‐regulation and not down‐regulation) to a transmembrane region that includes residues in both the inner and outer alpha helices. Moreover, we identified putative cholesterol binding sites that overlap with the gating hinge region in the transmembrane domains of both cardiac and neuronal channels. Together, our studies provide novel insights into the molecular mechanism that underlies cholesterol‐driven up‐regulation of Kir3 currents in the heart and in the brain.Support or Funding InformationScientist Development Grant 11SDG5190025 from the American Heart Association (to ARD)
Identification of Extracellular Residues Critical to the Epithelial Sodium Channel
Authors: Dismuke Taylor Y , Booth Rachell 
Published: November 2023

Disruption of homeostatic sodium reabsorption takes place in the kidney can result in various diseases such as osteoporosis, kidney stones, or hypertension. The protein channel found in the membrane of kidney cells that carries out sodium reabsorption is the epithelial sodium channel (ENaC). Identifying and characterizing residues that are critical to ENaC function are key to understanding ENaC's mechanism of action. Complete understanding of the structure‐function relationships within ENaC's extracellular loop are still rudimentary, but studies have demonstrated that it plays a role in channel regulation. Using random mutagenesis, mutations within the extracellular loop of βENaC were generated and incorporated into a yeast expression system. Qualitative screening using a survival pronging dilution assay with yeast expressing αβγENaC was used to monitor ENaC function. Yeast cells expressing αβMutγENaC demonstrated changes in growth compared to yeast expressing wild‐type αβγENaC. Future studies include sequencing to identify mutations and localization of αβMUTγENaC within yeast cells.Support or Funding InformationThis research was supported by National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under award number R15GM086798 (R. Booth and W. David) and R25GM10783 (K. Oyajobi, N. Blake, R. Booth, and R. Walter).
Hydrogen sulfide homeostasis and signaling in normal and neoplastic intestinal cells
Authors: Libiad Marouane , Sakamoto Naoya , Fearon Eric , Banerjee Ruma 
Published: November 2023

Hydrogen sulfide (H2S) is an important gas signaling molecule with effects on multiple physiological processes including neuromodulation, inflammation and cardiac function. Maintaining healthy levels of H2S in mammalian cells requires tight control between its biosynthesis and catabolism. The biogenesis of H2S involves two enzymes from the transsulfuration (cystathionine β‐synthase (CBS) and γ‐cystathionase (CSE)) and cysteine catabolic (mercaptopyruvate sulfurtransferase) pathways. The sulfide oxidation pathway requires the concerted action of the mitochondrial enzymes: sulfide quinone reductase (SQR), a persulfide dioxygenase (ETHE1), rhodanese and sulfite oxidase. The dominant sulfide oxidation products vary in a tissue‐specific manner and are sulfate in liver and kidney and thiosulfate in colonic mucosa. The mechanism of sulfide‐based signaling remains unclear. Persulfidation of proteins is postulated to be a major mechanism by which sulfide‐based signals are relayed. However, it is unclear whether H2S or a downstream product is involved in persulfidation modification.Herein, we report the analysis of human colon cancer biopsies and patient‐matched controls that reveal an up‐regulation at the protein level of SQR, ETHE1 and rhodanese in colon cancer tissues. We have also observed a differential distribution of these three proteins in colon cancer vs matched normal tissues. Colon cancer‐derived epithelial cell lines (HT‐29, LoVo, CaCo‐2, RKO and HCT116) also exhibited an increase in the protein levels of SQR, ETHE1 and rhodanese, compared with the nonmalignant colonic epithelial cells, HECE. Mercaptopyruvate sulfurtransferase is the major contributor to H2S production in the colon cells lines that were tested. Supplementation of the HT29 colon cancer cell line with exogenous thiosulfate enhances intracellular persulfide levels as visualized by SSP4 labelling.