bluuebunny/crossref_metadata_2025_split huggingface dataset:
Proteomics Analysis of Tea Tree Oil‐Selected Staphylococcus aureus Small Colony Variant
Authors: Torres Nathanial J , Hartson Steven D , Rogers Janet , Abdulhafid Khadija A , Gustafson John E 
Published: November 2023

Staphylococcus aureus small colony variants (SCV) are associated with chronic and recurring infections that are recalcitrant in antimicrobial therapy. SCVs demonstrate: slower growth rates; defective metabolism and electron transport; and reduced antimicrobial susceptibility. Tea tree oil (TTO) kills bacteria by denaturing proteins and disrupting membrane structure and TTO reduced‐susceptibility (TTORS) S. aureus mutants exhibit an “unique” SCV phenotype. Similar to previously described SCVs, all TTORS SCVs investigated were less susceptible to both the cell wall antibiotics vancomycin and oxacillin. A TTORS SCV mutant (TTORS‐1) harbored numerous mutations, including a mutation within acpP which encodes the acyl carrier protein (ACP) essential for fatty acid biosynthesis. Comparative proteomics revealed that TTORS‐1 demonstrated increases in 39 proteins and decreases in 74 proteins compared to parent strain SH1000. In TTORS‐1, the fatty acid biosynthesis proteins FabF (3‐oxoacyl‐synthase) and FabD (malonyl CoA‐acyl carrier protein transacylase) and the bifunctional phosphopantothenoylcysteine decarboxylase/phosphopantothenate‐cysteine ligase were found in greater abundance. This latter enzyme is required for the synthesis of 4′‐phosphopantetheine, which when linked to ACP acts as the anchor on which fatty acid biosynthesis takes place. Furthermore, RT‐PCR analysis revealed that the 5 gene fapR operon gene member plsX (encodes glycerol‐3‐phosphate acyltransferase), was up‐regulated in TTORS‐1. These findings indicate an alteration in fatty acid biosynthesis in TTORS‐1. S. aureus SCVs can result from the deletion of menB or cold shock protein cspB, and menB SCVs demonstrate a decrease in citric acid cycle activity. TTORS‐1 harbored less menaquinone biosynthetic protein MenB (1,4‐dihydroxy‐2‐naphthoyl‐CoA synthase), cold shock proteins CspB and CspC, ATP synthase subunit gamma, and proteins involved with the citric acid cycle. Collectively our data indicates that fatty acid biosynthesis is altered in TTORS‐1, as would be expected in an acpP mutant. We also demonstrate the reduced synthesis of certain proteins in TTORS‐1 mirroring what has been observed in previously described SCV phenotypes.Support or Funding InformationOklahoma Agricultural Experimental Station
There are still proteins in rat urine after 7‐day starvation
Authors: Zhang Fanshuang , Yuan Yuan , Ni Yanying , Gao Youhe 
Published: November 2023

Protein has been found in urine in all relevant studies. Why are there proteins in urine? If the protein in urine acts as a nutrient, should all proteins be reabsorbed into the blood during starvation to maintain the homeostasis of the internal environment? Are they toxic? Is there any protein in urine when the animal is starved? If the protein in urine is toxic or is discarded for the regulation of body's own cellular functions, it still must be released into the urine even when the animal is starved. Does the kidney need protein to maintain urine flow? If the protein is necessary to maintain urine flow, at least some of it should remain in the urine even after starvation. In this study, five Sprague Dawley rats were starved continuously for 7 days. The quantity, composition, and posttranslational modification of the urine proteome was studied before and after starvation. After 7‐day starvation, urinary protein concentration had no significant changes, even though the serum protein concentration decreased for about 10%. Only five and thirteen urinary proteins were significantly changed in the 4‐ and 7‐day starvation groups, respectively, compared with before starvation group. These findings indicate there were still proteins even after starvation which supports that urinary proteins may be necessary for kidney to maintain the urine flow or be toxic and discarded for the regulation of cellular functions. Not all potentially nutritious to the body will be reabsorbed and utilized. It seems that removing proteins in urine is even more important than maintain the homeostasis of the internal environment for the survival of the animals.Support or Funding InformationThis work was supported by the National Key Research and Development Program of China (grant number 2016 YFC 1306300); the National Basic Research Program of China (grant number 2013CB530805); and funds from Beijing Normal University (grant number11100704, 10300‐310421102)
Development of a high throughput extraction procedure for nuclei and mitrochondria from rat tissues
Authors: Easparro Brandon , Garrett Shari , Atwood James 
Published: November 2023

Comprehensive bottom up profiling of tissue proteomes is limited by the large dynamic range of protein expression and the ability of modern LC‐MS/MS instrumentation to adequately separate and detect peptides in such a complex mixture. Tissue proteomes are diverse and a common method for segregating the proteome is subcellular fractionation; isolating organelles which contain smaller subsets of proteins that can be more readily detected. Isolating tissues organelles is commonly performed manually using either a dounce or rotor stator homogenizer. However, this process is not amendable to high throughput quantitative proteomic studies as the reproducibility is low and the processing time is significant.In a previous study, we presented an automated bead mill procedure for extraction of nuclei from rat tissue. Here, we present a modified procedure to obtain high purity nuclei and mitochondria from rat tissue. We then compared purity, proteome coverage and protein extraction reproducibility to the extracts isolated by dounce and rotor stator homogenization. Following each extraction, organelles were isolated by centrifugation. Purity was then assessed by both microscopy and western blotting for known nuclear, mitochondrial and cytoplasmic protein markers. Proteins from each extraction method were then analyzed by LC‐MS/MS and protein extraction yields were quantified by spectral counting.
Global Proteomics Assay of Ubiquitin‐Knockout Strains of Yeast
Authors: Hanse Alexander , Minogue Catherine , Cooper Eric 
Published: November 2023

Ubiquitin is a small, polymerizable protein found universally in eukaryotic organisms. Ubiquitin polymerization serves as a signal for protein degradation, intracellular localization, and DNA repair, among many other cellular processes. Disruption of the ubiquitin system has been linked to many human afflictions, including neurodegenerative disorders and certain cancers (Mani, 2005)(Ciechanover and Brundin, 2003). Strains of S. cerevisiae incapable of producing specific ubiquitin polymers were developed using homologous recombination techniques and modern CRISPR/Cas9 editing technology for the ultimate goal of performing mass spectrometry‐based proteomics to look for proteins and metabolic pathways regulated by these polymers. Further revelations into the role of ubiquitin has implications within the field of molecular and chemical biology, as well as practical medicine.Support or Funding InformationThe Freedman Prize, Natural and Physical Sciences, 2016
Fyn Regulates Cyclic‐AMP Dependent Protein Kinase A Binding Interactions
Authors: Barritt Samuel A , Weir Marion E , Ballif Bryan A , Deming Paula B 
Published: November 2023

Cyclic‐AMP dependent protein kinase A (PKA) and Src family kinases (SFKs) are important for signal transduction in a multitude of cellular processes. While it is known that PKA positively regulates SFKs such as Src and Fyn through phosphorylation, we have discovered that Fyn can regulate PKA through a reciprocal phosphorylation event on the catatlytic subunit (PKA‐C). Here we report that PKA holoenzymes are physically associated with Fyn in HEK293 cells, suggesting the formation of a novel signaling complex. Interestingly, a Fyn mutant lacking the SH3 domain displayed markedly enhanced binding to the PKA catalytic subunit as compared to wild type (WT) Fyn. To determine whether Fyn regulates the interaction of PKA with intracellular binding partners, we employed stable isotopic labeling of amino acids in cell culture (SILAC) coupled with tandem mass spectrometry. Indeed, in cells overexpressing WT‐Fyn, PKA displayed enhanced binding to 40 intracellular proteins. The three most significantly enhanced binding partners (AKAP9, PDE4DIP, and CDK5RAP2) were A kinase anchoring proteins (AKAPs) known to function in microtubule nucleation and organization at the centrosome and Golgi apparatus. The ability of Fyn to modulate the physical interaction of PKA with these AKAPs was validated through coimmunoprecipitation and western blot analysis. Interestingly, this function of Fyn was independent of its kinase activity, as similar results were obtained in experiments using a kinase dead (KD) allele of Fyn. Taken together, our results suggest that Fyn complexes with PKA and directs its association with distinct AKAPs. Current studies aim to further characterize this novel molecular complex and identify downstream substrates of these kinases that may be targeted due to this interaction.Support or Funding InformationThis work was supported by the Arnold and Mabel Beckman foundation Beckman Scholars Program, a Scientist Development Grant from the AHA and an Endowed Professorship (to PBD) and the Vermont Genetics Network through U. S. National Institutes of Health Grant 8P20GM103449 from the INBRE program of the NIGMS.
Novel Molecular Interaction between Cyclic‐AMP Dependent Protein Kinase A Holoenzymes and La‐Related Protein 4
Authors: O'Toole Kayla T. , Deming Paula B. 
Published: November 2023

Cyclic‐AMP‐dependent protein kinase A (PKA) is a serine threonine kinase that is involved in regulating cellular processes such as migration, proliferation, metabolism and survival in response to a variety of environmental cues. PKA exists in cells as an inactive tetrameric holoenzyme, comprised of two regulatory (R) and two catalytic (C) subunits. Activation of PKA's enzymatic activity typically occurs in response to stimuli that induce increases in intracellular cyclic adenosine monophosphate (cAMP). In conducting a proteomics screen to identify novel binding partners of PKA, we found that the C subunit of PKA was consistently associated with the La‐related binding protein 4 (LARP4) in HEK293 cells. LARP4 is a member of the evolutionarily conserved family of La‐related proteins yet the functions of LARP4 are largely unknown. Therefore, we sought to further investigate this novel signaling complex. The binding of LARP4 to PKA was validated through co‐immunoprecipitation experiments. Results revealed that LARP4 was present in immunoprecipitates of the C and RII subunits, indicating that LARP4 interacts with PKA holoenzymes. Moreover, LARP4 immunoprecipitates demonstrated significant PKA kinase activity even in the absence of cAMP, suggesting that the PKA associated with LARP4 is active. Preliminary experiments conducted with mouse embryo fibroblasts (MEFs) from wildtype litter control or LARP4 knockout mice revealed differential phosphorylation of endogenous PKA substrates as measured by western blot analysis using a phospho‐PKA substrate antibody. This result suggests that the interaction of LARP4 with PKA may regulate downstream signaling targets. Current experiments are focused on characterizing the biophysical interaction between LARP4 and PKA and determining how this molecular complex influences cellular function.Support or Funding InformationThis work was supported by a Scientist Development Grant from the AHA (to PBD) and an Endowed Professorship (College of Nursing and Health Sciences, University of Vermont).
Spatial Organization of a cAMP/Ca2+‐Regulated Signaling Complex: A Solution Structural Small‐Angle X‐ray and Neutron Scattering Study of an AKAP79‐Scaffolded Complex Containing Type IIbeta PKA and Calcineurin
Authors: Hall James Ernest , Copps Jeff , Zhang Ping , Heck Albert , Blumenthal Don , Taylor Susan 
Published: November 2023

cAMP‐dependent protein kinase (PKA) regulates many cell processes including development, differentiation, and memory. In its resting state, PKA is a tetrameric holoenzyme consisting of a regulatory (R) subunit dimer and two catalytic (C) subunits. Four isoforms of the R‐subunit exist (RIα, RIβ, RIIα, and RIIβ). Although each isoform varies in sequence and quaternary structure, all share a common domain organization. The strength, duration, and specificity of PKA activity within the cell are mostly dependent upon isoform specific subcellular localization. The D/D domain interface of the R‐subunits serves as a docking site for A Kinase Anchoring Proteins (AKAPs) that target PKA to specific cellular locations. In cardiac myocytes and neurons, PKA is localized to Cav1.2 L‐type calcium channels by AKAP79 and up‐regulates calcium influx by phosphorylating the channel's cytosolic C‐terminal tail. AKAP79 also binds the Ca2+/calmodulin‐dependent protein phosphatase type 2B (PP2B, or calcineurin) at a site very close to that of PKA binding. Given the close proximity of the calcineurin and PKA binding sites on AKAP79, calcineurin has been suggested to play roles in regulation of calcium channels, along with regulation of PKA activity itself. Using a variety of biochemical and biophysical techniques, we have isolated and characterized a stable complex of the C‐terminal tail of AKAP79, RIIβ holoenzyme, and calcuneurin (“ARC” for short). We show a stoichiometry of 1:1:1 (AKAP79: RIIβ2C2 :CN) using native mass spectroscopy, and SAXS/SANS techniques confirm that this is a compact complex, suggesting a close interaction between PKA and calcineurin when bound to AKAP79. Furthermore, we have identified a functional interaction between RIIβ and calcineurin using peptide arrays. Altogether, we suggest beyond its function of localizing PKA and calcineurin in close spatial proximity to the Cav1.2 L‐type calcium channel, the ARC signalosome fosters extended protein‐protein interactions and regulations that suggest tight spatiotemporal control.Support or Funding InformationSupport for this work was provided through NIH GM034921
AMPK‐related kinase MPK38/MELK potentiates p21‐mediated apoptosis, cell cycle arrest, and inhibition of adipocyte differentiation via Thr55 phosphorylation
Authors: Ha Hyunjung , Seong Hyun‐A , Manoharan Ravi 
Published: November 2023

Murine protein serine‐threonine kinase 38 (MPK38) has been shown to interact with p53 and to stimulate p53 signaling. Here, we identify p21, a downstream target of p53, as another interacting partner of MPK38. The association between MPK38 and p21 is mediated through the C‐terminal domain of MPK38 and the CDK binding region of p21. MPK38 phosphorylates p21 at Thr55, implying a potential role for p21 phosphorylation in p21‐mediated activity. Indeed, MPK38 potentiates p21‐mediated apoptosis, cell cycle arrest, and inhibition of adipocyte differentiation in a kinase‐dependent manner by decreasing complex formation between p21 and its negative regulator Mdm2. These results suggest that MPK38 acts as a positive regulator of p21 activity.Support or Funding InformationThis work is supported by a National Research Foundation of Korea Grant (2015R1A2A2A01006098)
Cyclosporin A‐Induced Calcineurin Isoform Specific Matrix Metalloproteinases (MMP2 and MMP9) Expression in Renal Fibroblasts
Authors: Francis Cynthia E. , Bai Yun 
Published: November 2023

BackgroundIt has been shown that long‐term treatment with the potent immunosuppressive drug cyclosporin A (CsA) results in chronic nephrotoxicity. Its immunosuppressive properties are due to the results of the inhibition of the calcium‐ and calmodulin‐dependent phosphatase protein calcineurin A (CnA). The catalytic subunit of CnA has 3 isoforms: α, β and γ. The CnA α and β isoforms are expressed ubiquitously in a wide variety of tissues (particularly the kidney) while the CnAγ isoform is restricted to the testes and brain. Additionally, chronic nephrotoxicity has been associated with an imbalance of extracellular matrix (ECM) synthesis and degradation which may result in an accumulation of ECM molecules.ObjectiveThis study evaluates whether the expressions of matrix metalloproteinases (MMP2 and MMP9) induced by CsA is calcineurin isoform specific.Methods and ResultsWild‐type (WT), CnA α knockout (CnAα −/−) and CnA β knockout (CnAβ −/−) kidney fibroblast cell lines (an in vitro innovative tool that was previously created in our lab) were treated with CsA at 10 ng/ml for 24 and 48 hrs. Cells were lysed in TNESV buffer (1% NP40, 50mM Tris‐HCl pH 7.5, 100mM NaCl, 2mM EDTA, 10mM NaVO4) with protease inhibitor cocktail. Quantification of protein was performed using BCA protein assay. Then, western blots were probed with MMP9 and MMP2 antibodies. Western blot results demonstrated that CsA induced an increase in MMP9 protein levels in CnAβ −/− fibroblasts, but not in wild‐type and CnAα −/− fibroblasts. The protein levels were 2 to 2.5‐fold higher vs. basal levels in CnAβ −/− cultured in the absence of CsA. In contrast, CsA did not induce an increase in MMP2 protein levels in WT, CnAα −/− nor CnAβ −/− renal fibroblasts.ConclusionsThese results indicate that the CnAα isoform contributes to the CsA‐induced upregulation of MMP9 (gelatinase B) while the CnAβ does not. Interestingly, MMP2 (gelatinase A) may not play a role in the calcineurin‐mediated nephrotoxic changes in ECM that are induced by CsA treatment. As such, understanding the role of calcineurin A isoforms in the regulation of the homeostasis of ECM degradation in the kidney after long‐term CsA treatment needs to be further investigated.Support or Funding InformationNIH 1R15GM11312001
Disrupted BKCa Channel β1 Subunit Gene Contributes to Vascular Dysfunction in Pulmonary Hypertension
Authors: Barnes Elizabeth A , Chen Chih‐hsin , Lee Lori , Barnes Shayna L , Cornfield David N 
Published: November 2023

BACKGROUNDThe BKCa channel is a calcium‐sensitive potassium channel fundamental to the control of smooth muscle cell (SMC) tone. Loss of BKCa channel function leads to membrane depolarization and vasoconstriction. Previous reports have shown that mice with a disrupted BKCa channel β1 subunit have an increase in blood pressure and arterial tone in cerebral arteries. In addition, a gain‐of‐function β1 mutation, E65K, has been associated with low prevalence of diastolic hypertension, suggesting that the β1 subunit of the BKCa channel is a key molecular component involved in vasoregulation.HYPOTHESISIt is unknown as to the role of the β1 subunit in modulating pulmonary vascular tone. To assess the contribution of the BKCa channel β1 subunit in pulmonary vasoregulation, mice with a disrupted β1 gene (KCNMB1−/−) were examined.RESULTSKCNMB1−/− mice exhibited significantly higher right ventricular systolic pressure (RVSP) compared to wild‐type (WT) littermates with exposure to acute or chronic hypoxia in the absence of histologic evidence of accentuated vascular remodeling. The β1 subunit was predominantly expressed in pulmonary arteriole SMC. Isolated peripheral PASMC promoted collagen gel contraction irrespective of β1 expression. Isolated KCNMB1−/− PASMC exhibited significant increases in focal adhesions and Rho kinase expression compared to WT cells.CONCLUSIONSCompromised PASMC β1 function may contribute to the heightened vasoconstriction in the microvasculature that characterizes pulmonary hypertension.
Fluorescence Investigations of the Rate‐Limiting Step in the HCN Ion Channel Deactivation Pathway
Authors: Magee Kaylee EA , Claydon Thomas W , Young Edgar C 
Published: November 2023

Hyperpolarization‐ and cyclic nucleotide‐activated (HCN) ion channels support electrical oscillations of neuronal networks and help prevent absence seizures. The S4 helix of the voltage‐sensor (VS) region in HCN channels moves inwards upon hyperpolarization and this movement is coupled to pore opening. An open HCN channel can further be stabilized by cAMP binding to its C‐terminal region. Modifying the speed of the rate‐limiting step for HCN channel gating might help treat neuropathies, but the steps in the gating pathway are unclear. Specifically, it has been proposed that the depolarization‐dependent deactivation pathway of HCN channels includes a voltage‐independent pore closure step that becomes rate‐limiting at strong depolarizations. To clarify which step is rate‐limiting at different depolarizations, we performed voltage clamp fluorometry on mouse HCN2 channel derivatives expressed in Xenopus oocytes using a fluorophore attached to the N‐terminal region of the S4 helix. These results represent the first direct measurement of VS movements of an HCN2 channel. The fluorophore tracked depolarization‐dependent VS movements during deactivation that preceded pore closure significantly at strong depolarizations; the current decay rate was approximately 4‐fold slower than the fluorescence decay rate at +20 mV. We call this ratio of decay rates the “transmembrane coupling quotient”. At weaker depolarizations, VS movements still preceded pore closure, but less dramatically (transmembrane coupling quotient approximately 1.5‐fold at ‐60 mV). This voltage dependence of the transmembrane coupling quotient was cAMP‐independent, i.e., it remained similar even when cAMP binding was prevented by a mutation in the C‐terminal region. The voltage dependence of the transmembrane coupling quotient is consistent with a model of mammalian HCN channel deactivation where the VS undergoes movements that have a greater voltage dependence compared to pore closure movements. VS movements may serve as the rate‐limiting step for deactivation at weak depolarizations, but another step – perhaps pore closure – is the rate‐limiting step at strong depolarizations. The temporal association between VS movements and pore movements would thus be loose at strong depolarizations, and tight at weak depolarizations.Support or Funding InformationNatural Sciences and Engineering Research Council (NSERC) Postgraduate Scholarship to K.E.A.M; Natural Sciences and Engineering Research Council (NSERC) Discovery Grant to E.C.Y
Identification of Accessory Proteins Impacting the Function of the Epithelial Sodium Channel (ENaC)
Authors: Adewunmi Toyosi , Booth Rachell 
Published: November 2023

The epithelial sodium channel (ENaC) plays a crucial role in ensuring proper Na+ balance in the body. ENaC is a heterotrimeric channel located mainly in tight or high resistance epithelia and allows for the passive transport of Na+ into the epithelial cell from the lumen. In the nephron, ENaC regulates the final 3–5% reabsorption of Na+ and is a limiting factor for Na+ reabsorption in the kidneys. Because of its role in the kidneys, ENaC is thought to be a critical component in the control of blood pressure. Though there have been several accessory proteins known to regulate ENaC function, there are still questions to be answered about the role other proteins play in ENaC trafficking and assembly. Understanding the mechanisms behind ENaC regulation will potentially provide insight into certain defects in the trafficking or assembly of the channel, and identify possible therapeutic targets. Through survival dilution growth assays in yeast deletion strains, several genes were shown to effect ENaC function in a high salt growth media. Yeast deletion strains expressing αENaC but lacking ER degradation enhancing alpha‐mannosidase like protein 3 (EDEM3) or nuclear protein localization 4 (NPL4) showed an increase in the growth of yeast cells compared to wildtype indicating a loss in ENaC function. A syntaxin 3 yeast deletion strain expressing αENaC resulted in decreased cell growth indicating a gain in ENaC function. Preliminary studies on the knockdown of EDEM3 using a CRISPR/Cas9 knockout plasmid in a M‐1 mammalian cell line endogenously expressing αβγENaC, demonstrated a decrease of ENaC protein expression levels.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).