bluuebunny/crossref_metadata_2025 huggingface dataset:
Divergent Mechanisms of Allosteric Regulation of Pyruvate Carboxylase by Acetyl Coenzyme A
Authors: Liu Yumeng , St.Maurice Martin
Published: November 2023

Pyruvate carboxylase is a biotin‐dependent enzyme, composed of multiple functional domains on a single polypeptide chain, including the biotin carboxylase domain, the carboxyltransferase domain, the biotin carboxyl carrier protein domain and the allosteric domain. Acetyl coenzyme A is an allosteric activator of pyruvate carboxylase from a wide variety of species. However, the sensitivity to acetyl coenzyme A activation varies depending on the organism from which the enzyme was cloned or isolated. We are investigating the divergent allosteric activation of pyruvate carboxylase from two organisms, Rhizobium etli (RePC) and Aspergillus nidulans (AnPC). The maximum activity of RePC is accelerated ~10‐fold by acetyl coenzyme A, while AnPC is insensitive to acetyl coenzyme A activation, despite binding acetyl coenzyme A. Previous studies have structurally and kinetically characterized RePC in the presence and absence of acetyl coenzyme A. In order to investigate the divergent mechanisms of allosteric activation in AnPC and RePC, we have structurally and kinetically characterized the contributions of acetyl coenzyme A to catalytic turnover in AnPC. We have observed a highly divergent response of AnPC and RePC to acetyl coenzyme A activation in both the overall pyruvate carboxylation reaction and in the half‐reactions catalyzed at the individual catalytic domains. The binding of acetyl coenzyme A to RePC at the allosteric domain is primarily mediated by two conserved arginine residues. We generated mutations at the equivalent arginine residues in AnPC and determined that the Ka for acetyl coenzyme A was significantly increased in both mutations, indicating that acetyl coenzyme A most likely binds at the same binding site in AnPC as it does in RePC. To further clarify the mechanism of acetyl coenzyme A activation in pyruvate carboxylase, we investigated the catalytic turnover of hybrid tetramers composed of several combinations of inactivating mutations. Our data reveals that, in RePC, acetyl coenzyme A constrains the translocation of the biotin carboxyl carrier protein domain, limiting it to sample only two productive motions during catalytic turnover. However, in AnPC, acetyl coenzyme A does not alter the range of motions sampled by the biotin carboxyl carrier protein domain during catalysis. Thus, while acetyl coenzyme A binds at the same binding site in both AnPC and RePC, the divergent response of AnPC and RePC to acetyl coenzyme A activation arises, in part, from different innate carrier domain movements during catalytic turnover.

Support or Funding Information

This work is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number 1R15GM117540‐01
Probing the Role of N and C‐terminal Regions in Allosteric Regulation of Thermodesulfovibrio yellowstonii ADP‐Glucose Pyrophosphorylase
Authors: Yik Eric , Kaur Sandeep , Pushkarev Edward , Mercado Elizabeth , Collazo Michael , Cascio Dulio , Axelrod Herbert , Meyer Christopher R
Published: November 2023

Glucans such as starch serve as energy storage compounds and renewable and biodegradable carbon sources for use in industry. ADP‐Glucose Pyrophosphorylase (ADPG PPase) catalyzes the rate limiting step of glucan biosynthesis in plants and bacteria, respectively. The novel Thermodesulfovibrio yellowstonii (Td.y) ADPG PPase, from a thermophilic, sulfate‐reducing bacteria, has potential properties that could lead to engineering a very stable and highly active form to increase biomass yield. Sequence alignment data indicate that Td.y harbors varied amino acids in two conserved N‐terminal and C‐terminal regions that are important for regulation in other ADPG PPases that may be responsible for its altered effector specificity, including activation by PEP. To probe the roles of these regions, several variants (including F23R, S25A, K365G, and R397A) were generated and purified for kinetic analyses. The F23R demonstrated a 4 fold decrease in activity and a ~2 fold decrease in apparent affinity for the substrate ATP. However, in the presence of PEP, ATP affinity was restored to WT values. The variant S25 showed a Vmax ~3 fold higher than WT with a10‐fold higher apparent affinity for ATP as well as desensitization to effectors. The K365G enzyme displayed two fold lower activity than WT and desensitization to PEP. The R397A enzyme exhibited a ~20 fold activity reduction versus WT and PEP inhibition. Initial crystallization trials identified 0.2M di‐ammonium citrate (pH 5.0) and 20% polyethylene glycol as a condition that resulted in crystals of the S25A variant that diffracted; subsequently, an x‐ray structure was determined by molecular replacement to a nominal resolution of 2.9 Å, only the third known ADPG PPase structure. The structure indicates that the activated state of the S25A enzyme may result from an alteration in the positioning of Arg24 which participates in a salt bridge with Asp364, linking the N and C‐terminal domains by a functional relay switch mechanism. Initial results probing ligand binding with Isothermal Titration Calorimetry are promising, with good correlation between some kinetic parameters and KD values. Taken together, the kinetic data as well as the new structure and previous homology modeling confirm and extend understanding of the conserved regions for activity, regulation, and allosteric specificity and suggest additional mutagenesis targets.

Support or Funding Information

Supported in part by NSF BIO MCB grant #0448676 and NSF BIO DBI.
Phosphoinositides and Kinesin 13 Family Member Kif2A Cooperatively Regulate Arf GTPase Activating Protein AGAP1
Authors: Roy Neeladri Sekhar , Luo Ruibai , Randazzo Paul A.
Published: November 2023

ADP‐ribosylation factor GTPase‐activating proteins (Arf GAPs) are a large family of proteins that induce hydrolysis of GTP bound to Arf family proteins. Arf proteins are members of Ras superfamily of small GTPases involved in membrane traffic and actin cytoskeleton remodeling. AGAP1, a member of the AGAP subfamily of Arf GAPs, consists of G protein like domain (GLD), pleckstrin homology (PH), Arf GAP, and Ankyrin repeat domains. Kinesin Family Member 2A(Kif2A) belongs to the Kinesin 13 family of kinesins, which affect microtubule dynamics. In vitro studies in breast cancer and squamous cell carcinoma has correlated overexpression of Kif2A with increased invasion and cell migration. Recently, an interaction between AGAP1 and Kif2A has been reported. Kif2A was found to increase the GAP activity of AGAP1, which depended on phosphatidylinositol 4,5‐bisphosphate (PIP2), a membrane phospholipid.

Here we test the hypothesis that PIP2 is an allosteric modifier of Kif2A binding to AGAP1, with the consequence that PIP2 and Kif2A synergistically increases GAP activity of AGAP1.

As an intitial test of the hypothesis, we examined the effect of PIP2 and Kif2A on enzymatic power of AGAP1. PIP2 increased enzymatic power by approximatey 10‐ to 100‐fold depending on the concentration of PIP2. Kif2A in the absence of PIP2 had no effect, whereas in the presence of PIP2, increased enzymatic power by 3‐ to 5‐fold beyond the effect of PIP2 alone. The effect was due to a 3‐ to 5‐fold decrease in Km for myristoylated Arf·GTP and a 50 % increase in kcat. Further studies are designed to determine the molecular basis behind the allosterism.

To summarize, here we found two levels of allosterism: 1. PIP2 control of Kif2A interaction with AGAP1 and 2. PIP2 and Kif2A control of substrate (Arf1·GTP) interaction with AGAP1.

Support or Funding Information

This work was supported by the intramural program of the National Cancer Institute (Project BC007365).
Characterization of an Unprecedented Hybrid Pteridine‐Nonribosomal Peptide Synthetase‐Like Biosynthetic Gene Cluster
Authors: Perez Corey E , Park Hyun Bong , Barber Karl W , Rinehart Jesse , Crawford Jason M
Published: November 2023

The entomopathogen Photorhabdus luminescens is mutualistically associated with nematodes of the genus Heterohabditis while pathogenically associated with the nematode's larval prey. Upon invasion of an insect larva and exposure to its haemocoel, the nematode regurgitates P. luminescens. In this environment, an array of cellular processes are activated ultimately producing a plethora of small molecule metabolites that serve as antimicrobials, immunosuppressants, and cellular signals, amongst other functions. Given this nature, P. luminescens serves as an ideal source for the discovery of novel, bioactive small molecules as well as intriguing biochemistry. Utilizing genome synteny analysis, a technique capable of identifying atypical biosynthetic gene clusters that have been integrated into their host genomes via horizontal gene transfer, we identified a silent and previously uncharacterized biosynthetic pathway in P. luminescens that contains an unprecedented fusion of pteridine, pyruvate dehydrogenase‐like, and nonribosomal peptide synthetase (NRPS) biosynthetic machineries. Through the application of pathway‐targeted metabolomic analysis, a small family of metabolites, which we termed the “pepteridines,” were identified and structurally characterized. These molecules consist of a tetrahydropterin core functionalized with an acyl appendage at the N5 position. Through genetics, we further identified the individual genetic determinants for pepteridine biosynthesis. Seeking to better understand the coupling of the tetrahydropterin with its acyl substituent, a series of in vitro biochemical reactions were performed to assess the substrate scope of the atypical condensation domain present in the biosynthetic pathway. The resulting condensed metabolites demonstrate in vitro radical scavenging activity. And, utilizing a P. luminescens knockout strain, we demonstrated that the pathway serves to protect against oxidative stress. The pepteridines, as the first reported example of metabolites synthesized by a hybrid NRPS‐pteridine pathway, enhance our understanding of the combinatorial biosynthetic potential available in microbes.

Support or Funding Information

National Cancer Institute Grant (1DP2‐CA186575)National Institute of General Medical Sciences Grant (R00‐GM097096)Searle Scholars Program Grant (13‐SSP‐210) CEP was supported in part by a National Institutes of Health Chemistry Biology Interface Training Grant (5T32GM067543‐12)
Predicting Function of Class II Diterpene Cyclases in Bacterial Species Using a Sequence Similarity Network
Authors: Lemke Cody , Nett Ryan
Published: November 2023

Diterpenoids are natural products commonly derived from plants as well as bacteria and fungi. Many of these secondary metabolites have been shown to possess anti‐inflammatory, antimicrobial, insecticidal, and even antitumor properties making them of great interest in the pharmaceutical and agricultural industries. However, due to their complex structure, metabolic engineering is typically a necessary means of producing these compounds. For this reason, it is important to understand the function and mechanisms of the enzymes that often perform the committed step in diterpenoid biosynthesis, class II diterpene cyclases (DTCs). To better understand the breadth and diversity of bacterial DTCs, a protein sequence similarity network (SSN) was constructed from a previously characterized DTC involved in the synthesis of the plant hormone, gibberellin. This network established a representation of homology between putative DTCs in bacteria that could potentially be used to predict function of uncharacterized genes. Interestingly, this method also demonstrated that some bacterial DTCs have similarity to plant and fungal DTCs, suggesting a possible evolutionary relationship. To assess the SSN results, we cloned and expressed several putative bacterial DTCs in our metabolic engineering system, and in doing so have confirmed their functionality as DTCs. These newly characterized enzymes exhibit a wide range of functionality, and active site analysis suggests that only a few key amino acids may be involved in determining product outcome. Thus, our results demonstrate the efficacy of SSNs for identifying potential DTCs, along with providing insight into the mechanisms underlying the biochemical diversity exhibited by bacterial DTCs.

Support or Funding Information

Laboratory of Reuben Peters, Iowa State University
Mechanisms by which plant extracts delay aging in yeast by targeting certain signaling pathways and modulating lipid metabolism
Authors: Titorenko Vladimir , Lutchman Vicky , Medkour Younes , Arlia‐Ciommo Anthony , Dakik Pamela , McAuley Mélissa
Published: November 2023

The yeast Saccharomyces cerevisiae is a unicellular eukaryote that has been successfully used as a model organism for discovering signaling pathways and chemical compounds that modulate cellular aging, define organismal lifespan, and influence organismal fitness not only in yeast but also in various multicellular eukaryotes. Using a robust quantitative assay for measuring yeast chronological lifespan, we screened a library of natural products to discover six plant extracts (PE; i.e. PE4, PE5, PE6, PE8, PE12 and PE21) whose longevity‐extending efficiencies greatly exceed those of currently known natural anti‐aging compounds, such as resveratrol, rapamycin, spermidine, caffeine, and lithocholic acid. Our genetic, biochemical, and mass spectrometry‐based lipidomic analyses uncovered the following mechanisms by which these PE delay yeast chronological aging by targeting certain signaling pathways and modulating lipid metabolism: 1) PE4 attenuates the inhibiting effect of the pro‐aging TOR (target of rapamycin) signaling pathway on the anti‐aging AMP‐activated protein kinase (AMPK/Snf1), increases the intracellular concentration of phosphatidic acid (PA), and decreases the concentration of triacylglycerols (TAG; the major form of energy storage in yeast and other eukaryotes); 2) PE5 mitigates the pro‐aging cAMP/PKA (cAMP/protein kinase A) signaling pathway, greatly elevates the concentration of PA, reduces the concentration of TAG, and rises the concentration of cardiolipin (CL; a phospholipid synthesized only within mitochondria in yeast and other eukaryotes); 3) PE6 targets currently unknown pro‐ and/or anti‐aging pathways of longevity regulation, increases the concentration of PA and phospholipids other than CL, and decreases the concentration of TAG; 4) PE8 weakens the inhibiting effect of the pro‐aging cAMP/PKA signaling pathway on the anti‐aging AMPK/Snf1, enlarges the concentrations of PA, CL and all other phospholipids, and lessens the concentration of TAG; 5) PE12 activates the anti‐aging protein kinase Rim15 (on which the pro‐aging TORC1 and cAMP/PKA signaling pathways converge), and decreases the concentrations of CL and TAG; and 6) PE21 attenuates the pro‐aging protein kinase Sch9 (which is activated by the pro‐aging TOR and Pkh1/2 signaling pathways), elevates the concentrations of PA and phospholipids other than CL, reduces the concentration of TAG, and attenuate an age‐related form of liponecrotic cell death.

Support or Funding Information

Supported by NSERC of Canada.
Serological Diagnosis of Mycoplasma via Lipoprotein Specific Electrochemical Biosensor
Authors: Jacobs Jena , Bonham Andrew J
Published: November 2023

We have developed a novel electrochemical biosensor capable of detecting pathogenic Mycoplasma for use in academic, research, and clinical applications. Mycoplasma presents key health concerns; Mycoplasma pneumonia infects 2 million people every year, it is responsible for upper respiratory infections and “walking pneumonia”, while Mycoplasma genitalium, is responsible for a large amount of nongonococcal urethritis cases. Current methods of detecting such Mycoplasma strains, such as molecular‐based assays, PCR and immunological serological analysis, are time consuming, expensive, and minimally accurate. Improved serological analysis would be beneficial as effective serological analysis is not readily available in the United States and current analysis internationally only measures the host immune response and not the direct presence of the microorganism, which can produces false negative results. Here, we offer one solution to this challenge through means of direct detection of the Mycoplasma microorganism with a diagnostic electrochemical biosensor directed against a secreted 48 kDa lipoprotein, P48, which is common across many strains. Our aptamer‐based electrochemical DNA biosensor has been characterized and exhibits high specificity and sensitivity for P48 in human serum. This biosensor will allow us to potentially improve both prevention and diagnosis of Mycoplasma in patients who present a proposed infection.

Support or Funding Information

Support for this work comes from the Metropolitan State University of Denver Provost's and LAS Dean's offices.
A Novel and Robust Series of Organelle Fluorescent Probes
Authors: Rigden Gabrielle , Tourville Claudia , Lewis David , Hartsel Scott
Published: November 2023

We have developed and tested a series of organelle‐specific fluorescent probes based on the aminonaphthalimide fluorophore. The three probes presented here redistribute according to transmembrane voltage, pH or high cholesterol lipid domains. We have successfully used them to image mitochondria, lysosome/acidic endosomes and Golgi apparatus in multiple live mammalian cell lines as well as fungi including Candida biofilms by confocal microscopy. Compared to some of the most popular commercial probes, our probes possess several advantages: 1) they utilize the increasingly available but little used 405 nm laser for excitation yet due to a large Stokes shift can be visualized with green filter sets such as those used with GFP, 2) they equilibrate rapidly (seconds to minutes) with target organelles and respond rapidly to changing conditions such as mitochondrial depolarization, 3) they are extremely chemically stable and photostable allowing long‐term monitoring of live cell organelles and 3D confocal imaging as well as having a long shelf life, 4) they have low toxicity toward mammalian and fungal cells and, 5) they have high quantum yields with little self quenching allowing for quantitative and qualitative imaging in complete culture media with minimal interference from background and autofluorescence. These probes have the potential to be very useful in long‐term microscopic cell biology studies and other methods using live cells such as flow cytometry.

Support or Funding Information

Office of Research and Sponsored Programs‐ UW‐Eau Claire
Constructing Red‐Shifted Fluorescent Protein Sensors of Cellular Redox Status
Authors: Norcross Stevie , Trull Keelan , Snaider Jordan , Doan Sara , Tat Kiet , Huang Libai , Tantama Mathew
Published: November 2023

Redox status, which can vary over time and across subcellular compartments, is an important indicator of cell activity and health. Fluorescent protein sensors are particularly useful for the measurement of cellular redox dynamics because they are genetically encoded, they can be targeted to organelles, and they are typically non‐cytotoxic. For example, the reduction‐oxidation‐sensitive green fluorescent proteins (roGFPs) are sensors that measure cellular redox and can detect reactive oxygen species (ROS) [Hanson, G. T. et al., 2004, J. Biol. Chem. 279, 13044–13053]. The roGFP sensors encode two cysteines engineered in close proximity to the chromophore, and a change in fluorescence is observed upon reduction of the disulfide bond formed by the cysteines. Currently, these sensors emit green fluorescence. In order to enable detection of varying ROS dynamics across cellular compartments, red emitting variants are needed. Here, we describe efforts to develop roGFPs with red‐shifted emission via a Förster resonance energy transfer (FRET) relay strategy. Sensors were screened for FRET efficiency with steady‐state and time‐resolved fluorescence measurements. Functional sensors were characterized for efficient mitochondrial targeting and response to induced oxidative stress in live cells. We will present our progress towards demonstrating that these red‐shifted sensors and the green fluorescent roGFPs can be used to simultaneously monitor redox dynamics in different cellular compartments.
Development of an Arsenic Sensitive Bacterial Biosensor and San Diego Soil Testing
Authors: Magsumbol Alisson Mae , Magee Moriah S , Provost Joseph John
Published: November 2023

Arsenic occurs naturally and is found in either oxidation states of arsenite (As(III)) or arsenate (As(V)), depending on the amount quantity of dissolved oxygen that is present in the environments oil. Some A few options available for the detection of arsenic include liquid chromatography coupled with fluorescence detection, as well as various field kits, one of which will involve a reaction between mercuric bromide and a reduced form of arsenic containing water. The latter option will yield a color output proportional to the concentration of arsenic present. Another option that is available are bacterial biosensors. Bacterial biosensors are whole cells that can be engineered with wild‐type gene induced promoters to detect environmental contaminants. The vector can be further engineered to yield either a fluorescence or a color output, as well as have different sensitivities for the detection of the environmental contaminants. The detection sensitivity can depend on the type of vector used, the efficiency of the ribosome binding site(s), promoter region, and the substrate binding site(s). In this work, we attempted to create a biosensor that had an altered ribosome binding efficiency to output blue coloration in the presence of arsenic ranging 0.05–100 μg/L in potable water and soil. In addition, we examined two established biosensors using beta‐galactosidase and green fluorescence protein as the reporters (kind gifts from Dr. Jan Roelof van der Meer). To investigate the utility of each construct, we transformed DH5‐alpha and XL10‐gold bacteria with each construct and incubated the culture in a variety of time of incubations in several arsenic concentrations. We collected and analyzed soil from areas surrounding Mission Bay San Diego and Black Mountain San Diego, each reported to pose high levels of arsenic. Most sites were near detection limits; however, an increased presence of arsenic was determined for the Black Mountain site. This work shows the potential for biosensors and the ability for inexpensive field analysis of toxic compounds

Support or Funding Information

This research was supported in part by an NSF REU grant (CHE‐‐1460645; PI Talley).
High Dynamic‐Range LRET Biosensors of Rac1
Authors: Pham Ha Thi , Chen Ting , Miller Lawrence
Published: November 2023

Rac1, a member of Rho GTPase family, is well recognized for controlling a diverse range of cellular process, including cytoskeletal dynamics, cell migration and transcription. The diverse functions of Rac1 require a complex regulation of its subcellular location and interactions with multiple downstream targets. Over the past 10 years, rapid progress was made in understanding Rac1 signaling, in which FRET‐based biosensors using fluorescent proteins (FPs) play a vital role in quantifying the spatio‐temporal dynamics of the Rac1 nucleotide state in living cells. While proven, valuable research tools, FP biosensors of Rac1 and in general often have low signal‐to‐noise and/or dynamic range, which makes it very difficult to quantify small changes in activity. This problem can be overcome with lanthanide‐based FRET (LRET) biosensors that incorporate Tb(III) or Eu(III) complexes as energy transfer donors and FPs as acceptors. Lanthanide and lanthanide‐to‐FP sensitized emission signals have ms‐scale lifetimes that permit the use of time‐gated detection to eliminate non‐specific, ns‐scale autofluorescence and directly excited acceptor fluorescence. We prepared stably transformed NIH 3T3 fibroblast lines that express a single polypeptide consisting of the following elements (N to C): 1) EGFP; 2) p21‐binding domain (PBD); 3) a 30 nm α‐helix linker; 4) Escherichia coli dihydrofolate reductase (eDHFR); and 5) Rac1. The eDHFR moiety binds to a trimethorprim (TMP)‐Tb(III) complex heterodimer, completing the sensor. The presence of a rigid α‐helix linker keeps each end of the inactive sensor far apart, so there is a low baseline LRET signal. The single‐chain LRET sensors permit straightforward, two‐color ratiometric imaging of Rac1 activity. Moreover, a large difference in LRET signal (up to 25‐fold) between positive and negative controls was observed directly in sensor‐expressing fibroblasts grown in 96‐well plates. Single‐chain LRET affinity biosensors will permit both dynamic microscopic imaging and medium‐high throughput analysis of Rac1 or other protein activities in living cells.

Support or Funding Information

Funding provided by the National Institutes of Health, R01 GM081030.
Nicotine‐Induced Proteome of Arthrobacter nicotinovorans pAO1+
Authors: Mihasan Marius , Babii Cornelia , Darie Costel C
Published: November 2023

Nicotine alkaloid plays an important role in smoking addiction and has been found to be implicated in many biological processes. In addition, large quantities of waste with high concentrations of nicotine are generated during the tobacco‐manufacturing process and are simply discarded into the environment. The enzymes encoded by pAO1 megaplasmid of Arthrobacter nicotinovorans might offer a unique way of exploiting nicotine waste for the production of “green” chemicals and for understanding smoking addiction. Although the organization, function, origin and evolution of the pAO1 megaplasmid have been extensively studied, several key points such as regulation of the expression of the nicotine catabolic genes and nicotine transport or metabolic fate of the pyridine ring are still missing.

The current work attempts to identify all the nicotine induced proteins from Arthrobacter nicotinovorans pAO1+ by using using nano LC‐MS/MS. Arthrobacter nicotinovorans pAO1+ cells were grown to log phase on citrate medium and on citrate medium supplemented with 0,005% nicotine. The cells were lysed using the method described by Henne et al., J Proteome Res 2009, 8:1704–16. Cell free extracts were separated on 9–16% SDS‐PAGE maxi (20 × 20 cm) gradient gels and Commasie stained. The extracts from the bacteria grown on nicotine‐containing media showed several extra bands în the range of 60 and 30 kDa. One particularity of Arthrobacter nicotinovorans extract is the high abundance of small molecular weight protein of around 6.5 kDa. Full scale proteomic analysis is currently under way.

Identification of all the key regulators and enzymes of the nicotine catabolism in Arthrobacter nicotinovorans would allow a better understand smoking addiction, but to also a way to manipulate the nicotine catabolic pathway to treat tobacco waste and convert it into green chemicals.

Support or Funding Information

MM is supported by a Fulbright Senior Award from the the Romanian ‐ U.S. Fulbright Commission, 2017.
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 Information

Oklahoma 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 Information

This 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 Information

The Freedman Prize, Natural and Physical Sciences, 2016
Identifying the Structural Determinants of Negative Cooperativity in Phosphagen Kinases
Authors: Stock Katie Lauren , Kerwood Gentry , Fraga Dean Lauren
Published: November 2023

When an enzyme exists as a dimer, the two subunits of the dimer are able to cooperate in such a way where the binding of a substrate in one subunit affects the other subunit's ability to bind substrates. In the case of negative cooperativity, the binding of substrates in one subunit decreases the ability for the other subunit to bind substrates. Currently it is unknown what structural features within a specific dimer allow for negative cooperativity to occur. The enzyme family phosphagen kinases can be used as a model system to study negative cooperativity, as some phosphagen kinases possess negative cooperativity while others do not. In order to determine potential structural determinants of negative cooperativity, the protein sequences and structures of both negatively cooperative and non‐cooperative PKs were compared. The presence of a specific histidine residue in the N‐terminal was identified as a possible structural determinant of negative cooperativity in PKs. To examine whether this histidine residue is a genuine structural determinant of negative cooperativity, a series of mutants will be created and the cooperativity of each will be determined via Isothermal Titration Calorimetry. Successful discovery of the structural determinant(s) of negative cooperativity in phosphagen kinases will allow for a better understanding of how negative cooperativity functions as a whole.
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 Information

This 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 Information

This 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 Information

Support 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 Information

This 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

Background

It 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.

Objective

This study evaluates whether the expressions of matrix metalloproteinases (MMP2 and MMP9) induced by CsA is calcineurin isoform specific.

Methods and Results

Wild‐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.

Conclusions

These 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 Information

NIH 1R15GM11312001
STIM1 INTERACTS WITH THE VOLTAGE SENSOR (L‐CHANNEL) OF L6 SKELETAL MUSCLE CELL CULTURES
Authors: Pitake Saumitra , Ochs Raymond S
Published: November 2023

Store‐operated calcium entry (SOCE) is the process of external Ca2+ entry driven by depletion of intracellular (endoplasmic reticumum (ER)) Ca2+. Entry of Ca2+ is known to occur through the Orai1 channel, and the concentration of ER Ca2+ is sensed by the STIM1 protein. In L6 muscle cells, Ca2+ entry also occurs during stimulation (exciation contraction calcium entry, ECCE). This Ca2+ flow is known to occur through the L‐channel and is inhibited by diltiazem and dihydropyridines. Surprisingly, we found that these inhibitors also blocked SOCE. To further examine this, we conducted co‐immunoprecipitation studies to study the interaction of STIMI, Orail and the L‐channel. We found the L‐channel had significant association with Stiml when calcium was depleted. This interaction was prevented by the STIMI blocker, ML‐9, or by K‐depolarization. In addition, ECCE Ca2+ currents were attenuated in STIM1 knockdown myotubes, but not Orai1 knockdowns. Similarly, the STIM1 inhibitor ML‐9 inhibited ECCE Ca2+ currents, while the Orai1 inhibitor BTP‐2 had no effect. Both of these inhibitors blocked SOCE Ca2+ currents. We conclude that the L‐channel interacts with the STIM1 protein during SOCE, which was unexpected since the L‐channel is known to play a role only in excitation‐contraction calcium release and not the refilling of ER stores.