bluuebunny/crossref_metadata_2025_split huggingface dataset:
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 InformationSupported 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 InformationThis 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 InformationNational 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 InformationLaboratory 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 InformationSupported 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 InformationSupport 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 InformationOffice of Research and Sponsored Programs‐ UW‐Eau Claire
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 compoundsSupport or Funding InformationThis 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 InformationFunding 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 InformationMM is supported by a Fulbright Senior Award from the the Romanian ‐ U.S. Fulbright Commission, 2017.