bluuebunny/crossref_metadata_2025 huggingface dataset:
Gyrase Inhibition by Toxin‐Antitoxin Modules
Authors: Bourne Christina R , White John C , Dabadi Shivani , Muthuramalingam Meenakumari
Published: November 2023

Toxin‐Antitoxin (TA) modules are interacting cytosolic protein pairs found in most prokaryotes that can mediate metabolic adaptation by interacting with essential cellular machinery. TA modules are organized into families with highly similar structures yet poorly conserved sequences. Among these, the RelE/ParE superfamily has conserved protein folds but disparate cellular functions. Interactions of RelE‐type toxins with the ribosome, resulting in translational inhibition, have been well characterized. In contrast, the interactions of ParE toxins with their target, DNA gyrase, have remained elusive.

The objective of this study was to determine the potency of individual ParE toxin proteins with respect to gyrase inhibition. This was determined by changes in cell morphology when the toxins were over‐expressed, and directly using in vitro inhibition assays. Through these studies we have identified a severely truncated ParE toxin in Agrobacterium tumefaciens that maintains an ability to inhibit DNA gyrase. We have also discovered a sub‐family member within this organism that closely matches the E. coli YoeB translation‐inhibiting toxin, however, it effectively inhibits DNA gyrase. Continuing studies are evaluating the propensity of RelE/ParE superfamily members to interact with DNA gyrase. Among over‐expressed ParE toxins, characteristic patterns of filamentatous cell morphology are consistent with phylogenetic sequence analyses. In conclusion, these results unexpectedly revealed that selected members of this superfamily can carry out overlapping functions. Ongoing studies are pursuing shared sequence features dictating these resulting phenotypes, as well as the structural interactions between the toxins and their cellular target.

Support or Funding Information

This research was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103640.
Streptococcus mitis and Streptococcus oralis Mutate an ‘Essential’” Gene Upon Exposure to Daptomycin
Authors: Adams Hannah , Joyce Luke , Guan Ziqiang , Akins Ronda , Palmer Kelli
Published: November 2023

Viridans Group Streptococci (VGS) are Gram‐positive opportunistic pathogens which are part of the normal gastrointestinal and oropharyngeal flora. Clinically, VGS are causative agents of infective endocarditis and bacteremia in immuno‐suppressed and neutropenic patients. Multidrug resistance (MDR) is on the rise within the VGS, as many isolates are now resistant to ampicillin, fluoroquinolones and macrolides. As with most pathogens, novel treatment methods are needed to combat VGS infections. Daptomycin (DAP) is a lipopeptide antibiotic with bactericidal activity against Gram‐positive bacteria. DAP has been shown to be effective against MDR infections, including methicillin‐resistant Staphylococcus aureus (MRSA) and vancomycin‐resistant enterococci (VRE). Previously, Akins et al obtained DAP‐resistant VGS strains after overnight exposure to DAP in a simulated endocardial vegetation model. Using two Streptococcus oralis strains (1647 and 1648) and one Streptococcus mitis strain (1643), we demonstrated that DAP resistance up to 256 μg/mL could be obtained overnight in standard laboratory conditions with a single 4 μg/mL DAP exposure. Using whole genome sequencing, we identified loss‐of‐function mutations in the gene cdsA for all three VGS strains. These mutations reverted when the resistant strains were serially passaged until susceptibility was observed. cdsA is essential in better studied streptococci such as S. pneumoniae and S. pyogenes. CdsA is responsible for catalyzing the conversion of phosphatidic acid (PA) into cytidine diphosphate‐diacylglycerol (CDP‐DAG), which is a key intermediate in the biosynthesis of major phospholipids found in bacterial membranes, including phosphatidylglycerol (PG) and cardiolipin (CL). Using liquid chromatography/mass spectrometry (LC/MS), we confirmed CdsA loss‐of‐function mutations in each of the DAP‐resistant strains, as evidenced by the absence of PG and CL, and the accumulation of PA in these strains. In addition, we have identified a new glycolipid in our S. mitis strain. These data show that DAP resistance proceeds through a novel adaptive pathway in S. mitis and S. oralis as compared to MRSA and VRE; that cdsA is non‐essential in these VGS and that adaptation to DAP results in altered membrane lipid composition in these organisms.

Support or Funding Information

Start‐up funds from UT‐Dallas to KP and GM069338 and EY023666 from NIH to ZG.
Biochemical characterization of Type II Toxin‐Antitoxin module from Pseudomonas aeruginosa
Authors: Muthuramalingam Meenakumari , White John C , Bourne Christina R
Published: November 2023

To date numerous putative Toxin‐Antitoxin (TA) loci have been reported, and these consist of a dicistronic operon encoding two genes; one for antibacterial toxic protein and another for antitoxin which neutralizes its specific toxin (Leplae et al., 2011). TA systems have been reported to play numerous physiological roles, including formation of persister cells (Maisonneuve et al., 2011 & Li et al., 2016), stress resistance (Christensen et al., 2001), protection from bacteriophages (Koga et al., 2011) and regulation of biofilm formation (Wang et al., 2011). Further, overexpression of a Hig‐type toxin affects the virulence factor production that is crucial for modulating Pseudomonas infection (Wood TL, Wood TK. 2016).

The goal of this study is to characterize a highly conserved member of the ParE/RelE superfamily from Pseudomonas aeruginosa. Toxin and antitoxin genes were cloned into the pHERD shuttle expression vector for propagation in both E. coli and P. aeruginosa. Heterologous over‐expression of the toxin in E.coli promoted a filamentous phenotype and decreased viability based on enumerating the colony forming unit, while overexpression of the antitoxin did not affect the cell count or the phenotype. We developed purification strategies to purify RelE toxin in the absence of any binding partners, as well as the RelBE protein complex. Gel filtration purification revealed mixtures of dimers of both proteins in complex (RelB2E2) and also fractions with molecular weight matching the free RelE monomer mass. Upon examining the sequence of this RelE toxin, the longer helices associated with a ParE gyrase inhibiting toxin were evident. Experimental testing has clearly demonstrated the ability of this toxin to inhibit DNA gyrase, and studies are underway to evaluate potential ribonuclease function as expected from its annotation. However, based on the longer helices, the region important for specificity for cellular targets, we expect that this RelE toxin is instead a ParE toxin. Additionally we have been successful in obtaining crystals of RelE and evaluated their diffraction properties at the OU Macromolecular crystallography facility, which yielded weak reflections to 4.5 Å. Initial crystallization condition for the RelBE complex have been identified and current efforts are focused on optimizing these for analysis by crystallography. The determination of the structure of this TA module will be the first from P. aeruginosa. Our overall goal of combining structural features and functionality of this TA module will facilitate understanding its role in bacterial physiology, a promising approach for combating this multidrug resistant pathogen.

Support or Funding Information

This work was supported by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health.
The Structure of EmrE and Its Role in Antibiotic Resistance
Authors: Kenana Jaida , Langat Brenda , Kalicki Colin , Inthavong Emily , Kanna Arrihant
Published: November 2023

Antibiotic resistance occurs when bacteria evolve different methods of surviving in the presence of antibiotics such as quaternary cationic compounds (QCC). One mechanism that bacteria utilize to attenuate the impact of antibiotics is the use of efflux systems. These systems allow bacteria to expel toxins and other compounds. The bacteria Escherichia coli (E. coli) uses Ethidium multidrug resistance protein E (EmrE) as an efflux system. EmrE is a 110 amino acid protein classified as a small multidrug resistance transporter (SMR). It can be encoded on a chromosome, plasmid, or integron. EmrE transports substrates into the periplasm. In order to do that, EmrE exchanges protons to channel substrates. Those substrates can then be taken up by other efflux systems such as resistance‐nodulation‐division (RND) transporters to be exported to the outer membrane. EmrE is a dual‐topology membrane protein with the functional structure of a homodimer. However, the most functional orientation of the monomers, having either a parallel or antiparallel structure, within the dimer is under debate. Recent studies have concluded that the antiparallel structure is more stable. This structure consists of four transmembrane helical bundles. Helices 1, 2, and 3 reside at the core, where there is a single binding pocket in which all toxic molecules are transported. Some amino acids within the core are highly conserved, indicating that amino acids such as glutamate 14 and asparagine 104 of EmrE are present through several different bacteria. Glutamate 14 is essential to the function of EmrE because mutations at this position can result in differing rates of efflux. When glutamate 14 is mutated into cysteine, EmrE activity is abolished. Also, when glutamate 14 is mutated to asparagine, EmrE does not confer drug resistance. Instead, it slows the rate of EmrE and transports compounds to liposomes. Helix 4 of EmrE is lipid facing and protrudes from the core. Lipid facing amino acids are less conserved compared to those in helices 1, 2, and 3. Often times mutations to the lipid facing amino acids, such as tyrosine 40 being mutated to glycine, occur. This can result in EmrE having modified specificity to different substrates. Though the specific structure of EmrE has been controversial, its antiparallel structure and functionality has been confirmed to be vital in antibiotic resistance as an efflux system.

Support or Funding Information

This is a SMART Team project supported through the contributions of Dr. Joanna Slusky of the University of Kansas, the Milwaukee School of Engineering, and the Olathe Medical Professions 21st Century Academy.
Functional Analysis of Toxoplasma gondii cGMP‐Dependent Protein Kinase Isoforms Using an Auxin‐Inducible Degron System
Authors: Brown Kevin M. , Long Shaojun , Sibley L. David
Published: November 2023

Toxoplasma gondii is an important obligate intracellular pathogen and model apicomplexan parasite. To cause infection, T. gondii utilizes an active form of invasion that is regulated by cGMP‐dependent protein kinase (PKG). In T. gondii, two isoforms of PKG are produced from the PKG transcript due to alternative translation initiation methionines (M1, M103) (Donald and Liberator 2002). The full length 994 aa type I isoform (PKG I) associates with the plasma membrane via an N‐terminal dual acylation motif. The 892 aa type II isoform (PKG II) lacks the first 102 amino acids and is cytosolic. Whether these isoforms are both required and if they are redundant or functionally distinct has not been determined. To date, functional analyses of T. gondii PKG have relied on small molecule inhibitors of its activity. However, PKG inhibitors are not isoform‐specific and may have off‐target effects in both the parasite and host (Donald et al 2006). To study PKG and other essential genes, we have adapted the auxin‐inducible degron (AID) system (Nishimura et al 2009) for use in T. gondii. Using this system, we are able to target AID‐tagged proteins for proteasomal degradation by addition of auxin (indole‐3‐acetic acid) in strains that heterologously express the plant auxin receptor TIR‐1. Using CRISPR/CAS9 genome editing, we introduced a miniature‐AID (mAID) tag to the C‐terminus of PKG, allowing us to rapidly deplete PKG‐mAID isoforms simultaneously with auxin, resulting in parasite death. By complementing the PKG‐mAID strain with PKG isoform‐specific plasmid constructs, we were able to define unique roles for PKG I and II. Importantly, PKG I is necessary and sufficient for parasite fitness while PKG II is only partially able to support growth and is fully dispensable in the presence of PKG I. Interestingly, these roles could be reversed based the presence or absence of the 15 amino acid N‐terminal dual acylation motif of PKG I. Taken together, our results provide strong evidence that PKG I activity at the plasma membrane is required for T. gondii infectivity. Furthermore, the auxin/AID system offers a new and robust avenue for exploring essential gene functions in T. gondii.

Support or Funding Information

Postdoctoral Fellowship (KMB), American Heart Association, (15POST22130001)
Genesis of Antibiotic Resistance XXIII: The River Ganga (RG)‐Segment IVB, secede Hardy‐Weinberg‐Castle Equilibrium (HWCE), and concoct as cataclysmic epicenter of Antibiotic Resistance Pandemic (ARP)
Authors: Martinez Francisco , Fuentes Esai , Montoya Hector , Flores Jorge , Carbajal Carlos , Wickham Cheyenne , Lopez Christian , Johnson Dennie , Kannan Subburaj
Published: November 2023

An effort has been made to compare the water quality of the City of Eagle Pass Water Works and RG to determine the mechanism of induction of antibiotic resistance (AR) in bacteria (e.g. Fecal coliform bacteria (FC)) known to cause AR infectious diseases. For this purpose, a correlation – regression analysis was carried out to determine the dependency among the following variables: i. Nephelometric Turbidity Units (NTU), ii. Saprobic index (SI) Pantle and Buck 1955) and iii. Incidence of Fecal Coliform (MPN/100ml), F.C 160,000 and iv. Morbidity and Mortality rate due to AR pathogen induced infectious diseases. According to water quality data of RG Segment‐IVB[1], a positive correlation coefficient was observed between increases in NTU (~95.53NTU) paralleled with higher FC count (F.C. (MPN/100ml): ~ higher than 3000 – 6.5×104). Regression coefficient analysis (where y= a + bx) showed a slope=1. This phenomenon is attributed to the above normal Biological Oxygen Demand (BOD) ~ 95.04TPD due to the influx of an untreated sewage 1780 million liter/ day (MLD) (54 sewage drains) along with 22 Grossly Polluting Industries (GPI) discharging an average of 87 MLD(15.6 BOD), pose unknown quantities of mutagenic agents. SI, S=5.44[2](strongly polluted water) showed a positive correlation with FC count and NTU. Data on mortality and morbidity rate (M&M) due to incidence of AR pathogen induced infectious diseases in RG‐IVb by both Government of West Bengal and Government of India is incoherent. As a result, no correlation was found. Data analysis from this study shows that an alarming trend of ever increasing pollution loads with an unknown mutagenic potential. Taken together, in comparison to CEPWW water quality data, it is concluded that the aforesaid factors in RG IVb mount a cause for a serious concern as an a major contributing factor prompting an “Infinite allelic” model in AR gene(s), rustling a “Founder Effect” in FC with an allelic fix accentuating an ARP. Data analysis to be presented in EB 2017.

Support or Funding Information

Professional development funds to Subburaj Kannan
Genesis of Antibiotic Resistance XXV: Mitigation of antibiotics resistance pandemic (ARP) by immediate moratorium on effluent discharge practices from antibiotics manufacturing pharmaceutical industry
Authors: Carbajal Carlos , Fuentes Esai , Wickham Cheyenne , Lopez Christian , Carrizales Diana , Carrizales Tania , Jauregui Suzzette , Thomas Ryan , Lechler Raul , Beautnagel Joanathan , Johnson Dennie , Kannan Subburaj
Published: November 2023

To date, no substantive coefficient and regression analysis has been performed to explore and determine correlative data that is concerned with the cause and effect relationship of the incidence of antibiotic resistance in bacteria and the concentration of antibiotics in the effluents from antibiotic manufacturing processes in the pharmaceutical industry. In order to actualize correlative data, four variables were identified and compared to ascertain the cause and effect relationship as may contribute to ARP. The following variables were prosecuted: j Concentration of the Active Pharmaceutical Ingredients (API), break down of intermediate / metabolite(s), decomposition isomer(s) in the effluents discharged by the antibiotics manufacturing industries (Global Scale: India, China, United States, Turkey, France, Nigeria, and Brazil); k Evolution of AR resistance pathogen as determined by Culture‐Sensitivity(C‐S lab) (phenotypic/genotypic); l Epidemiological data on morbidity‐mortality rate due to the incidence of AR pathogen(s) induced infectious diseases (county‐village/city/health district/state/country/continental); and m Detection of API, decomposition isomers (active vs. inactive@ based on Fenton Reaction) in the water resources [e.g.: ground water table, surface, stagnant, storm water runoff, bioremediation by chlorination/membrane filtration/ Ozonization / Flocculation / advanced oxidation / portable water – purified / nonpurified]. Based on the correlation coefficient analysis, a linear – positive correlation was observed between variable j and k. Though the data for variable l was limited, no correlation was observed between variable j and l (where r = 0). A similar trend was observed between the variables j and m (where r = 0). The regression coefficient among the variables was not symmetric as bxy≠byx. Data sources were Appl Envir Micro 64(9)3499–3502(1998); 76, 3444–3451(2010); Water Res. 42(1–2) 307–317(2008); Env. Toxi.Chem. 28(12)2522–2527(2009). Until the “Best Available Techniques” for detoxification and reduction in the discharge of API from Ab manufacturing industries are implemented on a global scale, current practices pose plausible cause for concern for API. Data analysis to be presented in EB2017.

Support or Funding Information

Professional development funds to Subburaj Kannan
O‐GlcNAc is Critical for the Regulation of Energy Metabolism
Authors: Tan Ee Phie , McGreal Steven , Graw Stefan , Tessman Robert , Koppel Scott , Dhakal Pramod , Zhang Zhen , Machacek Miranda , Soares Michael J , Zachara Natasha , Koestler Devin , Peterson Kenneth R , Thyfault John P , Swerdlow Russell H , Kasturi Partha , Apte Udayan , Slawson Chad
Published: November 2023

O‐linked N‐acetylglucosamine (O‐GlcNAc) is the attachment of a single β‐N‐acetylglucosamine to serine/threonine amino acid residues of nuclear, cytoplasmic, and mitochondrial proteins. The modification is sensitive to changes in the cellular environment and is dynamically regulated by the opposing functions of two specific enzymes; O‐GlcNAc transferase (OGT) adds the modification, whereas, O‐GlcNAcase (OGA) removes it. Disruptions in O‐GlcNAc cycling contribute to diseases such as neurodegeneration. Dysfunctional mitochondria lead to neurodegenerative diseases, and importantly, O‐GlcNAcylation regulates mitochondrial function. Previously, we found in a proteomic‐based study that OGT or OGA overexpression disrupted mitochondrial protein expression, including electron transport chain (ETC) and TCA cycle proteins. Here, we hypothesized that prolonged elevations in cellular O‐GlcNAc levels would decrease mitochondrial function. We elevated cellular O‐GlcNAc levels by either treating SH‐SY5Y cells with low levels of Glucosamine (GlcN), the metabolic substrate of OGT, or OGA inhibitor Thiamet‐G (TMG). We found that mitochondrial respiration and ATP production rates are lower in these cells. Additionally, these cells produce significantly less reactive oxygen species (ROS). We found that mitochondria were longer and mitochondrial fusion/fission protein expressions were decreased. Next, we performed RNA‐Seq analysis and found that 21 mitochondrial‐encoded genes are affected by the treatment. We found the nuclear factor erythroid 2 (NRF2) pathway that controls cellular ROS response was down regulated in these O‐GlcNAc elevated cells. Next, we found that C57B/6J mice dosed with TMG display a similar phenotype as SY5Y cells. Mitochondria isolated from mice brain had lower respiration and ROS levels, and decreased NRF2 response compared to control. Altogether, our results point towards prolonged elevations of O‐GlcNAc modulating mitochondrial function by influencing mitochondrial transcriptional programs, and that O‐GlcNAc affects ROS production likely via adjusting ETC activity and skewing its substrate (NAD+/NADH) availability. Furthermore, prolonged elevations of O‐GlcNAc impacts mitochondrial ROS production influencing the NRF2 antioxidant response. Importantly, these findings demonstrate the critical role for O‐GlcNAc cycling in the regulation of metabolism and will provide new insights into metabolic diseases such as Alzheimer's.

Support or Funding Information

Biomedical Research Training Program Fellowship, Mabel A. Woodyard Fellowship, National Institute of Diabetes and Digestive and Kidney Diseases R01DK100595 to C. Slawson
Inflammatory stress induces a biphasic Nrf2 activation in neuronal cells
Authors: Krepel Stacey A , O'Hara Lars , Mangum Sally , Hogan Mary B , Kuhn Thomas B
Published: November 2023

Persistent inflammatory and oxidative stress (IOS) is largely responsible for the progressive loss of neuronal integrity and connectivity underlying the continuous decline of cognitive function associated with most chronic CNS disorders as well as normal aging. The transcription factor Nrf2 plays a pivotal role in the antioxidant defense of non‐neuronal and neuronal cells. Nrf2 remains sequestered in the cytosol of cells under basal conditions tightly bound to its repressor Keap1. Upon oxidative stress, Nrf2 dissociates from Keap1 and translocates into the nucleus to stimulate transcription of antioxidant defense mechanisms. It is not yet clear how duration and extent of IOS impact Nrf2 activation and nuclear translocation in neuronal cells. Exposure of SH‐SY5Y human neuroblastoma to 200 ng/ml TNFa caused a time dependent Nrf2 translocation (2 fold increase) into the nucleus peaking 4 h after addition. However, nuclear Nrf2 decreased and remained at 50% of peak maximum for a prolonged time period. A presence of 50 ng/ml TNFa resulted in the highest accumulation of Nrf2 in the nucleus—giving a 53% increase of translocation relative to the control—compared to higher concentrations (100 to 400 ng/ml) even over prolonged periods of time. Immunocytochemistry against Nrf2 corroborated these findings. Additionally, prolonged exposure of SH‐SY5Y cells to TNFa (24 h) at concentrations 200 ng/mL or higher resulted in significant cell death due to oxidative stress. At high concentrations of 200 ng/mL and 400 ng/mL, even short treatment times of 1 hour resulted in the respective 52% and 112% reduction in Nrf2 translocation relative to the peak translocation achieved at 50 ng/mL. Together these findings suggest that Nrf2‐mediated antioxidant defenses in response to high concentrations of either chronic or acute TNFa exposure were insufficient and even depressed. One of the many health benefits of nutrition rests on the capacity of distinct botanicals, in particular polyphenols, to directly act on biochemical mechanisms rather than through passive antioxidant capacities. We demonstrated that supplementation of SH‐SY5Y neuroblastoma cells with extracts obtained from Alaska wild bog blueberries (5 mg/ml) increased neuronal viability in the prolonged presence of 200 ng/mL TNFa. Surprisingly, blueberry extracts prevent Nrf2 nuclear accumulation upon exposure to TNFa. Understanding the regulation of Nrf2 through botanicals could provide vital insight for developing neuroprotective strategies applicable to an array of neurodegenerative diseases.

Support or Funding Information

Supported in part by NIH grants RL56M118990, 1UL16M18991, TL4GM118992, and generous donations from the Alzheimer's Resources of Alaska. The work is solely the responsibility of the authors and does not necessarily represent the official view of the National Institutes of Health
Exploring the Role of Trehalose‐6‐phosphate Synthase in Oxidation and Desiccation Stress Tolerance of Fusarium verticillioides
Authors: Oberlie Nicole R. , McMillan Sarah D. , Pierson Paige N. , Bonich Nicholas S. , Brown Daren W. , McQuade Kristi L.
Published: November 2023

Fusarium verticillioides is a pathogenic filamentous fungus that primarily affects maize. We are exploring stress response mechanisms in F. verticillioides, particularly the role of the disaccharide trehalose. Trehalose‐6‐phosphate synthase, coded for by the TPS1 gene, catalyzes the first of two steps in trehalose synthesis. Past work in our lab indicated that an F. verticillioides Δtps1 mutant produces no trehalose and has reduced pathogenicity against maize. We report here that the Δtps1 mutant is sensitive to both oxidative and desiccation stress, suggesting a role for trehalose in stress tolerance. To determine if the stress sensitivity observed in this strain is due to the absence of trehalose or to the lack of a secondary function(s) of the enzyme, we are exploring stress response and sensitivity of a deletion mutant complemented with a mutant of TPS1 encoding a form of the enzyme expected to be catalytically inactive.
Lipid Stress Alters Cell Distribution, Traffic, and Desensitization Properties of Melanocortin‐4 Receptor, a GPCR Involved in Appetite Control
Authors: Cooney Kimberly , Molden Brent , Russell Susan , Baldini Giulia
Published: November 2023

Melanocortin‐4 Receptor (MC4R) is a G‐protein coupled receptor expressed in the brain hypothalamus where it regulates food intake and energy expenditure. MC4R signals in response to its natural agonist, α‐MSH, by increasing intracellular cAMP. Obesity is often associated with dyslipidemia, a condition where the concentration of circulating Non‐Esterified Fatty Acids (NEFAs), such as palmitic acid, is increased. It is not clear how a high‐fat diet effects the function of MC4R. We have modeled effects of lipid stress on MC4R function by exposing neuronal Neuro2A cells and immortalized hypothalamic mHypoE‐42 neurons to elevated concentrations of palmitate within the physiological range. Using confocal microscopy we found that lipid stress changes the cell shape of Neuro2A cells and inhibits internalization of transferrin receptor both in these cells and in the mHypoE‐42 neurons. Super‐resolution fluorescence microscopy indicates that exposure to lipid stress inhibits transit of tagged HA‐MC4R‐GFP to early endosomes by halting exit of the receptor from clathrin‐coated pits. In addition, lipid stress blunts desensitization of MC4R in response to the synthetic agonist Melanotan II (MTII). A single intraperitoneal injection of MTII decreases weight in obese male C57BL/6J mice exposed to high fat diet, but not in lean mice. These studies provide evidence that lipid stress may unexpectedly promote an increased response to anti‐obesity treatments targeted to MC4R by changing traffic of the receptor.

Support or Funding Information

This work was funded by NIH Grants R01‐DK102206 (to G.B.) and by Intramural Funding Support from the UAMS Research Council
′Ome is where the Wound is: Biomarkers of Healing in Chronic Diabetic Foot Ulcers
Authors: Glucksman Marc , Philibert Keith , Shao Xiaomeng , Yang Charlie , Ortiz Jacque , Wu Stephanie
Published: November 2023

Of the approximate 30 million Americans that suffer from Type 2 diabetes, 25% will develop a foot ulcer during their lifetime that often exhibit impaired healing with a cost of diagnosis and treatment exceeding $250 billion per year. These chronic wounds require continuous care and treatment to promote healing and prevent infection. To provide insight into the biochemical and molecular mechanisms underlying diabetic foot ulceration that are currently unclear, we sought to define biomarkers as an objective biologic measurement and evaluation of the normal versus pathological biological processes leading to wound healing. The objective is to discover and validate proteins that may predict what patient will heal or not. These biomarkers can thus be used for diagnosis, prognosis and for targeted therapies. Our approach was to obtain patient plasma samples from current diabetic and non‐diabetic patients with foot ulcers and utilize both genomic and proteomic approaches to discover lead molecules.

Debridement of the wound yielded the sample, blood collected (aliquot of whole blood or serum was for RNA analysis), centrifugation and protease inhibitors added, aliquoted and frozen. One aliquot was used for Luminex 18 flow cytometry‐based cytokine analyses. Another aliquot was immunodepleted of the 12 most abundant proteins comprising 95% of total plasma protein content and separated and identified by electrospray ionization mass spectrometry. Bioinformatics was then used for protein identification and quantification of samples. Patients returned bimonthly for medical assessment: wound size, cardiovascular, diabetes, and physical data with follow‐up samples obtained and processed accordingly.

The proteomics and genomics discovery phases yielded an increase in RANTES (CCL5), CXCL2 (GRO2), and Leucine Rich Alpha‐2‐ glycoprotein in non‐healing patients whereas there was an increase in interleukin IL‐8, granulocyte colony stimulating factor (G‐CSF), colony stimulating factor‐2, matrix metalloprotease 7, Alpha‐2‐HS‐glycoprotein, and Kininogen 1 in healing patients. We are currently validating these results in archived patient samples and assaying more patients to yield a further understanding of the factors for prediction of wound healing in diabetic foot ulcers. Identification of biomarkers of wound healing in DFU will allow for better clinical diagnosis, treatments and new targets, to prevent complications and non‐traumatic amputations to improve patient quality of life and reduce financial burdens.

Support or Funding Information

Supported by a Pilot Grant from RFUMS/DePaul University and NIH OD010662 (MJG).
Regulation of Hepatic Inflammation by Folic Acid in Non‐alcoholic Fatty Liver Disease (NAFLD)
Authors: Sid Victoria , Shang Yvette , Siow Chris , Karmin O
Published: November 2023

Non‐alcoholic fatty liver disease (NAFLD) is a multifaceted liver disorder that often exists with the comorbidities of obesity and type 2 diabetes. The histopathology of NAFLD is characterized by steatosis, inflammation, fibrosis, and liver injury. Chronic hepatic inflammation is an important pathogenic mediator of NAFLD that contributes to disease severity. The progression of NAFLD may be associated with development of cardiovascular disease which is the primary cause of death in patients. Dietary supplementation of vitamins is important for health maintenance, and has been suggested to be a beneficial strategy for NAFLD management. Folic acid is a water soluble B vitamin that has been demonstrated to have lipid‐lowering and anti‐oxidant effects. The objective of our study was to investigate the effects of folic acid supplementation on hepatic inflammation and to identify the underlying mechanisms. Male C57Bl/6J mice were fed a control diet (10% kcal fat), a high‐fat diet (60% kcal fat), or a high‐fat diet supplemented with folic acid (26mg/kg diet) for a 5 week period. High‐fat diet feeding induced a significant body weight gain and increased aggregation of inflammatory foci in the liver. Folic acid supplementation did not alter the body weight of mice fed a high‐fat diet. However, folic acid supplementation reduced the number of inflammatory foci as well as lipid vacuoles in the liver of high‐fat diet fed mice. This correlated with reduced expression of pro‐inflammatory cytokines mediated through attenuation of NF‐κB transcription activity, an inflammatory transcriptional regulator. Our results suggest that folic acid supplementation can alleviate the hepatic inflammatory response induced by chronic consumption of high‐fat diets, which may contribute to the hepatoprotective effect by folic acid.

Support or Funding Information

This study was supported, in part, by NSERC and CIHR.
Charecterization of a Glycerophosphocholine Acyltransferase (Gpc1), a Novel Enzyme in Phosphatidylcholine Biosynthesis in Saccharomyces cerevisiae
Authors: Anaokar Sanket , Lager Ida , Glab Bartosz , Banas Antoni , Stymne Sten , Patton‐Vogt Jana
Published: November 2023

Phosphatidylcholine (PC) is the major phospholipid found in most eukaryotic membranes. The maintenance of membrane composition through regulated synthesis and turnover of phosphotidylcholine is crucial to cell functioning. We have identified a novel enzyme glycerophosphosphocholine acyltransferase (Gpc1) that expands our understanding of PC biosynthesis and remodeling. Gpc1 acylates glycerophosphocholine (GPC), a product of complete deacylation of PC to Lysophosphotidylcholine (LysoPC), which can subsequently be converted to PC by lysophospholipd acyltransferase, Ale1. The yeast knock‐out collection was screened in order to find the Gpc1‐encoding gene. In vitro assays confirm that Gpc1 is an acyltransferase involved in PC biosynthesis. In addition, metabolic labeling studies performed in various strain backgrounds in which the established pathways for PC biosynthesis were attenuated indicate an in vivo role for Gpc1 in PC biosynthesis. Factors and growth conditions that regulate GPC1 expression are being explained. Gpc1 belongs to a new protein family as it shows no similarities to the known acyltransferases or transacylases. Gpc1 homologs are found in multiple organism including plants and medically important pathogenic fungi, but they are absent in vertebrates. Overall, we have successfully identified Gpc1, a glycerophosphocholine acyltransferase encoding gene that contributes to PC biosynthesis in the cell.

Support or Funding Information

National institute of Health Grant NIH R15 GM104876
The Nem1‐Spo7/Pah1 Phosphatase Cascade is Negatively Regulated by Protein Kinase A
Authors: Su Wen‐Min , Han Gil‐Soo , Carman George M
Published: November 2023

Yeast Pah1 phosphatidate phosphatase, which dephosphorylates phosphatidate to generate diacylglycerol, catalyzes the penultimate step in the synthesis of triacylglycerol. The translocation of Pah1 from cytosol to ER membrane is essential for its function and is strictly dependent on the nuclear/ER membrane‐associated Nem1‐Spo7 protein phosphatase complex. Dephosphorylation of Pah1 by Nem1‐Spo7 enables its membrane association, and also stimulates its phosphatidate phosphatase activity. Previous work has shown that protein kinase A phosphorylates Pah1 to inhibit its function in lipid metabolism. Here, we showed that Nem1, the catalytic subunit of the protein phosphatase complex, is also phosphorylated by protein kinase A. The kinase phosphorylated Nem1 on multiple serine residues and caused a 35% decrease in its protein phosphatase activity for phospho‐Pah1. The Nem1 synthetic peptides KRNRGSNASEN (Vmax/Km = 2.2 μmol min−1 mg−1 mM−1) and RPRSYSKSELS (Vmax/Km = 94.9 μmol min−1 mg−1 mM−1) that contain Ser‐140 and Ser‐210, respectively, were phosphorylated by protein kinase A. Site‐direct mutagenesis of Ser‐140 and Ser‐210 in Nem1 followed by phosphopeptide analysis showed that Ser‐210 was the major protein kinase A target site. The alanine mutation of Ser‐210 caused a 50% reduction in the in vivo phosphorylation state of Nem1. These data indicated that protein kinase A plays a negative regulatory role on the activity of the Nem1‐Spo7/Pah1 phosphatase cascade.

Support or Funding Information

Supported by NIH grant GM050679
Crystal Structure of LCAT Bound to a Small Molecule Allosteric Activator Reveals its Active Conformation
Authors: Manthei Kelly A. , Yang Shyh‐Ming , Chang Louise , Freeman Lita A. , Baljinnyam Bolormaa , Shen Min , Maloney David J. , Remaley Alan T. , Jadhav Ajit , Tesmer John J.G.
Published: November 2023

There is an inverse relationship between risk for coronary heart disease (CHD) and the level of high density lipoprotein cholesterol (HDL‐C). As CHD is one of the leading causes of death worldwide, there is great interest in mechanisms to raise HDL‐C for treatment of CHD. Lecithin:cholesterol acyltransferase (LCAT) catalyzes the transfer of an acyl chain from phosphatidylcholine to cholesterol to create a cholesteryl ester (CE) in the process of reverse cholesterol transport. The formation of the more hydrophobic CE leads to the maturation of HDL from discoidal to spherical and promotes the clearance of cholesterol from arterial plaques. Mutations in LCAT are known to cause two genetic diseases: fish eye disease (FED) and familial LCAT deficiency (FLD). Individuals with either FED or FLD have low HDL‐C and corneal opacities, while FLD patients ultimately develop renal failure. Recombinant LCAT and LCAT‐activating compounds are being investigated as treatments for both CHD and FLD. However, only recently has any structural information on LCAT been available to guide development of these therapies. Based on recent crystal structures and hydrogen deuterium exchange data, it is now apparent that LCAT has a dynamic lid that is able to protect the LCAT active site from solvent. This lid likely plays a critical role in activation of LCAT by apolipoprotein A‐I, the major protein component of HDLs. We report on a new structure of LCAT bound to an LCAT‐activating compound as well as an acyl intermediate state mimic, isopropyl dodecylfluorophosphonate. The combination of these two ligands promotes a lid‐open conformation. We confirmed that the compound binds to an allosteric site via site directed mutants that both eliminate binding and increase the thermal stability of LCAT. These studies pave the way for the design of rational drugs or improved biotherapeutics that can be used to treat CHD and FLD and augment reverse cholesterol transport. The new structure also allows for a better understanding of the acyl binding site of LCAT and its catalytic mechanism.

Support or Funding Information

This work was supported by US National Institutes of Health (NIH) grants HL122416 and Postdoctoral Fellowship F32HL131288 (K.A.M.).
Energy substrate levels and metabolic changes in skeletal muscle underlie increased activity and improved exercise performance in liver fatty acid‐binding protein null mice
Authors: Xu Heli , Gajda Angela , Zhou Yin Xiu , Fatima Anam , Storch Judith
Published: November 2023

Liver fatty acid‐binding protein (LFABP, FABP1) binds long chain fatty acids with high affinity, and is abundantly expressed in the liver and small intestine. Although LFABP is thought to function in intracellular lipid trafficking, studies of LFABP null (LFABP−/−) mice have also demonstrated a role in regulation of systemic energy homeostasis. We and others have reported that LFABP−/− mice become more obese than wild‐type (WT) mice upon high fat feeding. Despite increased body weight and fat mass, however, and in marked contrast to the drastic decrease in exercise capacity in high fat‐fed WT mice, LFABP−/− mice were able to maintain their endurance exercise performance, showing an approximate doubling of running distance compared with the WT mice. Therefore, to understand this surprising exercise phenotype, in the present studies we focused on the metabolic alterations in the skeletal muscle secondary to LFABP ablation. The results indicate significantly increased triglyceride (TG) content and glycogen content in the skeletal muscle of LFABP−/− mice. In addition, higher mitochondrial enzyme activities, in both the tricarboxylic acid (TCA) cycle and the electron transport chain, suggest that LFABP−/− muscle has greater energy substrate availability and greater capacity for substrate oxidation compared with WT muscle. Moreover, consistent with the findings that their muscle Pgc1α and Pparα mRNA levels were upregulated, LFABP−/−mice had an increased fatty acid oxidation rate in the skeletal muscle compared with WT mice. Interestingly, however, substrate utilization during exercise showed no differences between LFABP−/− and WT mice, as examined by respiratory exchange ratio, exercise‐dependent decreases in muscle TG and glycogen stores, and 5′AMP‐activated protein kinase activation levels. Thus, the LFABP−/− and WT mice appear to have similar energy production during exercise, indicating that the markedly improved exercise capacity in the LFABP−/− mice is related to their higher muscle substrate levels prior to exercise. Overall, the results show that although it is not expressed in muscle, LFABP ablation in the liver and intestine is associated with alterations in skeletal muscle energy metabolism and energy substrate levels. These changes may be due, in part, to liver‐and/or intestine‐derived mediators that contribute to the greater TG and glycogen availability and increased mitochondrial function, thereby preventing the high fat feeding‐induced decrease in exercise capacity.

Support or Funding Information

This work was supported by National Institutes of Health Grant DK‐38389 from NIDDK, and by funds from the New Jersey Agricultural Experiment Station (to J. S.).
Myopalladin's Role in Cardiac Muscle Function and Disease
Authors: Kadarla Vinay Kumar , Bigge Brae Marie , Beck Moriah Rene
Published: November 2023

Decades of research have provided fundamental insight into the human heart's structure and function. Yet, most cardiac malformations remain a mystery as scientists and clinicians continue to examine how inherited mutations and aging affect the normal biological functions of proteins associated with cardiac dysfunction. Recently, mutations in the muscle protein myopalladin have been linked to the pathogenesis of cardiomyopathy. Myopalladin is a sarcomeric protein that is thought to have an important role in maintaining sarcomere structure, signaling and regulation of gene expression in response to muscle stress. Myopalladin and palladin belong to a family of closely related immunoglobulin (Ig)‐domain‐containing proteins that have essential, but unclear roles in organizing the actin cytoskeleton. Recent work in the Beck lab has shown that the C‐terminal Ig domains of palladin bind directly to actin and increases both the rate of actin polymerization and the stability of actin filaments. The fact that a number of mutations in myopalladin are located within the analogous actin‐binding region suggests that a disruption in actin regulation may occur in cardiomyopathy. Thus, we hypothesized that myopalldin also binds directly to actin and increases both the rate of actin polymerization and the stability of actin filaments. To study the capability of myopalladin to bind and crosslink actin filaments, co‐sedimentation assays were performed between purified F‐actin and myopalladin Ig domains at various concentrations. The results suggest that Ig3 domain of myopalladin is the minimal domain required for both the binding and bundling of F‐actin. Pyrene fluorescence was used to monitor the polymerization rate of G‐actin in the presence of increasing concentrations of the various Ig domain of myopalladin. Our data reveals that myopalladin increases both the rate and extent of actin polymerization. From our data, we propose that myopalladin is involved in sarcomere structure and function through regulation of actin dynamics. We suggest that myopalladin may act both as a scaffold, binding directly to actin and other actin binding proteins at the Z‐disc, and by regulating actin thin filament turnover.

Support or Funding Information

This project was supported by the Kansas INBRE under grant number P20 GM103418; The WSU Carl and Rozina Cassat Regional Institute on Aging Grant (2016); and Wichita State University.
The Effect of Intermolecular Interactions in the Elongation Rates of Actin Filament by Formins
Authors: Aydin Fikret , Courtemanche Naomi , Pollard Thomas D , Voth Gregory A
Published: November 2023

Formins are large proteins (120–220 kDa) with multiple domains that regulate the elongation of actin filaments. The FH2 domains (~400 amino acids) form the core structure of formins and bind the growing barbed end of actin filaments, which slow the rate of barbed end elongation depending on the formin bound to it. We use multiscale simulation techniques to investigate the interactions of three formins FH2 domains (fission yeast Cdc12, budding yeast Bni1p, mouse mDia1) and the barbed end of the actin filament to understand these formins polymerize actin at different rates. The simulations demonstrated that the elongation rates of formins can be affected by the dissimilarities in their structures and intermolecular interactions with the barbed end. In addition, we identified critical regions of formins that are significant for regulating the configuration of the barbed end of the filament. The interactions between FH2 domains and actin filament in these regions differ among the formins (such as salt bridge formation), which can contribute to variation in modulating the barbed‐end configuration. The coarse‐grained simulations predicted that the FH2 domains have different stabilities at the barbed‐end, thus they have different degrees of steric interference to the addition of actin monomer. This work can help to resolve the underlying molecular mechanisms used by different formins to assemble actin networks.
Designed Proteins Induce Formation of Nanocage‐containing Vesicles
Authors: Votteler Jörg , Ogohara Cassandra , Yi Sue , Hsia Yang , Nattermann Una , Belnap David M , King Neil P , Sundquist Wesley I
Published: November 2023

Complex biological processes are often performed by self‐organizing nanostructures comprising multiple classes of macromolecules. For example, enveloped virus are built by self‐assembling proteins that package other proteins and nucleic acids, and acquire a lipid envelope by budding through limiting membranes. Viral structural proteins contain the membrane binding, assembly and budding activities necessary to create these extracellular virus particles, which can transduce new cells and deliver their cargoes. These activities underlie the efficacy of viral vectors, which have been explored extensively as therapeutic delivery vehicles. However, the use of viral vectors for therapy can be restricted by their limited packaging capacity, safety concerns, and immunogenicity.

To expand the toolbox of candidate delivery systems and to test our understanding of the fundamental requirements for viral biogenesis, we have designed protein nanocages that direct their own release from human cells inside small vesicles in a manner that resembles viral assembly and release pathways. We refer to these vesicles as enveloped protein nanocages (EPNs). Robust EPN assembly and release required three elements: membrane binding, self‐assembly, and recruitment of the cellular ESCRT pathway (endosomal sorting complexes required for transport). We have identified 16 different combinations of membrane binding, self‐assembly, and ESCRT recruiting elements that can produce EPNs efficiently, indicating that the strategy is very general. Biochemical and cryo‐electron microscopic analysis of one EPN design, termed EPN‐01, revealed that the protein nanocages precisely matched the structure of the designed 60‐subunit self‐assembling scaffold. EPN‐01 was released from cells within small membrane enclosed vesicles that each contained multiple nanocages. EPNs that incorporated the vesicular stomatitis viral glycoprotein (VSV‐G) were able to fuse with new target cells, thereby transferring their cargos from one cell to another, as assayed by successful packaging and delivery of a β‐lactamase‐Vpr fusion protein. We are now using these systems to analyze how different variables affect nanocage envelopment and release, and as a starting point for creating new synthetic delivery systems that are based on the viral assembly principles.

Support or Funding Information

NIH P50 082545