bluuebunny/crossref_metadata_2025 huggingface dataset:
Metal Induced Conformational Changes of Alpha‐Synuclein and the Role of Ambient Oxygen
Authors: Lucas Heather R
Published: November 2023

Hypotheses surrounding the native conformational states of α‐synuclein (αS), which is known to be a key player in Parkinson's Disease (PD) pathology, have evolved in recent years to adopt both a monomeric and tetrameric form. Traditionally, the conformation of αS was described as an intrinsically disordered monomer that aggregates to form β‐sheet fibrils, potentially through oligomeric intermediates. Upon interaction with membranes, this monomeric conformation of αS is known to shift from its dynamic unfolded state to α‐helical. Tetrameric αS has been characterized as both α‐helical and aggregation resistant, likely a passive storage form of the protein. The pathological conformation of αS remains controversial but each functional fold and dysfunctional misfold must originate from the monomeric protein. Membrane‐protein interactions are thought to facilitate the transition of monomeric αS to the α‐helical tetramer due to the similarity in folding patterns.

Physiological levels of cerebral biometals, such as copper and iron, change in various regions of the brain with aging and with advanced disease states. Metal dyshomeostasis has long been linked to PD but most of the work predated the discovery of the tetramer and the acceptance of αS as being post‐translationally acetylated at the N‐terminus. The latter has shown biophysical variation in regards to both αS conformational dynamics and metal coordination. The presented research will highlight metal‐induced changes in the folding pattern of αS and the distinguishable differences observed between various metal oxidation states. Unique changes are observed in the protein secondary structure and the hydrodynamic radius upon the introduction of stoichiometric quantities of individual metal ions based on circular dichroism and dynamic light scattering analyses respectively. Under oxidatively stressed conditions, metal‐bound αS can promote the generation of reactive oxygen species. Immunoblotting techniques have revealed that metal‐bound αS can follow distinct pathways towards toxic oligomers or β‐sheet fibrils depending on the metal. Furthermore, a clear involvement of ambient oxygen in the production of post‐translational modifications has been observed. Such metal‐induced conformational changes result in altered membrane interactions, which could have physiological implications. Taken together, these results contribute to the ongoing conversation within the field about the role of metals and αS in PD progression.

Support or Funding Information

Supported by Virginia Commonwealth University, College of Humanities & Sciences
Optimization of the Paired Enzyme Assay Synthesizing UDP‐Xylose from UDP‐Glucose
Authors: Cook Matthew D , Culbertson Alan , Zabotina Olga
Published: November 2023

UDP‐xylose (UDPX) is the substrate used for xylosylation in most organisms. Xylosyl residues are components of vital molecules in the cell walls of plants and bacteria and in the extracellular matrices of animals. Despite the proliferation of xylosylated compounds, UDPX is a markedly expensive chemical which limits research potential into these areas. It is known that the pair of enzymes UDP‐glucose dehydrogenase (UGD) and UDPX synthase (UXS) can catalyze the set of reactions leading from UDP‐glucose, a much cheaper chemical, to UDPX. This project optimized the enzyme assay of both UGD and UXS to improve the efficiency and rate of UDPX synthesis in order to develop an economical means of producing UDPX. Additionally, the kinetics of the previously uncharacterized UGD and UXS isoforms used in the assay were investigated.

Enzyme assays were performed, systematically comparing the rates of reaction and final UDPX‐production efficiency under different assay compositions and component concentrations for both enzymes individually and then together. The UGD assay was optimized first. As a result of reacting UDPG to the product UDP‐glucuronic acid, two equivalents of NAD+ were reduced to NADH. The generation of NADH could be analyzed spectrophotometrically at a 340 nm wavelength. Results from optimization have assay efficiency approaching 100% and the rate of reaction has improved appreciably. The earliest assays reacted with a maximum rate of 1.4 × 10−5 umol UDPG/min/ug UGD. Optimized assays have improved to 1.0 × 10−3 umol UDPG/min/ug UGD, a nearly 100‐fold improvement. Work on UXS proceeds.

Support or Funding Information

NSF‐MCB Grant # 1121163
Development of a Thermostable ATP Hydrolysis Coupled Enzyme Activity Assay
Authors: Cumpian Kayleah L , Canny Marella D , Latham Michael P
Published: November 2023

Rad50, when complexed with Mre11 and Nbs1 (MRN complex), is an important early response protein for finding and initiating the repair process of DNA double strand breaks. If DNA breaks are not repaired, genomic instability and cancer can arise. Mre11 is a 3′‐ to 5′‐exonuclease, while Rad50 is an ATP binding and hydrolysis protein. When Rad50 binds ATP, it dramatically alters the global structure and therefore the activity of the MRN complex. A major focus of the laboratory is studying Mre11, Rad50 and the MR complex using solution state nuclear magnetic resonance (NMR) spectroscopy in an effort to decipher structure‐function relationships. Due to the large size of Mre11, Rad50, and the MR complex, it is advantageous to use high temperatures during NMR experiments to ensure optimum signal. Thus, heat stable Rad50 and Mre11 proteins from the thermophilic archaea P. furiousus, a common model system used in the field, are used for the structural studies. In order to fully understand structure‐function relationships in Rad50 and the MR complex, the ATP hydrolyzing (ATPase) activity of Rad50 is also being studied. This ATPase activity is crucial for facilitating double stranded DNA break repair. The gold standard method for monitoring ATPase activity is via an ATP hydrolysis coupled enzyme activity assay, where the activity of an ATPase is coupled to pyruvate kinase (PK) and lactate dehydrogenase (LDH) activities. Thereby, ATP hydrolysis is coupled to NADH oxidation, which can be monitored via fluorescence at 340 nm. To perform this kinetic assay at the high temperatures required for our thermostable Rad50 to function, LDH and PK must also be heat stable. Currently the only commercially available LDH and PK are from rabbits and other mammals which denature at high temperatures. To acquire heat stable LDH and PK, the two enzymes were cloned from the DNA of hyperthermophilic bacteria T. maritima into protein expression vectors, overexpressed in E. coli, and purified by standard chromatography techniques. The coupled enzyme activity was then optimized at > 60 °C in terms of substrates and enzyme concentrations before applying it to P. furiosus Rad50. Here we describe the high temperature ATPase assay development and optimization process and our survey of several Rad50 mutants.
Reactivity of Neuroglobin with H2S
Authors: Ruetz Markus , Kumutima Jacques , Filipovic Milos , Lehnert Nicolai , Banerjee Ruma
Published: November 2023

H2S is toxic at high concentrations due to the inhibition of cytochrome c oxidase. However, it is also an important signaling molecule. Inside the cell, H2S is mainly produced by three enzymes, cystathionine β‐synthase (CBS), cystathionine γ‐lyase (CSE) and 3‐mercaptopyruvate sulfur transferase (MST). The low steady‐state levels of H2S in tissues are maintained by the efficient oxidation of H2S by the mitochondrial sulfide oxidation pathway that converts H2S to thiosulfate and sulfate.[1]

Recently, we reported that H2S can be cleared by a noncanonical sulfide oxidation reaction catalyzed by hemoglobin, the oxygen carrier in blood.[2] In this study, we report that neurogloin, a relatively recently discovered member of the globin family also exhibits the capacity for sulfide oxidation. In contrast to hemoglobin and myoglobin, the heme cofactor in neuroglobin is coordinated by two histidine residues. The function of neuroglobin, which is primarily expressed in neuronal tissue, is still unclear. It has been suggested that neuroglobin plays a role in protecting neuronal cell from cell death.

In this study, we have elucidated the reactivity of human neuroglobin with H2S. Under aerobic conditions, H2S is oxidized by neuroglobin to hydropolysulfides and thiosulfate. EPR and resonance Raman spectroscopic analyses reveal that the heme cofactor is reduced during the reaction, following the initial formation of a ferric sulfide intermediate. To determine the effect of the bis‐histidine coordination on sulfide oxidation, the distal histidine was substituted by alanine by site directed mutagenesis to create a myoglobin‐like mutant. The H64A mutant shows a 4000‐fold increase in the bimolecular rate constant for sulfide binding as well as in the rate of formation of the oxidation products, thiosulfate and hydropolysulfides.
Spectroscopic insight into the mechanism of nickel‐substituted rubredoxin, a bioinspired hydrogenase mimic
Authors: Stevenson Michael James , Slater Jeffrey Worthington , Marguet Sean Conrad , Shafaat Hannah S
Published: November 2023

Nickel‐substituted rubredoxin (NiRd) has been shown to serve as a functional mimic of nickel‐containing hydrogenases, able to produce hydrogen gas with high catalytic rates. In the native enzyme, the nickel center is coordinated by four cysteine thiolates. A similar environment is seen in NiRd, which represents the first functional model to reproduce this coordination motif. This 52‐residue protein is stable under a wide range of conditions, making it well‐suited for catalytic hydrogen production or in future fuel cells. Although much is known about the structure and function of native rubredoxin and its variants, the catalytic mechanism for hydrogen generation remains unknown. It has been postulated that NiRd may follow a similar mechanism to the well‐studied [NiFe] hydrogenase, with a terminal, rather than a bridging, hydride on the nickel(III) center. Using multiple spectroscopic methods, structures of key catalytic intermediates have been elucidated and compared to those reported for the native [NiFe] hydrogenase. This study of NiRd not only resolves the mechanistic details of an efficient, robust, bioinspired catalyst, but also provides insight into proposed intermediates of the [NiFe] hydrogenase.

Support or Funding Information

The Ohio State University, The Institute for Materials Research, The National Science Foundation
Investigation of the Substrate Specificity of L‐Idonate Dehydrogenase By Site‐directed Mutagenesis
Authors: Steiner Steve , Terpening Allison , McCurdy Chelsea
Published: November 2023

The enzyme L‐idonate dehydrogenase (L‐IdDH) is an important enzyme in the pathway for the biosynthesis of tartaric acid from ascorbic acid in grapes. L‐IdDH is an NAD+/NADH‐dependent enzyme which catalyzes the conversion of L‐idonate to 5‐keto‐D‐gluconate. Analysis of the amino acid sequence indicates that L‐IdDH from Vitis vinifera is a member of the family of medium chain dehydrogenases/reductases and is 79% identical with the putative sorbitol dehydrogenase (SDH) from Arabidopsis thaliana. Despite the high degree of homology, each enzyme is specific for its substrate with neither enzyme showing activity toward the substrate of the other. One of the notable differences in the amino acid sequences of L‐IdDH compared to SDH is at position 42 where His is located in L‐IdDH in contrast to Tyr in SDH. It is possible that the cationic His in L‐IdDH is essential to the binding of the anionic substrates L‐idonate and 5‐keto‐D‐gluconate. For this study, site‐directed mutagenesis was performed to convert 42H to 42R and to 42Y in L‐IdDH. Assay results indicate that mutation of His (positive charge) to Arg (positive charge) causes a significant reduction in activity toward 5‐keto‐D‐gluconate and a mutation to Tyr (uncharged) at this position completely eliminates activity. These finding illustrate the critical role of 42H in the activity and substrate specificity of L‐IdDH.

Support or Funding Information

This work is supported by a grant from the Indiana Academy of Science.
Substrate specificity of the novel serine hydrolase, LipN, implicated in the virulence of Mycobacterium tuberculosis
Authors: Schemenauer Daniel , Johnson R. Jeremy
Published: November 2023

Tuberculosis (TB) remains one of the most prevalent diseases in the world, infecting one third of the world's population. The causative agent, Mycobacterium tuberculosis, relies on a plethora of lipases to maintain an infection in its host. With their central roles in infection, lipases have become a viable target for drug development, especially as drug targets for the dormant state of M. tuberculosis. This research aimed to determine the substrate specificity, biochemical properties, and potential structural information of LipN, one proposed mycobacterial lipase. Initially, wild type LipN was expressed in E. coli, purified to homogeneity, and its substrate specificity characterized against a library of 35 latent fluorophore substrates. Wild type LipN demonstrated the highest catalytic efficiency against small carbon chains with a kcat/Km over 105 M−1s−1 for its best single acetyl ester substrate and maintained high activity (kcat/Km > 104) with small polar groups, including esters and oxazole rings. LipN's preference for short nonpolar substrates (three carbons or less) and polar substituents suggests its physiological role as an esterase rather than a lipase. This specificity also suggests hydrogen‐bonding capabilities within the active site of LipN. To categorize the structural factors controlling this substrate specificity, nine variants of LipN were made with substitutions in predicted binding pocket and active site residues and their relative kinetics against the top three ester substrates were characterized. Based on this analysis, the serine and histidine residues of the catalytic triad were positively identified, with 100‐fold decreases in catalytic activity upon their substitution. The aspartate residue in the catalytic triad was tentatively assigned, as multiple aspartate substituted variants showed similar reductions in catalytic efficiency. Future work will be aimed at definitely assigning the catalytic triad as well obtaining a three‐dimensional structure of LipN to confirm the structural features controlling its strict substrate specificity.

Support or Funding Information

Funded by NIH 1R15GM110641‐01A1
Elucidating the Mechanism of Thiol Oxidase Activity of a B12‐trafficking Protein
Authors: Li Zhu , Shanmuganathan Aranganathan , Ruetz Markus , Lesniak Nick , Koutmos Markos , Banerjee Ruma
Published: November 2023

CblC is involved in an early step in cobalamin (or B12) processing following entry of the cofactor into the cytoplasm. CblC catalyzes glutathione‐dependent dealkylation of alkylcobalamin and reductive decyanation of cyanocobalamin (CNCbl). Recently, we found that Caenorhabdiitis elegans CblC (ceCblC) exhibits a robust thiol oxidase activity, converting GSH to GSSG accompanied by O2 scrubbing. This futile oxidative cycle is suppressed in wild‐type human CblC (hCblC) but significant in two of the pathogenic mutants, R161G and R161Q. In this study, we sought to understand the catalytic mechanism of the thiol oxidase activity of ceCblC using biochemical and structural analyses. We propose the following mechanism: i) the binding pocket of ceCblC is tighter than hCblC, which facilitates formation of a thiol‐ligated glutathionyl‐cob(II)alamin, (GS‐Co(II); ii) GS‐Co(II) is oxidized by superoxide ion to form glutathionyl‐cob(III)alamin which undergoes GSH‐dependent dethiolation catalyzed by ceCblC, iii) the product of dethiolation, cob(I)alamin, is subsequently oxidized in air to cob(II)alamin, and thereby, the redox cycling continues.

Support or Funding Information

This work was supported in part by the National Institutes of Health (DK45776) to RB and the American Heart Association (13SDG14560056) to MK
The Regulatory Subunit Type Iα of Protein Kinase A: A Study of Carney Complex and Acrodysostosis Mutations
Authors: Del Rio Jason C , Nielsen Cole R. , Taylor Susan S.
Published: November 2023

cAMP‐dependent Protein Kinase A (PKA), a tetrameric holoenzyme comprised of two Catalytic (C) and one dimer of Regulatory (R) subunits, is the prototypical kinase in response to intracellular cAMP. Of the four functionally non‐redundant R subunits (RIa, RIb, RIIa, RIIb), RIa is the dominant, ubiquitously expressed isoform and is the only R subunit which is embryonic lethal in homozygous mouse knockout models due to defective cardiogenesis at E8.5. Recently, two rare phenotypically dichotomous diseases have been linked to mutations in the same gene encoding RIa. Hindering the capacity of RIα to bind and respond to cAMP results in overregulation (inhibition) of the C subunit, a rare condition called Acrodysostosis type I (ACRDYS1), where patients display severe skeletal defects, mental retardation, and hormonal resistance. In contrast, patients with gain of function mutations result in Carney Complex (CNC) disease, caused by under‐regulation of C subunit activity, and present with lentigenosis, recurrent cutaneous and cardiac myxomas, and a Cushings‐like endocrinopathy. While the majority of CNC mutations result in happloinsufficiency via nonsense mediated decay (NMD), the mutations which survive NMD implore multiple mechanisms for disease manifestation. Using these disease mutations with functional effects of opposite magnitude will provide insight into the allosteric communication networks present within the RIa subunit necessary to tightly regulate the critical and finely tuned PKA signaling node for biological processes. Furthermore, structural studies of these two rare diseases will provide a pharmacological platform for rational drug design in their treatment.

Support or Funding Information

JCDR was supported in part by the UCSD Graduate Training Program in Cellular and Molecular Pharmacology through an institutional training grant from the National Institute of General Medical Sciences, T32 GM007752
Relationship in Broccoli Between Genetic Mapping and Small Molecule Profiling Using NMR Spectroscopy
Authors: Knagge Kevin , Yousef Gad , Winnike Jason , Blankenship Kyle , Thomas Aswathy , Brown Allan
Published: November 2023

Broccoli was analyzed using both genetic mapping and small molecule profiling by NMR spectroscopy. 125 separate families of a broccoli population were utilized for the analysis. Broccoli families were analyzed using NMR spectroscopy to obtain small molecule profiles. These profiles were generated by quantifying a number of compounds in over 600 broccoli spectra. The profiles were compared to genetic mapping data generated for each broccoli family to determine possible QTLs (quantitative trait loci) associated with variation in the small molecules detected. Variation in some sugars and amino acids ranging up to 15% could be accounted for by genetic variation and traced to certain QTLs.
Development of Algorithmic Techniques for Designing Electrochemical DNA Biosensors
Authors: Bonham Andrew J , Bulow Aviva J
Published: November 2023

Oligonucleotide‐based, electrochemical (E‐DNA) biosensors have been shown to be effective tools for detecting heavy metals, small molecule drugs, and protein targets. There is thus considerable interest in moving these biosensors from the lab bench to applied fields, such as medical, environmental, and food safety diagnostics. A significant hurdle to this transition is the development of effective methods to rapidly and effectively adapt E‐DNA biosensors to the detection of new targets of interest. While great strides have been made in computationally predicting the folded structures of DNA and RNA‐based biosensors, interpreting whether such predicted structures would be effective biosensors and using that knowledge to design functional E‐DNA biosensors ab initio remains challenging. To overcome this we have designed a tool, Fealden, which models potential E‐DNA biosensors using the framework of graph theory and automates the process of structure interpretation and sorting. Using this tool, a researcher can provide a core sequence of interest (such as a DNA recognition element or aptamer) and the software will generate optimized, functional E‐DNA biosensor designs for the sensitive detection of that target. For example, our approach has allowed order‐of‐magnitude improvement in the affinity of biosensors directed towards the antibiotic tobramycin, without human intervention in the design process. This software incorporates literature knowledge of oligonucleotide biosensor design constraints, such as the proximity of appended chemical moieties and predicted folding free energies, and can traverse a search space of thousands to millions of possible sequences to determine the “best candidate” biosensor. This work provides insights into applying an algorithmic approach to utilize the wide pool of existing aptamers as E‐DNA biosensors in practical applications spanning such diverse fields as food safety, environmental monitoring, and clinical diagnostics.

Support or Funding Information

Support for this work comes from the Metropolitan State University of Denver Provost's and LAS Dean's offices.
An Inexpensive and Effective Aptamer‐Based Evanescent Wave Biosensor
Authors: Mazin Ilia , Bonham Andrew J
Published: November 2023

We have developed an inexpensive and effective evanescent wave biosensor utilizing 3D printed scaffolding and readily available electronics. This biosensor consists of a 3D printed brace, an inexpensive LED light source and cylindrical lens to direct the light in an elliptical pattern, and an acrylic slide on which DNA is immobilized. This scaffold allows light to enter the slide at above the critical angle for total internal reflectance, ensuring that the immobilized DNA experiences excitation by an evanescent wave. If the immobilized DNA is an aptamer‐based biosensor with a fluorescent reporter tag, binding of target that is washed across the slide surface will result in characteristic and measurable fluorescence changes. The utility of this inexpensive DIY setup was evaluated with image‐analysis software to create dose‐response curves for the response of both a botulinum toxin serotype A‐binding aptamer biosensor and a uranium ion‐binding aptamer biosensor, and demonstrates the potential utility of these convenient evanescent wave biosensors.

Support or Funding Information

Support for this work comes from the Metropolitan State University of Denver Provost's and LAS Dean's offices.
Lanthanide‐based FRET biosensors for time‐gated imaging and detection of protein‐protein interactions in live mammalian cells
Authors: Chen Ting , Pham Ha , Miller Lawrence
Published: November 2023

Förster resonance energy transfer (FRET) using fluorescent proteins (FPs) as donors and acceptors is commonly used to microscopically image protein‐protein interactions in living cells. However, overlapping FP spectra lower signal‐to‐noise because the FRET‐sensitized acceptor emission signal is convoluted with donor bleedthrough and directly excited acceptor emission, or crosstalk. Quantitative FRET imaging is even more difficult when the local concentrations of donor‐labeled and acceptor labeled proteins are substantially different. We used the well‐studied interaction between FK binding protein 12 (FKBP12) and the rapamycin binding domain of m‐Tor (FRB) as a model system to show that lanthanide‐based biosensors can overcome the limitations of conventional FP‐FRET. The essential features of this approach include the use of luminescent Tb(III) complexes as FRET donors and the elimination of non‐specific fluorescence background that comes from time‐gated detection of long‐lived (~ms) Tb(III) and Tb(III)‐to‐FP emission signals. We prepared stably transformed NIH 3T3 fibroblast lines that express a single polypeptide consisting of the following elements (N to C): 1) FRB; 2) Escherichia coli dihydrofolate reductase (eDHFR); 3) a linker sequence; 4) GFP; and 5) FKBP12. A Tb(III) complex (TMP‐Lumi4) that selectively binds to eDHFR completes the sensor. The linker comprises alternating repeats of four glutamic acid residues followed by four arginine or lysine residues (ER/K) that form a rigid alpha helix in solution. Stochastic breaks in the helix permit close approach and binding of affinity elements positioned at each end. The effective concentration of each binding element depends on linker length and not solution concentration. Consequently, the fraction of two elements in the closed (high‐FRET) state depends on KD and linker length. We tested constructs with ER/K linker lengths of 10, 20 and 30 nm. Following addition of cell permeable TMP‐Lumi4 and rapamycin to culture medium, two‐color, time‐gated images showed significant, ~3‐fold increases in acceptor denominated FRET ratios relative to baseline (without rapamycin). The high sensor dynamic range permitted robust, time‐gated detection (Z'‐factor > 0.8) of the FKBP12/FRB interaction and its inhibition by ascomycin in cells grown in 96‐well plates.

Support or Funding Information

NIGMS R01 081030
Effect of Zhibai Dihuang Granule on anti‐inflammatory proteins associated with Yin deficiency heat syndrome
Authors: Mao Liangen , Liu Changming , Jiang Tingting , Wang Chong , Chen Zhongliang , Li Jicheng
Published: November 2023

This study aims to explore the effect of Zhibai Dihuang Granule (ZDG) on anti‐inflammatory proteins in the serum of Yin deficiency heat syndrome rats. The Yin deficiency syndrome rat model was established by triiodothyronine, and differentially expressed proteins in the serum of rats were analyzed by iTRAQ. The levels of anti‐inflammatory proteins such as Gelsolin (Gsn), Plasminogen (Plg), Fibrinogen alpha chain (Fga), and Thymosin beta‐4 (T‐β4) were significantly increased in the ZDG treated model rats, compared with the control rats. However, the level of Coagulation factor XIII A chain was significantly decreased in the ZDG treated group. Gsn has been found to remove the damaged tissue and release actin, thereby inhibiting cell apoptosis. Gsn was significantly increased in the ZDG treated group, indicating that ZDG may affect Gsn to inhibit cell apoptosis. Colla2 and Plg have been shown to regulate the blood rheology in the inflammatory response. Colla2 was significantly decreased and Plg wes significantly increased in the ZDG treated group, suggesting that ZDG can regulate the blood rheology of the inflammatory response by down‐regulating the Colla2 and up‐regulating Plg. Gsn and T‐β4 have been shown to promote the healing of inflammatory damage by regulating cytokines. Gsn and T‐β4 were significantly increased in the ZDG treated group, indicating that ZDG could inhibit apoptosis by regulating cytokines.

Conclusion

ZDP can treat Yin deficiency heat syndrome by removing damaged tissue, releasing actin and lipopolysaccharide, inhibiting cell apoptosis, regulating inflammatory factors and the blood rheology of inflammation, and promoting the healing of inflammatory damage.

Support or Funding Information

This work was supported by grants from the National Basic Research Program of China (No.2014CB543002), the National Natural Science Foundation of China (No.81573709, No.81273882)
Screening and identification of novel serum potential biomarkers for pulmonary tuberculosis by iTRAQ‐2D LC‐MS/MS
Authors: Jiang Tingting , Shi Liying , Li Xiang , Wei Liliang , Yang Su , Wang Chong , Liu Changming , Chen Zhongliang , Tu Huihui , Li Zhongjie , Li Jicheng
Published: November 2023

Pulmonary tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) poses great threat to human health. Delay in the diagnosis of TB may increase the severity and mortality, as well as increase the risk of transmission of the disease. Therefore, early diagnosis and treatment are important for the control of TB spread. The aim of this study was to discover novel biomarkers for TB. Differentially expressed proteins in the serum of patients with TB were screened and identified by iTRAQ‐2DLC‐MS/MS analysis. A total of 47 abnormal proteins were discovered in TB patients, compared with healthy controls, pneumonia and chronic obstructive pulmonary disease (COPD) patients. The levels of serum amyloid A (SAA), vitamin K‐dependent protein Z (PROZ), and C4b‐binding protein β chain (C4BPB) were significantly higher in TB patients (n=136) than healthy controls (n=66) (P<0.0001, P<0.0001, and P<0.0001, respectively), whereas, significantly lower levels of SAA, PROZ, and C4BPB were observed in TB patients compared with pneumonia patients (n=72) (P<0.0001, P<0.0001, and P<0.0001, respectively) and COPD patients (n=72) (P<0.0001, P<0.0001, and P=0.0016, respectively). After 6 months of treatment, the levels of SAA and PROZ were significantly increased (P=0.022 and P<0.0001, respectively), while the level of C4BPB was significantly decreased (P=0.0038) in treated TB cases (n=72). Significantly increased SAA and PROZ levels were observed in TB patients with cavitary lung lesions (P=0.006 and P=0.03, respectively), compared with TB patients with non‐cavitary lung lesions. Meanwhile, the serum concentrations of SAA were higher in TB patients with double lung lesions than TB patients with single lung lesions (P=0.003). The serum SAA levels were also found to be increased in smear positive TB patients than smear negative TB patients. Decision trees of TB were constructed, and receiver operating characteristic curve analysis revealed that the area under the curve value (AUC) of the diagnostic model combined by SAA, PROZ, and C4BPB was more accurate than the single serum protein. The AUC was 0.972, 0.928, 0.957, and 0.969, to discriminate the TB group from the healthy controls, pneumonia, COPD, and cured TB groups, respectively. Therefore, the results suggested that SAA, PROZ, and C4BPB may serve as potential biomarkers for TB. Our study could provide experimental data for the differential diagnosis of TB.

Support or Funding Information

This work was supported by grants from the National Basic Research Program of China (No.2014CB543002), the National Natural Science Foundation of China (No.81573709, No.81273882), and the scientific research fund project of South China University of Technology School of Medicine (NO. yxy2016006).
Investigation of Induced Obstructive Sleep Apnea (OSA) in Rat Atria using Mass Spectrometry based Proteomics
Authors: Channaveerappa Devika , Lux Jacob , Wormwood Kelly L. , McLerie Meredith , Panama Brian K. , Darie Costel C.
Published: November 2023

Obstructive sleep apnea (OSA) affects up to 24% of the adult population and is associated with several atrial diseases. It is associated with daytime sleepiness, headache, depression, hypertension, obesity, arthritis, type 2 diabetes, and heart disease. OSA is characterized by transient cessations in respiration lasting >10 seconds as a result of narrowing or occlusion of the upper airway during sleep. OSA severity is assessed by the apnea‐hypopnea index (AHI), or the average number of apneas (complete obstructions) or hypopneas (partial obstructions) per hour. An estimated 20% of adults have mild OSA (AHI 5–15) and 7% of adults have moderate (AHI 15–30) to severe (AHI>30) OSA, with 85% of patients remaining undiagnosed. Although clinical evidence linking OSA to proarrhythmaic atrial changes is well known, the specific molecular mechanisms by which OSA causes atrial disease remain elusive. To study the OSA‐induced cardiac changes, we have initiated a recently developed rat model which closely recapitulates the characteristics of OSA.

Male Sprague Dawley rats, aged 50–70 days, received surgically implanted tracheal balloons which were inflated to cause transient airway obstructions. Apnea groups experienced 60 apneas per hour of either 13 seconds (moderate apnea) or 23 seconds (severe apnea) for 2 weeks and 8 hours per day. Control rats received surgeries but no inflations. Proteomics analysis was done on the rat atria homogenates to identify dysregulated proteins in moderate and severe apnea when compared to control. SDS‐PAGE was performed on the homogenates to separate the proteins and the gel bands were trypsin digested to obtain the peptide mixtures. The peptides were analyzed by a Nano Acquity UPLC coupled with Xevo G2 Mass Spectrometer. Data analysis was done using ProteinLynx Global Server (PLGS 2.4), Mascot server and Scaffold 4.1 software.

The proteomics analysis revealed that 3 of the 9 enzymes in glycolysis and 2 proteins related to oxidative phosphorylation were down‐regulated in the severe apnea group. In contrast, several structural and pro‐hypertrophic proteins were up‐regulated with chronic OSA. The data suggests the chronic OSA causes proteins changes which lead to cessation of glycolysis, a diminished capacity to generate reducing equivalents (i.e. NADH) as well as promotion of cardiac hypertrophy.

Support or Funding Information

Clarkson University
Temporal quantitative proteome analysis reveals dynamic change of cellular and secreted protein profiles of Clostridium cellulovorans depending on carbon sources
Authors: Shunsuke Aburaya , Wataru Aoki , Kouichi Kuroda , Mitsuyoshi Ueda
Published: November 2023

An anaerobic bacterium Clostridium cellulovorans secretes a large cell‐surface enzyme complex called cellulosome and this can degrade and metabolize cellulose, hemicellulose, and pectin which constitute plant cell wall. Furthermore, C. cellulovorans optimizes profiles of cellulosomal enzymes and non‐cellulosomal enzymes, which are secreted to media, depending on carbon sources. [1] Under the degradation of each carbon source, dynamical changes of composition of metabolic proteins occurred. [2] C. cellulovorans recognizes carbon sources in media and optimizes cell surface, secreted, and metabolic proteins to adapt to environment.

It is very interesting to reveal a dynamic adaptation mechanism of cellular and secreted protein profiles depending on carbon sources. In addition, temporal microbial profiling of cellular and secreted protein derived from the one‐pot culture has not been reported. In this research, we cultured C. cellulovorans in 5 carbon sources and took cellular and secreted samples temporally. After that, quantitative proteome analysis using tandem mass tag was carried out. From this analysis, we attempted to clarify temporal dynamic changes of protein profiles depending on carbon sources and culture times.

After the cultivation and sampling of each time point in glucose, cellulose, xylan, pectin, and galactomannan, the cellular proteins were extracted and the secreted proteins were concentrated. Each protein was alkylated after reduction, and digested by trypsin. Peptides were labeled with the stable isotope label and subjected to the LC‐MS/MS system equipped with unique super‐long monolithic silica capillary column. [2]

We could totally identify about 1500 proteins of C. cellulovorans in one‐shot proteome analysis, and these profiles were significantly dependent on each substrate and time point. Obtained data were analyzed statistically and changing proteins depending on substrates and time points was significantly identified. Focusing on central metabolic enzymes, we could clarify dynamic changes of these enzymes with time‐ and substrate‐depending manners. Cellulosomal and non‐cellulosomal proteins derived from each fraction are also dynamically changed as carbon sources were degraded. From these results, we considered adaptation and degradation mechanisms of C. cellulovorans with various carbon sources. [3]
Proteomic research on the therapeutic mechanism of Zhibai Dihuang Granule for treating hyperthyroidism yin‐deficiency rats
Authors: Liu Changming , Mao Liangen , Yang Su , Jiang Tingting , Wang Chong , Chen Zhongliang , Tu Huihui , Li Zhongjie , Li Jicheng
Published: November 2023

Zhibai Dihuang Granule (ZDG) is a classical traditional Chinese medicine that has been used to treat yin‐deficiency‐heat (YDH) syndrome. However, the therapeutic mechanism of ZDG is still unclear. The aim of this study was to observe the therapeutic effect and mechanism of ZDG in hyperthyroidism yin‐deficiency rats by detecting the serum proteome expression. Triiodothyronine (T3) was used to induce the hyperthyroidism yin‐deficiency model of rats, and the rats were treated daily with ZDG by gastrogavage. In the proteomics experiment, a total of 93 differentially expressed serum proteins (49 up‐regulated proteins and 44 down‐regulated proteins) were identified by iTRAQ labeling coupled with two‐dimensional liquid chromatography‐tandem mass spectrometry (2D LC‐MS/MS) analysis (fold change >1.50 or <0.60, p < 0.05) between model rats and control rats. After ZDG treatment, 16 proteins among up‐regulated proteins showed obvious downward trend, and 10 proteins among down‐regulated proteins showed obvious upward trend. Bioinformatics analysis of the proteins which returned to normal level revealed that 10 of these proteins were involved in immune response, especially in complement and coagulation cascades. After ZDG treatment, L‐selectin, C‐reactive protein (CRP), and Zinc‐alpha‐2‐glycoprotein (Azgp1) levels were significantly increased (p=0.0002, p=0.0003, p=0.0051, respectively). Therefore, we concluded that ZDG may treat YDH syndrome by regulating immune response, especially complement and coagulation cascades, and L‐selectin, CRP and Azgp1 may act as potential biomarkers to evaluate the therapeutic efficacy of ZDG. Our work provides important experimental basis to understand the mechanism of ZDG for treating YDH syndrome, and provide potential serum biomarkers for evaluating therapeutic efficacy of ZDG.

Support or Funding Information

This work was supported by grants from National Basic Research Program of China (No.2014CB543002), National Natural Science Foundation of China (No.81573709, No.81273882).
Alcohol's Effect on the Metabolome and CCL2
Authors: Berndt Calvin Wayne , Schumann Peter , Dunn Alyssa , Bray Jennifer , Lawrence Jim
Published: November 2023

When alcohol enters the central nervous system it disrupts the physiological balance, producing a plethora of negative effects on the nervous system, specifically learning and memory. It has been shown that the neuroimmune protein CCL2 perturbs the negative effects alcohol has on the hippocampus, the region of the brain associated with learning and memory. Although the signaling cascade initiated by CCL2 is currently unknown, elucidation of the pathway is possible through the use of a quadrupole time‐of‐flight mass spectrometer (Q‐TOF LC/MS). Using the previously specified instrument a molecular fingerprint can be produced by unambiguous and untargeted identification of metabolites located in hippocampus of CCL2‐transgenic mice. By comparing the fingerprint of non‐transgenic mice with that of CCL2‐transgenic mice one can identify dysregulated metabolites.

Over a time course of ten days mice of varying ages consumed 6g/kg of 20% ethanol every day. After the binge the left‐hemisphere hippocampus was extracted for metabolite analysis and the right‐hemisphere hippocampus was used for quantification of CCL2 concentration. This ensured identical biological samples for cross‐comparison between protein expression and the effects this had on the metabolome of the hippocampus. ELISA was used to quantify CCL2 concentration while the Q‐TOF LC/MS was used to identify metabolites. Statistical analysis (PCA) of mass spectra was done through XCMS Online, a cloud‐based metabolic data processor. Tentative assignment of m/z to compound names was done through built‐in Agilent software (MassHunter Qualitative and ProFinder) using METLIN databases.

Metabolites and their regulation tell a story about the physiology of the cell. Metabolic pathways can become temporarily dysregulated by external factors, such as alcohol consumption. By using transgenic mice, we can study the differing effects of alcohol on the metabolome by controlling a single molecular variable, such as CCL2 levels. By altering conditions of transgenic versus non‐transgenic mice, we can develop a broader knowledge base about the molecular fingerprint for alcoholism in the mammalian brain and understand the mechanisms of certain neurological disorders, such as alcoholism.

Support or Funding Information

University of Wisconsin ‐ Stevens Point URSCA Grant ($1,000)ASBMB Undergraduate Research Grant ($1,000)
O‐GlcNAcylation of the Human Kinome
Authors: Liu Xin , Han Guanghui , Pandey Akhilesh , Hart Gerald
Published: November 2023

O‐GlcNAc, first characterized more than 30 years ago, is an O‐linked β‐N‐acetylglucosamine moiety attached to the side chain hydroxyl of a serine or threonine residue. O‐GlcNAc has been found on over 4,000 cytoplasmic and nuclear proteins. The addition of O‐GlcNAc to proteins is catalyzed by O‐GlcNAc transferase (OGT) while its removal is catalyzed by O‐linked N‐acetyl‐β‐D‐glucosaminidase (OGA). This dynamic and reversible modification is emerging as a key regulator of various cellular processes, such as signal transduction, transcription, cell cycle progression and protein‐protein interactions. O‐GlcNAc plays important roles in human diseases, such as cancer, diabetes and neurological disorders.

Protein phosphorylation is a post‐translational modification that serves as a rapid and reversible means to modulate protein activity and transduce signals, the regulation of phosphorylation is a central mechanism in cell health and disease. The addition and removal of phosphorylation is through kinases and phosphates respectively, but little is known about O‐GlcNAcylation of kinases.

We have done kinase microarray which indicate that 297 kinases are substrates for OGT and then we used home‐made kinase inhibitor beads to pull down the kinases in Hela cells to further confirm the results in vivo. More than 200 kinases were identified by mass spectrometry. Currently we are using click chemistry followed by O‐GlcNAc enrichment to map the sites for all the O‐GlcNAcylated kinases we have pulled down.

Support or Funding Information

Supported by NIH P01HL107153, R01DK61671 and R01GM116891. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody, which are managed by JHU.
Cancer‐Related Mutations of ERK3 Promote Cancer Cell Invasiveness
Authors: Alsaran Hadel , Elkhadragy Lobna , Long Weiwen
Published: November 2023

Extracellular signal‐Regulated Kinase 3 (ERK3) is an atypical member of the mitogen‐activated protein kinase (MAPK) family. Recent studies have shown that ERK3 is highly upregulated in multiple cancers, such as lung cancer and colon cancer. In addition, ERK3 promotes cancer cell migration and invasion, at least partly by upregulating matrix metalloproteinase genes' expression. Gene mutation is a major cause of human cancers. However, little is known about ERK3 mutations in cancer development and progression. By reviewing COSMIC database of gene mutations in cancers, we found two ERK3 mutations L290P and L290V which exist in cancers of lung, large intestine and skin. Notably, ERK3 L290 residue is located in the kinase domain of ERK3 and is conserved in ERK1/2/3/4. In order to characterize these mutations, we generated plasmids for ERK3 with each of these point mutations, and overexpressed them in H1299 and A549 lung cancer cells. We found that in comparison with wild type (WT) ERK3, both of these cancer‐related mutants had greatly increased ability in promoting cancer cell migration and invasion. To elucidate the underlying mechanism by which ERK3 L290 mutants increased cancer cell invasiveness, we examined the kinase activity of ERK3 mutants by in vitro kinase assay. We found that ERK3 L290P and L290V mutants have similar kinase activity to WT ERK3. ERK3 protein is known to shuttle between the nucleus and the cytoplasm, which may alter its function. Interestingly, we found that both L290P and L290V mutations greatly increased the cytoplasmic localization of ERK3 proteins, whereas WT ERK3 is mostly nuclear. We are currently trying to elucidate the molecular mechanisms by which L290P (or V) mutations increase the cytoplasmic localization of ERK3 and its ability in increasing cancer cell migration and invasion. In conclusion, our present study has identified previously unstudied cancer‐related ERK3 mutations and their importance in cancer cell invasiveness.

Support or Funding Information

NCI 1R01 CA193264‐01 to Weiwen Long and “Saudi Culture mission” support to Hadel Alsaran.
The Phosphatase PRL‐3 as a Novel Drug Target in T‐Cell Acute Lymphoblastic Leukemia
Authors: Wei Min , Jing Huang , Liu Jinpeng , Wang Chi , Blackburn Jessica
Published: November 2023

PRL‐3, phosphatase of regenerating liver 3, is a member of a subclass of dual specific phosphatases. The up‐regulation of PRL‐3 is associated with progression of gastric, breast, liver, ovarian carcinomas, oral, and cervical caners and correlates survival in patients with colorectal, gastric, and breast cancers. However, it has not been associated with T cell acute lymphoblastic leukemia (T‐ALL). We have found that PRL‐3 was a top hit among genomic amplifications acquired in T‐ALL, concurrently with increased relapse potential, in an unbiased large‐scale cell transplantation experiment using zebrafish Myc‐induced T‐ALL model. Zebrafish injected at the one‐cell stage with rag2:Myc;rag2:GFP;rag2:PRL‐3 develop T‐ALL with a shorter latency and higher penetrance, compared with the fish injected with rag2:Myc; rag2:GFP alone. Growth after transplantation at low cell number was also significantly faster in the PRL3 expressing T‐ALL. Analysis of gene expression microarray data of 119 patient samples reveals that PRL‐3 was highly expressed in 58% of the patients. In addition, PRL‐3 had the highest expression in the most aggressive and treatment resistant subgroups of T‐ALL, TAL/LMO and Immature subtypes. PRL‐3 mRNA and protein expression is also highly expressed in human T‐ALL cell lines using quantitative RT‐PCR and immunoblotting, respectively. The treatment of T‐ALL cells using a small molecule 2‐cyano‐2‐ene‐ester compound (Analog 3), resulted in growth inhibition of T‐ALL cells. Annexin V staining revealed that Analog 3 treatment led to strong apoptosis in a subset of T‐ALL cells. Cell cycle analysis using DAPI staining showed that cell cycle distribution of the cells treated with Analog3 is not affected. In summary, our study provides evidence for a role of PRL‐3 in T‐ALL malignancy, and suggests that PRL3 may be a useful drug target in the treatment of this disease. Our future studies are focused on identifying the mechanisms by which PRL3 promotes T‐ALL progression and survival.

Support or Funding Information

NCI 4R00CA181500

NIGMS P30GM110787

The V Foundation for Cancer Research