bluuebunny/crossref_metadata_2025 huggingface dataset:
CACN‐1 and its Interacting Proteins are Important Regulators of Somatic Development in Caenorhabditis elegans
Authors: Hugunin Julie
Published: November 2023

With its easily visible U‐shaped gonad arm, Caenorhabditis elegans provides an excellent model to study germline development. This hermaphroditic nematode species has developed a mechanism to regulate the differentiation of its germline stem cells throughout development. C. elegans produce sperm until the onset of adulthood, when the worm permanently switches to producing oocytes. The Cram lab has identified the highly conserved protein CACN‐1 as an important player in proper germline differentiation. Loss of CACN‐1 leads to a masculinization of the germline where gonad arms are either exclusively filled with sperm, or contain a few irregular oocytes in addition to sperm. Previously, the lab identified CACN‐1 interacting proteins using tandem affinity purification and proteomics (TAP)1. CACN‐1 interactors grouped into a novel network suggest that CACN‐1 interacts with the spliceosome1. Depletion of some of these interactors in wild type animals also caused germline defects in differentiation and development comparable to CACN‐1. DAPI, a nuclear stain, revealed that gonad arms that lacked CACN‐1 and many of its interactors exhibited endomitotically duplicating oocytes (Emo phenotype) in a significant number of gonad arms containing abnormal oocytes. Due to the link between the Emo phenotype and ablated sheath cells2 an actin stain was used to examine sheath cell presence and germline phenotypes when these proteins were depleted via RNAi. As expected depletion of CACN‐1 and its interacting proteins lead to defects in sheath actin staining that correlated with Emo and sperm containing gonad arms. In addition, loss of these proteins in a worm strain that is only susceptible to RNAi in the somatic cells caused germline defects comparable to those observed in wildtype animals. Our data suggests that this novel network of proteins plays an essential role in somatic gonad development that has a direct effect on germline differentiation and maintenance. Ultimately a better understanding of CACN‐1 and its network of proteins in C.elegans may elucidate the role of splicing and translational regulation of gene expression in other processes, including disease and development, across species.

Support or Funding Information

A special thanks is extended to Alyssa Cecchetelli, Michael Doherty and Erin Cram, as well as the entire Cram Lab, Northeastern University's Biology Department for supporting this research, and the NIH (R01 GM85077) for funding.
Transcriptional complexes involving PAX3‐FOXO1
Authors: Weatherspoon Amber , Cherlin Tess , Johanson Kelly
Published: November 2023

Rhabdomyosarcoma is a rare soft tissue cancer that most often affects children and young adults but can occur at any age. Of the three types of Rhabdomyosarcoma, Embryonal, Alveolar, and Anaplastic, we focus on Alveolar Rhabdomyosarcoma (ARMS). Patients with ARMS have a chromosomal translocation that results in the production of a fusion protein, PAX3‐FOXO1. Although there are other translocations present in ARMS patients, PAX3‐FOXO1 is the most common and is liked with a more aggressive disease phenotype. A search of an updated ChIP‐seq screen of 3881 PAX3‐FOXO1 putative binding sites revealed that 36% contained both a PAX3 paired domain and FOXO1 recognition sequence within close proximity. With the knowledge that protein‐protein interactions involving FOXO1 have been shown to contribute to DNA targeting, we then examined the regions surrounding these dual motifs for the presence of common transcription factor binding sites. A number of common recognition sites, including those for MYOD, AP4R, and NEUR were overrepresented as compared to the expected distribution of these sequences in the genome. We are currently examining the contribution of these and other transcription factors to PAX3‐FOXO1 DNA‐binding through several in vitro assays. Identification of PAX3‐FOXO1 binding partners will give more detail about the mechanism of PAX3‐FOXO1 targeting and gene activation.

Support or Funding Information

NIH SC2GM095430

UNCF Stem Scholars Program (USSP)

NIH‐BUILD (TL4MD009637)
Novel Protein Binding Kinetics Measurements in Complex Biological Samples using AGILE Research
Authors: Goldsmith Brett , Walker Amy , Pan Deng , Lerner Mitch , Barron Francie
Published: November 2023

AGILE Research is a graphene based electronic binding assay that is an extension of recent advancements in the material science community. Protein binding kinetics measurements are increasingly common as tools allowing for effective binding measurements to gain wider adoption. However, use of these tools is often limited to concentrations and sample types outside of what is practical in a research environment. For example, many kinetic measurement techniques require an optically clear, homogeneous sample, leading to significant purification work and loss of material during sample processing. Electronic binding measurements represent a novel approach to obtain kinetics, which leverages advances in nanoelectronic sensors. In the past, electronic protein binding tools would require significant dilution of buffers to non‐physiologic levels, greatly limiting their utility in understanding native protein‐protein interactions. We demonstrate that new commercially available tools using graphene sensors overcome this challenge. Data from AGILE Research demonstrates protein‐protein binding kinetics measurements directly in complex media such as FBS and cell culture supernatant.
Competition Between Tamm‐Horsfall Protein and Heparin in Ligand Interactions
Authors: Haskell Matthew , Rhodes Diana C.J.
Published: November 2023

Tamm‐Horsfall protein (THP), also called uromodulin, is an 86kD acidic, single‐chain, glycoprotein produced in the kidney and excreted into the urine. THP binds various molecules of the immune system including immunoglobulins (Ig), complement 1q (C1q), and factor H (fH). Heparin is a negatively‐charged, sulfated polysaccharide that binds to a wide range of proteins, including C1q, fH, antithrombin III (ATIII), basic fibroblast growth factor (FGF2), and interleukin‐10 (IL‐10). The purpose of this study was to determine if heparin competed with THP for binding to proteins that interact with both THP and heparin (i.e. C1q and fH), and to screen other heparin‐binding proteins for previously unidentified interactions with THP. Dot blots were performed by immobilizing 1 μg of C1q, fH, and bovine serum albumin (BSA) to 4 strips of nitrocellulose (NC). One NC strip was stained with amido black and the other 3 NC strips were incubated with either THP (10 μg/ml), THP (10 μg/ml)/heparin (20 μg/ml) (sodium salt from porcine intestine), or no THP/heparin, before incubation with sheep anti‐THP antiserum and then donkey anti‐sheep IgG ‐ HRP. These blots showed that THP bound strongly to C1q and fH, and that this binding was inhibited by heparin. Heparin also inhibited the background binding of the antisera to C1q. ELISAs were used to quantify the ability of heparin to inhibit C1q (5 μg/ml) binding to immobilized THP (40 μg/ml). A steep inhibition curve was obtained, with no inhibition of C1q binding to immobilized THP occurring with 0.02 units/ml heparin, 50% inhibition of binding occurring with between 0.04 and 0.05 units heparin/ml, and 90% inhibition occurring with 0.1 unit heparin/ml (170 units/mg heparin). Control wells showed that heparin's inhibition of C1q binding to THP was not due to inhibition by heparin of the antisera's binding to C1q. Additionally, dot blots were used to evaluate binding of soluble THP to ATIII, IL‐10, and FGF2, three of the many proteins that heparin has been shown to bind. These blots demonstrated that THP bound to FGF2 and IL‐10, and this interaction was blocked by the addition of heparin. THP did not bind ATIII. In summary, heparin inhibited THP's binding to NC‐immobilized C1q and fH, as well as inhibiting immobilized THP's binding to soluble C1q in ELISAs. Protein‐protein interactions between THP and FGF2 and IL‐10, that were sensitive to heparin inhibition, were discovered. Heparin and THP coexist in the urine, especially after intravenous heparin administration or when heparin is instilled into the bladder as a therapy for interstitial nephritis. Mast cells are prominent in the wall of the urinary bladder in interstitial cystitis, so competition of heparin and THP for binding to other proteins may be important in this disease. Further studies to assess the physiological significance in THP/heparin competition for binding to various proteins is warranted.

Support or Funding Information

Funded by the PNWU Research Seed Program.
Investigating the Activation of the Tm2‐2 Immune Receptor in Response to the Tobamovirus Movement Protein
Authors: Sandoval Carina , Sacco Melanie A.
Published: November 2023

Effector‐triggered immunity (ETI) is a plant defense mechanism that evolved to detect pathogen effector proteins and initiate immune responses. Effectors are recognized by a direct or indirect interaction with plant immune receptor proteins. ETI often limits pathogen proliferation by promoting hypersensitive response (HR), a type of programmed cell death. In tomato (Solanum lycopersicum), the Tm2‐2 immune receptor provides resistance to Tobamovirus 30kDa movement protein. Tm2‐2 belongs to the CC‐NBS‐LRR class of resistance proteins, for which activation is mediated by specific intramolecular domain interactions. To study Tm2‐2 activation, mutations were introduced into the full‐length and truncated CC (coiled‐coil) domain clones. Highly conserved threonine or serine residues at positions 7, 8 and 13 were identified as potential phosphorylation targets and were mutated to aspartic acid (T7D, S8D, S13D) or to alanine (T7A, S8A, S13A). Nicotiana benthamiana was used for transient expression of constructs to observe for the effects of mutations on HR. Expression of the truncated CC domain clones with T7A or T7D mutations did not induce the HR in the presence or absence of the viral effector protein. The CC domain of the immune receptor with the effector protein is not sufficient to activate the plant's immune response. The full‐length T7D clone failed to elicit HR in the presence of the effector protein, showing loss of function. Unexpectedly, the full‐length T7A clone retained function and triggered HR in the presence of the effector protein. This evidence supports our hypothesis that position 7 of Tm2‐2 is a phosphorylation target that negatively regulates the activation of the Tm2‐2 immune receptor such that Tm2‐2 is inhibited in the charged state, but when neutral, Tm2‐2 is competent for activation of plant defenses. Understanding the mechanism of activation for Tm2‐2 can inform future engineering of efficient immune receptors that confer resistance against various pathogens.

Support or Funding Information

This project is supported by the Howard Hughes Medical Institute undergraduate research grant.
Mass Spectrometry Based Proteomic Analysis of Saliva from Children with Autism Spectrum Disorder (ASD) and Matched Controls
Authors: Wormwood Kelly L , Charette Laci , Ryan Jeanne P , Woods Alisa G , Darie Costel C
Published: November 2023

Approximately 1/68 children are believed to be affected by Autism Spectrum Disorder (ASD). A survey study suggests numbers as high as 1/50. With the increased diagnosis of ASD, treatment and understanding of the disorder is a pressing health concern. Protein biomarkers found for ASD may be used for ASD diagnosis, subtyping, monitoring, treatment, identifying therapeutic targets and may also provide clues about the causes of ASD. Here, saliva samples from 14 children with ADOS‐2 diagnosed ASD and 14 age and gender matched controls were analyzed using a combination of gel electrophoresis, in gel digestion or in solution digestion and nanoliquid chromatography‐tandem mass spectrometry (nanoLC‐MS/MS) to investigate differences between the proteomes of ASD subjects and matched controls. These results will hopefully shed light on possible causes of ASD and confirm our previous findings. In a preliminary 6 vs. 6 analysis using saliva from males, in solution digestion and analysis identified numerous proteins involved in immune responses to be dysregulated. Two of these proteins were lactotransferrin, involved in gastrointestinal antimicrobial activity, and prolactin inducible protein, involved in immune responses, which were upregulated in ASD compared to controls. Additional dysregulated proteins were also detected in blood serum, suggesting an enhanced response to environmental toxic factors. This investigation is ongoing.

Support or Funding Information

ASPIRE Fellowship, Shire, T. Urling and Mabel Walker Research Fellowship Program, SUNY Plattsburgh Presidential Research Award
The Potential Urinary Aging Markers from 20‐month‐old Rats
Authors: Li Xundou , Gao Youhe
Published: November 2023

Different organs may have different aging processes. Is it possible to find markers of aging from different organs?

Biomarker is measurable changes associated with physiological and/or pathophysiological conditions. Urine can be better source for biomarker discovery because it accumulates the changes of body while body removes them from blood by homeostatic mechanisms.

The urinary proteome is influenced by various factors, which is a major challenge in urinary biomarker discovery. To circumvent these problems, simpler systems, such as animal models, should be used to establish direct associations between physiological or pathological conditions and changes in the urinary proteome.

In this study, the urinary proteome of young (2‐month‐old) and old rats (20‐month‐old; 9 in each group) were analyzed using LC‐MS/MS (liquid chromatography‐mass spectrometry) and quantified using the Progenesis LC‐MS software. A total of 371 proteins were identified, 194 of which were shared between young and old rats. Based on the criteria of a fold change ≥ 2, P < 0.05 and being identified in each rat in the high abundance group, 33 proteins were changed (15 up‐regulated and 18 down‐regulated in old rats). By adding a more stringent standard (protein spectral counts from every rat in the higher group greater than those in the lower group), 8 proteins were changed consistently in all rats of between the groups, 2 of which are also altered in the urinary proteome of aging humans. There are no shared proteins between our results and the previous aging plasma proteome. Twenty of the 33 (60 %) changed proteins have been reported to be disease biomarkers, which implies that aging may share similar urinary changes with some diseases. The 33 proteins corresponded to 28 human orthologs, which are strongly expressed in the kidney, intestine, cerebellum and lung, according to the human protein ATLAS. Therefore, the urinary proteome may reflect aging conditions in these organs. More age groups should be included in the study if more mass spectrometry time could be available for this study.

Support or Funding Information

This work was supported by the National Basic Research Program of China (2012CB517606, 2013CB530805, 2014CBA02005 and 2013FY114100).
New serum biomarkers of blood coagulation function for pulmonary tuberculosis using iTRAQ‐coupled 2D LC‐MS/MS
Authors: Jiang Tingting , Wei Li‐Liang , Shi Li‐Ying , Wang Chong , Xu Dan‐Dan , Chen Zhong‐Liang , Ping Ze‐Peng , Liu Chang‐Ming , Li Ji‐Cheng
Published: November 2023

This study aimed to discover the novel protein biomarkers of blood coagulation function for pulmonary tuberculosis (TB) patients. Differentially expressed proteins of TB patients were screened and identified with the application of iTRAQ‐coupled 2D LC‐MS/MS technique. Clinical data of 204 TB cases, and their correlation with serum abnormal proteins were analyzed by statistical methods. A diagnostic model was constructed, and the accuracy was analyzed by receiver operation characteristic (ROC) curves. A total of 79 differentially expressed proteins were identified in the serum of TB patients, including 50 up‐regulated proteins and 29 down‐regulated proteins. Proteins closely related to clotting such as serum amyloid A (SAA) (P<0.0001), vitamin K‐dependent protein Z (PROZ) (P<0.0001), and C4b‐binding protein β chain (C4BPB) (P<0.0001) were significantly higher in TB patients than the healthy controls. Clinical analysis showed that coagulation indexes of TB patients, including PT, PT%, INR, FIB, APTT, and TT were significantly different with healthy controls. Correlation analysis revealed that PROZ was significantly correlated with PT% and INR, and C4BPB was significantly correlated with FIB. ROC curve analysis showed that the area under curve (AUC) value of the diagnostic model combined by SAA, PROZ, and C4BPB was 0.977 with 93.0% sensitivity and 93.1% specificity to discriminate TB patients from healthy controls. The results suggest that SAA, PROZ, and C4BPB may be potential biomarkers of blood coagulation function for TB.

Support or Funding Information

This work was supported by grants from National Basic Research Program of China (No.2014CB543002), National Special Sci‐Tech Projects (No. 2012ZX10005001‐006), and National Natural Science Foundation of China (No.81273882, NO. 81573709).
Analysis of Fluorescent Proteins for the Development of an In Vivo System for Protease Evolution
Authors: Selby Thomas Lee
Published: November 2023

The objective of this study is to develop a robust in vivo system using E. coli cells for screening protease activity based on intracellular Förster (fluorescence) resonance energy transfer (FRET). Toward this end, we expressed and purified the fluorescent proteins GFP, Orange‐FP (OFP), Clover, Ruby2, and td‐Tomato as single polypeptides, and as fusion partners linked by protease target sequences, to evaluate their stability, fluorescence intensity, chromophore maturation, kinetics, and FRET properties under various expression and reaction conditions. GFP and Clover showed similar spectroscopic properties when produced by constant induction, or mid‐log phase induction. However, the spectroscopic properties of OFP, Ruby2, and td‐Tomato varied significantly based on their induction conditions, cell density, and growth media. These differences appear to be related to the degree of maturation of the chromophore, which required optimization to achieve maximum FRET, but resulted in aggregation for some of the proteins under certain conditions. The rate of chromophore maturation in vitro varied depending on conditions and/or the presence of a fusion partner. In fusion constructs, the fully matured, fluorescent proteins demonstrated a constant decrease in FRET during proteolysis of the target sequence. However, interpretation of the kinetic profiles in vitro was complicated by samples with incomplete maturation. This complication is believed to be due a simultaneous decrease in FRET signal upon proteolysis, as well as an increase in FRET occurring during chromophore maturation. While the Clover‐Ruby2 fusion protein demonstrated a FRET efficiency that was superior to the GFP‐OFP protein, the hydrophobic Ruby2 protein was prone to aggregation under certain reaction conditions, resulting in a loss of fluorescent signal, challenges during purification, and difficulty interpreting kinetic results. Td‐Tomato, a less hydrophobic protein, gave lower yields during purification, and matured in vivo at a much slower rate than Ruby2 and OFP. In conclusion, these results revealed variable fluorescent properties for different fusion partners investigated in vitro, while providing data for comparison when optimizing fluorescent protein expression, and kinetics, during in vivo screening, with the ultimate goal of developing a system that will utilize fluorescent activated cell sorting (FACS) to evolve proteases with novel specificities.

Support or Funding Information

This work was supported by the National Institutes of Health under grant GM080691 and Mercer University School of Medicine, Division of Basic Medical Sciences.
Peptide‐induced inhibition of ATP Synthase as an alternative approach to combat microbial infections
Authors: Ahmad Zulfiqar
Published: November 2023

Background

ATP synthase is the fundamental means of cellular energy production. Inhibition of ATP synthase can deprive cell of required energy resulting in cell death. A wide variety of cationic α‐helical peptides are known to bind and inhibit ATP synthase. Our lab has studied the inhibitory effects of a variety of peptides with anti‐microbial and anti‐tumor properties on Escherichia coli ATP synthase. Peptide‐induced inhibition of E. coli ATP synthase establishes that the anti‐microbial and anti‐tumor effects of peptides are at least in part linked to the blocking of ATP synthesis in tumor and bacterial cells.

Method

Wild‐type E. coli strain, pBWU13.4 growth properties were checked on fermentable glucose and non‐fermentable succinate carbon sources before harvesting cells in minimal media to isolate ATP synthase. Peptide‐induced inhibitory studies were performed on membrane bound F1Fo ATP synthase. An additional positive charge in the form of a c‐terminal NH2 group was added to augment the extent of inhibition. Abrogation of cell growth was tested in presence and absence of peptides along with null control pUC118 strain.

Results

Peptides caused variable degree of inhibition. Potency of inhibition was found to be related to the complexity and number of positively charged residues. It was observed that the presence of a c‐terminal NH2 group on the peptides enhances the inhibitory potency on molar scale. Our studies also demonstrated that peptides follow a differential pattern of inhibition. These results confirm that incremental addition of positively charged residues can augment the inhibitory effects of peptides by about 100‐fold. Moreover, growth of wild‐type E. coli was fully abrogated while no effect was observed on the growth of null cells in presence of peptides. Thus, making ATP synthase a potent molecular drug target for anti‐microbial and anti‐tumor peptides. Currently, we are studying the inhibitory effects of venom peptides from different sources along with synergetic inhibitory effects of peptides on ATP synthase.

Conclusions

We conclude that cationic α‐helical peptides are potent inhibitors of E. coli ATP synthase. Our data suggests that the inhibitory potency of peptides can be enhanced by inserting additional positive charges including c‐terminal amide group. Finally, the selective inhibition of bacterial ATP synthase is an alternative novel approach to comate microbial infections.

Support or Funding Information

National Institutes of Health Grant GM085771
N‐terminal domain of Bothrops asper Myotoxin II Enhances the Activity of Endothelin Converting Enzyme and Neprilysin
Authors: Kuruppu Sanjaya , Rajapakse Niwanthi , Kleifeld Oded , Small David H , Whisstock James , Smith Alexander Ian
Published: November 2023

Neprilysin (NEP) and endothelin converting enzyme (ECE) are two enzymes that clear amyloid beta in the brain. Currently there are no molecules to stimulate the activity of these enzymes. Here we report, the discovery and characterisation of a peptide referred to as K49‐P1‐20, from the venom of Bothrops asper which directly enhances the activity of both ECE and NEP. This is evidenced by a 1.8‐ and 5.6‐fold increase in the Vmax of ECE and NEP respectively. The K49‐P1‐20 concentration required to achieve 50% of maximal stimulation (AC50) of ECE and NEP was 4.2 ± 0.2 and 2.9 ± 0.3 ng/μL respectively. Increase in activity is the result of a conformational change in the enzyme that occurs upon K49‐P1‐20 binding. Therefore, K49‐P1‐20 is both an excellent research tool to study enzyme activation, and a potential novel drug lead in the fight against Alzheimer's disease.

Support or Funding Information

This work was supported by funding made available through 1) the strategic grant scheme administered by the Monash University's Faculty of Medicine Nursing & Health Sciences; 2) Monash University's Interdisciplinary Research Funding Scheme; and 3) ANZ National Medical Foundation
Effect of the heavy metals cobalt and cadmium on LINE1 endonuclease activity
Authors: Ahmad Idrees , Smith Michaela , Meyers Jade , Russell Brittany , Dorrah Kimberly , DeFreece Cecily
Published: November 2023

Long interspersed element ‐1 (LINE‐1) is a mobile element, or jumping gene, which employs a “copy and paste” method called retrotransposition to insert itself into the human genome. This retrotransposition mechanism utilizes an enzyme called the L1 endonuclease to initiate the double strand breaks necessary for LINE‐1 insertion. Studies have found that a significant portion of the human genome is composed of mobile elements, which have likely played a crucial evolutionary role in the current genetic variability observed in humans. However, in some instances the mutations that have arisen from increased LINE‐1 retrotransposition, have been linked to genetic diseases such as colon cancer, breast cancer, and have been linked to genome instability due to increased double strand breaks. Previous research suggests that heavy metals such as Ni2+, Co2+, and Cd2+, which occur in our industrialized environment as pollutants, potentially affect levels of LINE‐1 retrotransposition. These toxic metals have been observed to compete with Mg2+ as cofactors for proteins, and have been observed to inhibit essential reactions in DNA repair. Competition with Mg as a cofactor suggests these heavy metals likely have an effect on levels of LINE‐1 endonuclease activity in vitro since it also utilizes Mg2+ as a cofactor for enzymatic activity. We focused on the effects that the heavy metals cobalt and cadmium have on the LINE‐1 endonuclease activity to determine whether or not these metals also influence levels of double strand breaks in vitro. Preliminary results indicate that the metal cobalt does not prevent LINE‐1 endonuclease activity at doses up to 100uM.

Support or Funding Information

This work was funded by an Institutional Development Award (IDeA) from the NIGMS under grant number P20GM103424 and from the Louisiana Cancer Research Consortium, the NIH‐RCMI grant #8G12MD007595‐05.
Structural and inhibitor screening studies of a fungal form of aspartate semialdehyde dehydrogenase from Cryptococcus neoformans
Authors: Dahal Gopal P , Viola Ronald E
Published: November 2023

Aspartate semialdehyde dehydrogenase (ASADH) functions at a critical junction in the aspartate biosynthetic pathway and represents a validated target for antimicrobial drug design. This enzyme catalyzes the NADPH‐dependent reductive dephosphorylation of β‐aspartyl phosphate to produce the key intermediate aspartate semialdehyde. Production of this intermediate represents the first committed step for the biosynthesis of essential amino acids in fungi and in bacteria. The absence of this enzyme in humans and other mammals will allow selective targeting of pathogenic microorganisms. We have accumulated significant structural and mechanistic information about the bacterial ASADHs, but have only limited knowledge of their fungal counterparts. The structure of a new fungal form of ASADH from Cryptococcus neoformans has been determined to 2.6 Å resolution. The overall structure of CnASADH is similar to that of its bacterial orthologs, but with some critical differences both in biological assembly and in secondary structural features that can potentially be exploited for the development of species selective drugs with selective toxicity against only certain infectious organisms. A customized fragment library has been screened against several fungal ASADHs and initial hits have been identified with inhibition constants (Ki) in the low millimolar range. The ASADH obtained from this fungal species have shown different inhibition selectivity when compared to this enzyme from bacterial species and also differences between fungal species. Docking studies with the CnASADH structure are being used to guide the design and development of potent inhibitors. The long term goal of this project is to develop drugs that selectively target this enzyme and block the aspartate biosynthetic pathway.

Support or Funding Information

This work is supported by funding from the NIH (AI077720).
Functional Studies of Thiosemicarbozone Compounds Against Topoisomerase IIα
Authors: Brown Anthony Ray , Ngo Lana , Morris William , Medawala Wathsala , Khoo Vivien , Lisic Edward , Jiang Xiaohua
Published: November 2023

Topoisomerase IIα is known to play a vital role in cell proliferation of all cells, especially cancer cells. It has been a popular target for the production of new drugs to combat cancer. This study looks at the inhibition of Topoisomerase IIα with a variety of thiosemicarbazone, such as ATZ ligands with different metals complexes. We studied three groups of ATZ compounds including ATZ‐ligand without metal, ATZ‐Copper, and ATZ‐Palladium complexes. In each of those groups, six different modifications on the side chain are examined. The compounds were tested using cleavage assays for stimulation of the relative DNA cleavage by Topoisomerase, which is a very important indicator for the potentiality to be an anticancer drug. Compounds that showed promising stimulation were selected and tested further in a dose dependent cleavage assay. Our results show that ATZ‐Copper complexes exhibited greater stimulation on Topoisomerase II. And Cu(ATZ‐ETSC)Cl is twice as effective as Etoposide‐ a leading anticancer drug in targeting Topoisomearse II.

Support or Funding Information

This project is funded by URECA! Undergraduate research program at Tennessee Tech University
Investigation of Solvent Hydron Exchange in the Reaction Catalyzed by the Antibiotic Resistance Protein, Cfr
Authors: Bauerle Matthew Richard , Grove Tyler L. , Booker Squire J.
Published: November 2023

Cfr is a radical SAM methylase that catalyzes the radical‐mediated appendage of a methyl group at C8 of adenosine 2503 in 23S ribosomal RNA (rRNA). Methylation of C8 by Cfr confers resistance to several classes of antibiotics that bind to the peptidyl transferase center of the bacterial ribosome. RlmN, a homologous RS methyltransferase, exclusively methylates C2 of A2503, which tunes the ribosome's interaction with nascent peptide chains. Cfr can also catalyze methylation at C2 in vitro, but it is much slower than methylation at C8. The Cfr and RlmN reactions take place via a cross‐linked intermediate between the terminal carbon of a methylcysteine residue on Cfr or RlmN and the carbon atom undergoing methylation on the RNA. This intermediate is resolved by abstraction of the C8 proton during C8 methylation or the C2 proton during C2 methylation. However, the identity of the active site base that mediates this abstraction at C8 has not been determined. EPR and crystallographic studies on the C118A variant of RlmN provided evidence that C118 is the operative base in the RlmN reaction. Previous studies using deuterium‐labeled substrates showed that the C2 proton is transferred to the nascent methyl group without isotope dilution during C2 methylation, while partial solvent hydron exchange takes place during C8 methylation. These observations led to the proposal that a polyprotic base is responsible for abstracting the C8 proton to initiate resolution of the cross‐linked intermediate. Herein, using deuterated substrates in conjunction with mass spectrometry and EPR we provide evidence that C105 of Cfr, the cognate residue to C118 of RlmN, deprotonates C8 of A2503.

Support or Funding Information

This work was supported by NIH Grant GM 101957 (S.J.B.)
Systematic proteomics and biochemical approaches to identifies the function of UBXN10 in p97‐associated process
Authors: li xiaolong , li shan , Chou Tsui‐Fen
Published: November 2023

The VCP AAA‐ATPase (also known as p97), an evolutionarily conserved ATP‐driven ‘segregase’, is a central regulator of protein quality control and ubiquitin‐mediated signalling. VCP functions in diverse processes such as endoplasmic reticulum (ER)‐associated degradation (ERAD), macro and selective autophagy and DNA damage responses(DDRs). These functions are dependent on association with cofactors that are specific to the different biological pathways p97 participates in. The UBX‐protein family (ubiquitin regulatory X) is an evolutionary conserved protein family and determine the specificity of p97/VCP/Cdc48p function by binding as its adaptors. constitutes the largest known group of p97 cofactors. However, few general or specific function of them has been revealed. Especially UBXD3, During the course of understanding not only their function but also specified function of p97, we investigated how the UBXD3 and the protein bind with UBXD3 worked in this study. The steady‐state kinetic study demonstrates that UBXD3 increases the catalytic efficiency (Kcat/Km) of both WT and the D2‐active mutant, but does not affect the D1‐active mutant. Thus, UBXD3 mainly regulates the D2 ATPase activity of p97. We used the co‐immunoprecipitation (1P) and LC‐MS/MS quantitation approaches to analyze interacting proteins of p97 and UBXD3. Analysis of UBXD3—Flag IP from transient transfection with 293T and U20S cells demonstrated that UBXD3 could bind with MOB1B and STK3. Indicate the function of UBXD3 is related to the Hippo signaling pathway.
Cleavage of proglucagon by the prohormone converting enzymes PCSK1 and PCSK2 is not dictated by the amino acid sequence immediately adjacent to the cleavage site
Authors: Mackin Robert B
Published: November 2023

The peptide hormones glucagon, glucagon‐like peptide‐1, and glucagon‐like peptide‐2 are all derived from proglucagon (PG), a 160 amino acid polypeptide containing multiple potential cleavage sites designated by either single or paired basic amino acids. Initial cleavage of PG takes place on the C‐terminal side of the K70‐R71 dibasic site, which liberates the intermediates glicentin and major proglucagon fragment (MPGF). My laboratory is interested in examining the specificity of the prohormone cleaving enzymes PCSK1 and PCSK2 using both PG and proinsulin (PI) as model substrates. PG is a useful model because it has one cleavage site (the glicentin/MPGF junction) recognized almost equally well by both enzymes and multiple potential cleavage sites that are not recognized by either enzyme. PI is a good model because it has one cleavage site (the B chain/C peptide junction) preferentially recognized by PCSK1 and a second cleavage site (the C peptide/A chain junction) preferentially recognized by PCSK2. My lab has previously described the expression and purification of these two model substrates. However, the purification of PG has presented a number of problems because of the presence of contaminants that obscure detection and participate in co‐precipitation during early stages of purification. I have now devised an improved, two‐step scheme using step elution from SP Sepharose as a capture step, followed by gradient elution from Q Sepharose as the major purification step. This method resolves the problems posed by the contaminants and generates PG samples suitable for analysis of the PCSK‐mediated cleavage reaction. An in vitro cleavage assay was then set up so that the PG substrate (~3 μM) incubated with either PCSK1 or PCSK2 (~100 nM at pH 6.0 or 5.4, respectively) results in ~30% cleavage at the glicentin/MPGF junction using either enzyme. As noted above, in contrast to the situation with PG, the normal maturation pathway for PI exhibits a marked preference for PCSK1‐mediated cleavage at the B chain/C peptide junction. To examine whether this cleavage selectivity is due to amino acids in the immediate vicinity of the cleavage site, the sequence EAED from the C‐terminal side of the B chain/C peptide cleavage site in PI was cloned into the C‐terminal side of the K70‐R71 cleavage site in PG. In contrast to expectations, this substitution resulted in a nearly 50% decrease in PG cleavage by PCSK1. Equally surprising was that this substitution had almost no effect on the efficiency of PCSK2‐mediated cleavage of the altered site, despite the fact that in the original context of the PI substrate, this sequence is recognized much less efficiently by PCSK2 than by PCSK1. Taken together, these results suggest that three‐dimensional features (secondary or tertiary structure) play a significant role in the cleavage site specificity exhibited by both PCSK1 and PCSK2.
Search for New Pro‐apoptotic agents in Indigenous Antitumor Plants in Nigeria
Authors: James Ayorinde Babatunde , Saibu Gbemisola Morounke , Magbagbeola Olubunmi Abiola , Omilabu Sunday Aremu , Meyer Mervin
Published: November 2023

Due to severe cytotoxicities exacerbated by cytotoxic drugs, newer therapies from natural products are desirable. This work aimed to partially purify pro‐apoptotic agents from indigenous natural products and to investigate the mechanism of action of the selected plant fractions.

Sixteen indigenous medicinal plants were collected and fractionated into hexane (HF), chloroform (CF), ethylacetate (EAF), methanol (MF) and water (WF) fractions using solvent‐solvent partitioning and polyamide adsorption chromatography. The resultant ninety‐six test fractions were screened for lethality potential using brine shrimp lethality (BSL) assay. Fractions with LC50 less than 10 ug/ml were subjected to Water Soluble Tetrazolium (WST‐1) cytotoxicity assay and apoptosis evaluations using: morphological assessment, flow cytometric analysis of phosphatidylserine externalization (Annexin V) and propidium iodide (PI) staining, caspase 3 enzyme assay, immunoblot assessment of Poly ADP‐Ribose Polymerase‐1 (PARP‐1) Cleavage, carboxymethyl‐2′,7′‐dichlorodihydrofluorescein‐diacetate (CM‐H2DCFDA) fluorescent microscopic/microplate analysis of reactive oxygen species generation, and Terminal deoxynucleotidyl transferase (Tdt) mediated dUTP‐biotin Nick End Labelling (TUNEL) assessment of DNA fragmentation.

Following BSL assay, C. afer (EAF), P. guineense (HF), P. guineense (CF), A. virides (EAF), C. roseus (CF), Z. zanthoxyloides (HF) and Z. zanthoxyloides (EAF) had LC50 values < 10 μg/ml. WST‐1 Cytotoxicity testing of these hit fractions on HeLa (Tumourigenic) and KMST‐6 (Non tumourigenic) cell Lines confirms their cytotoxic effects with the exception of Z. zanthoxyloides (HF). Significant (P<0.05) selective cytotoxic preference for HeLa to KMST‐6 Cell lines was exhibited by P. guineense (CF) and C. roseus (CF). Membrane blebs were observed in HeLa cells treated with the hit fractions in comparison to 17.42 μg/ml(50μM) Camptothecin. Annexin V‐PI analysis reveals that Z. zanthoxyloides (EAF) and P. guineense (HF) showed similar apoptotic effect in comparison to 21.8% apoptotic population in Camptothecin. A significant (P<0.05) increase in caspase‐3 enzyme activity was observed in C. roseus (CF) and Z. zanthoxyloides (EAF) in comparison with the negative control. PARP‐1 cleavage was confirmed in all the test groups except for A. virides (EAF) treated cells.

Findings in this study suggest that all the hit test fractions elicited apoptotic effects but at varying degrees with Z. zanthoxyloides (EAF) showing the most prominent effect. While A. virides (EAF) elicits its cytotoxic effect via necrosis.
Evolution of Metal Ion and Purine Nucleotide Effects and Cooperativity in Glutamate Dehydrogenase: The Role of Conformational Flexibility
Authors: Tran Sirena , Bell Jessica , Bell Ellis
Published: November 2023

Higher eukaryote glutamate dehydrogenases are highly allosterically regulated by a wide variety of compounds including purine nucleotides and metal ions. Of the purine nucleotides ADP and GTP are the most potent allosteric heterotropic effectors while NAD(P)+ shows negative homotropic allosteric effects involving communication between active sites. To investigate potential evolutionary factors we have compared a variety of available structures and homology models of glutamate dehydrogenases that either show allosteric effects or lack allosteric interactions to elucidate the formation of either the purine heterotropic sites or the metal binding sites of the enzyme using HINT computational analysis of contributions to binding. Local Spatial Pattern alignments have identified potential conformationally flexible spines that could form pathways of conformational changes. The effects of purine nucleotides and various metal ions on overall stability have been determined by both heat and chemical denaturation using CD and fluorescence spectroscopy. Inhibition is correlated with decreased flexibility while activators enhance flexibility. Similar studies with inhibitory or non‐inhibitory meal ions and limited proteolysis experiments using immobilized trypsin and tandem mass spectrometry to identify initial cleavage sites provide more detailed spatial information are in progress.
Effects of Purine Nucleotides on the Stability of Glutamate Dehydrogenase Studied using a Fluorescence Based Thermal Shift Assay
Authors: Smith Andrew , Bell Jessica , Bell Ellis
Published: November 2023

Mammalian Glutamate Dehydrogenase is unique in that it utilizes both NAD+ and NADP+ in the oxidative deamination of L Glutamate, and like many higher eukaryote glutamate dehydrogenases is highly regulated with GTP being a potent inhibitor and ADP, depending upon pH and substrate concentrations being either an activator or an inhibitor. Previous studies using Thermal Melts followed by CD at 222nm have indicated that while GTP enhances global flexibility, ADP makes the enzyme more rigid. Due to absorbance in the 220–250nm region it is difficult to use these nucleotides at high concentrations or in combination with cofactors. To overcome this problem we have used a Fluorescence Based Thermal Shift assay to screen both the concentration dependence of the effects of ADP, GTP, NAD+, NADP+, NADH and NADPH individually and with various combinations of nucleotides, with and without glutamate or 2 oxoglutarate, on the stability of the enzyme to establish which binding events predominate effects on stability. More detailed information on the effects are being investigated using limited proteolysis with immobilized trypsin and chymotrypsin combined with tandem mass spectrometry to identify the time course of cleavage and the effects of purine ligands in the presence or absence of the substrate glutamate or 2 oxoglutarate.
Cell‐rounding compounds inhibit components of the bacterial cytoskeleton
Authors: Lammers Matthew , Santos Thiago , Hurley Katherine , Mitchinson Timothy , Weibel Douglas
Published: November 2023

The bacterial cytoskeleton is a highly organized complex consisting of actin, tubulin, and intermediate filament protein homologs. Particularly, the actin homolog MreB is important for chromosome segregation, cell wall morphogenesis, and maintenance of rod‐shape morphology. A22 is a small molecule bacterial inhibitor of MreB that interferes with normal growth and produces rounded cells. In this study, we identify the mechanism of six novel bacterial inhibitors that produce a similar rounded phenotype with varying degrees of chemical similarity to A22. We characterize the activity of these cell‐rounding compounds (CRCs) against diverse bacterial species and perform toxicity testing in mammalian cells to test the viability of these compounds as therapeutic agents. Furthermore, we characterize phenotypic aberrations by quantifying cellular aspect ratio and localization of MreB in treated cells via flow cytometry and structured illumination microscopy, respectively. To test the hypothesis that the CRCs bind the MreB ATPase catalytic center in a similar fashion to A22, we test A22‐resistant mutants for cross‐resistance. We also perform a multi‐copy suppression screen to determine cytoskeleton gene products which confer reduced susceptibility to these compounds and generate spontaneous mutants to identify mutations which provide resistance. Finally, we report a convenient chemical synthesis for the CRCs. This study will help identify the mechanism of action of new bioactive molecules and serve as a starting point for the development of a novel class of therapeutic agents.
Progress Towards Design of a Light Activated COX‐2 Inhibitor
Authors: Paulishak Wyatt , Armstrong Christopher , DeBerardino Maryrose , Rous Clarissa , Mertz Pamela , Streu Craig
Published: November 2023

Controlled inhibition of enzymes is a therapeutic mechanism that has long been sought after in medicine. Much of modern day therapeutic research has been aimed at targeting specific cell or tissue types with limited success. However, light activated molecules could provide a safe, targetable alternative to these therapeutics. This could allow for drugs with side effects deemed too harmful for use previously to be revisited and modified, allowing for new avenues of treating diseases. Cyclooxygenase‐2 (COX‐2) is an enzyme that synthesizes prostaglandins from arachidonic acid and is responsible for pain and inflammation. Ibuprofen and aspirin act as inhibitors of COX‐2, which has become a well‐understood enzyme usable for testing entirely new types of inhibitors. Previous research on other types of compounds has demonstrated photo‐switchable organic groups that could allow controlled inhibition for site‐specific therapy instead of whole body treatment. Here we discuss the replacement of key groups within known COX‐2 inhibitors and analyze their ability to perform light controlled inhibition of COX‐2.
Biophysical and Structural Studies of Chelator Fragment‐Library Candidates as Inhibitors of Metallo‐β‐lactamases
Authors: Williams Cameron , Tierney David L , Crowder Michael W , Cohen Seth , Fast Walter , Page Richard C
Published: November 2023

Antibiotic resistance is a serious worldwide health problem. In the 2013 Antibiotic Resistance Threat Report, the CDC estimates that over 2 million people suffer from antibiotic‐resistant infections every year in the U.S. and 23,000 of these individuals die as a result. Metallo‐β‐lactamases are a class of enzymes that catalyze the hydrolysis of almost all β‐lactam antibiotics and, as a result, confer dangerous antibiotic resistance to the bacteria expressing them. Gram‐negative bacteria possessing metallo‐β‐lactamases are of utmost concern because these enzymes also break down carbapenems, a class of antibiotics commonly used as a last resort to treat antibiotic‐resistant bacterial infections. Enterobacteriaceae with carbapenemase activity are becoming increasingly widespread. This is a major problem because these gram‐negative bacteria of the intestinal flora are easily spread between humans and commonly found in clinical settings. Metallo‐β‐lactamase inhibitors are of particular interest because they could potentially restore the efficacy of current generation β‐lactam antibiotics. A library of chelator fragments has been developed to inhibit metallo‐β‐lactamases by taking advantage of the active site Zn2+ ions critical for proper enzymatic function. Our in vitro activity assays have identified several potent candidates from the library for further study. VIM‐2, a variant of Verona integron‐encoded metallo‐β‐lactamase (VIM), was overexpressed and purified for the purpose of studying these chelator fragments. We utilized differential scanning fluorimetry to demonstrate that high concentrations of chelators destabilize VIM‐2 by removing Zn2+ cofactors. Co‐crystallization and crystal soaks of VIM‐2 with several chelators was attempted in order to gain insight into the structure of the protein‐ligand complex but x‐ray diffraction data suggests that the chelators are not present in the crystal structure. Future work will involve binding affinity studies by isothermal titration calorimetry (ITC) and structural studies with advanced crystallographic techniques. Additional studies will be employed to examine the importance of a loop near the active site of all metallo‐β‐lactamases. Overall, these studies will contribute to the development of more effective chelators.

Support or Funding Information

This work was supported by NIH grant R01 GM111926.