bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein‐protein interactions reduce Ca2+‐requirements for Transglutaminase 2 mediated post‐translational modifications of α‐synuclein
Authors: Zeczycki Tonya Nicole , Viscomi Jessica S 
Published: November 2023

The combined projected increase in the incidence of Alzheimer's and Parkinson's disease is nearly 40% over the next ten years as the “baby‐boomer” population advances in age. In the majority of late onset neurodegenerative diseases, the underlying cause of neuron damage is the accumulation of toxic protein aggregates. The Ca2+‐dependent, post‐translational modification of tau, α‐synuclein and β‐amyloid by transglutaminase 2 (TG2) contributes to the development of protein aggregates in the diseased brain. Vulnerable to the toxic effects of aggregated proteins, damaged neurons exhibit increased Ca2+‐influx, further activating TG2. Degeneration leads to neuronal death, which gives rise to cognitive decline and dementia. The pathological TG2 protein cross‐linking activity in neuronal cells is actually beneficial to normal cellular apoptosis when regulated properly. The advancement of TG2 as a therapeutic target is hampered by the lack of fundamental understanding of how TG2 cross‐links pathogenic protein substrates. In contrast to previous in vitro mechanistic studies, which rely heavily on the use of artificial substrate analogs (i.e. Cbz‐Gln‐Gly), we present here the kinetic characterization of the TG2‐mediated, Ca2+ deamidation and polyamidation of recombinant α‐synuclein. The apparent Km for α‐synuclein is nearly 10‐orders of magnitude lower than that for Cbz‐Gln Gly and Ca2+ requirements for activation (KA ~0.34 μM) are significantly reduced. In addition, we explore the effects the protein‐protein interaction between TG2 and CRT, a low affinity, high capacity Ca2+ binding protein, have on Ca2+‐activation and protein cross‐linking activity.Support or Funding InformationOffice of Research and Graduate Studies, Brody School of Medicine
Avoiding the Carbapenem Trap: KPC‐2 β‐lactamase Sequence Requirements for Carbapenem Hydrolysis
Authors: Mehta Shrenik C , Samanta Moumita , Chow Dar‐Chone , Palzkill Timothy 
Published: November 2023

β‐lactamases are bacterial enzymes that mediate antibiotic resistance through hydrolysis and inactivation of β‐lactam antibiotics. The hydrolysis reaction is a two‐step process involving cleavage of the β‐lactam amide bond to form a covalent acyl enzyme intermediate followed by deacylation through an activated water molecule. Traditionally, the carbapenem class of β‐lactam antibiotics formed the last line of therapy for treating bacterial infections. β‐lactamase enzymes, such as TEM‐1, that are responsible for resistance to most β‐lactam antibiotics are inefficient at providing resistance to carbapenem antibiotics because they become trapped in a deacylation deficient conformer. The emergence of β‐lactamases capable of hydrolyzing carbapenems, such as KPC‐2, in the bacterial population has resulted in increased mortality rates and healthcare costs. While TEM‐1 and KPC‐2 share a high degree of structural similarity (RMSD = 0.9 Å), KPC‐2 can evade the inhibitory trap and hydrolyze carbapenem antibiotics. In the absence of a KPC‐2/carbapenem co‐crystal structure, the molecular basis of carbapenem hydrolysis is not well understood. In this study, steady‐state and pre‐steady state kinetic analysis on several KPC‐2 active‐site mutants was used to decipher the mechanism of carbapenem hydrolysis. The results indicate that the sequence constraints for hydrolysis of imipenem and meropenem (carbapenem substrates) are more stringent as compared the requirements for hydrolysis of ampicillin (penicillin substrate) and cephalothin (cephalosporin substrate). Active site residues T235 and T237 were found to be critical for both imipenem and meropenem hydrolysis and mutating them to alanine resulted in inefficient or reduced carbapenem turnover. Residue R220 was found to be critical for meropenem hydrolysis but not imipenem hydrolysis. Surprisingly, mutating positions R220 and T237 to alanine resulted in a catalytic efficiency for cephalothin hydrolysis similar to the wild‐type enzyme suggesting that these positions are not important for cephalosporin hydrolysis. However, the T235A mutant was found to be critical for cephalothin hydrolysis. In addition, for ampicillin hydrolysis, each mutant displayed an increase in catalytic efficiency as compared to wild‐type enzyme suggesting the wild type residues at these positions are detrimental for penicillin hydrolysis. Thus, the KPC‐2 β‐lactamase active site has evolved to hydrolyze carbapenem antibiotics at the cost of its penicillinase activity.Support or Funding InformationWork supported by NIH RO1 A132956 to T.P.
The Role of Lys42 on the Catalytic Efficiency of the ADP‐glucose Pyrophosphorylase from Escherichia coli
Authors: Mahaffey Angela L , Aiyash Saleh , Ballicora Miguel A 
Published: November 2023

The synthesis of α‐1,4 polysaccharides (glycogen and starch) via NDP‐glucose pyrophosphorylases (NDP‐Glc PPases) is pertinent for energy storage in prokaryotic (bacteria), eukaryotic (plants) and mammalian organisms. Of this NDP‐Glc PPase superfamily, the ADP‐glucose Pyrophosphorylase (ADP‐Glc PPase) enzyme functions in the “first committed step” of glycogen synthesis for Escherichia coli, in the presence of a divalent magnesium ion (cofactor), ATP (substrate) and Glc‐1P (substrate), by production of the glucosyl donor (ADP‐glucose). The 200 kDa homotetrameric (α4) enzyme differs from the heterotetrameric (α2β2; S‐subunits and L‐subunits; 50–52kDa; 60kDa) plant counterpart as it has a conserved Lysine residue (Lys42 in E. coli ADP‐Glc PPase), present in the bacterial ADP‐Glc PPases and bacterial NDP‐Glc PPases, but not the Large subunit (non‐catalytic) of the eukaryotic plant ADP‐Glc PPase. This prompted our investigation into any catalytic properties of the Lys42 residue in E. coli ADP‐Glc PPase. By site‐directed mutagenesis, enzymes characterization and homology modelling, we found (1) Lys42 plays a critical role in the Mg2+ (cofactor) binding network (Lys42/Asp142/Asp276) – observed by both modelling and kinetics ‐a decreased cooperativity of the Mg2+ binding curves of the mutants (2) Modelling indicates the Lys42 amine side chain interacts with the oxygen between the α and β‐phosphate groups of the ATP. Possibly via a “general acid‐catalysis” mechanism the PPi product is stabilized becoming a better “leaving group” and (3) Mutagenesis of this site lowers the catalytic efficiency (kcat/S0.5) for the ADP‐Glc PPase enzyme substrates by 3–4 orders of magnitude. Conclusively, we postulate Lys42 is critical for the catalysis of ADP‐Glc PPase in E. coli.
kcat and kcat/S0.5 for ATP and Glc‐1P (substrates) of WT and Lys42 mutant E.coli ADP‐Glc PPase enzymes, in the synthesis direction.

Enzymea
kcatb
ATP
Glc 1P

‐fold decreasec
kcat/S0.5
kcat/S0.5

s−1

s−1mM−1
s−1mM−1

WT
103 ± 11
1
515
3814

K42Q
0.140 ± 0.006
736
0.7
2

K42A
0.098 ± 0.002
1051
0.2
2

K42R
0.053 ± 0.001
1932
0.2
0.9

K42L
0.041 ± 0.002
2512
0.5
0.6

K42C
0.040 ± 0.002
2575
0.3
0.6

K42E
0.032 ± 0.002
3312
0.3
0.7

K42T
0.026 ± 0.002
3962
0.3
0.2

Enzymes were purified to homogeneity and assays were performed at 37 °C as described under “Experimental Procedures.”
kcat values were calculated from curves via assays performed in the presence of saturated substrates (ATP and Glc‐1P) and activator (FBP). Assays were averaged from three independent experiments.
Fold decrease was calculated using wildtype enzyme activity as a reference.
Structure of a Key Intermediate in the Reaction Catalyzed by the Dual Specificity Methylase RlmN
Authors: Schwalm Erica Lynne , Grove Tyler , Boal Amie , Booker Squire 
Published: November 2023

Both rRNA and tRNA are highly modified, contributing to their structural and functional integrity. RlmN is a dual‐specificity RNA methylase that modifies C2 of adenosine 2503 (A2053) in 23S ribosomal RNA and C2 of adenosine 37 (A37) is several Escherichia coli tRNAs, representing one of only two enzymes known to act on both rRNA and tRNA. Cfr, a related methylase, modifies C8 of A2503, conferring resistance to more than five classes of antibiotics. These two methylases employ a unique radical mechanism to catalyze these difficult transformations. While a structure of RlmN was previously solved, it lacked the RNA substrate. As shown previously, a Cys118Ala substitution in RlmN results in the capture of a covalent intermediate in which the protein is cross‐linked to the RNA substrate at the C2 carbon of the target adenosine. The 2.4 Å resolution structure of this key intermediate formed using a tRNAGlu substrate was solved, providing key insight into the mode of substrate recognition and unresolved mechanistic details. RlmN contacts the entire length of the tRNA, similar to tRNA synthetases, but forms few nucleotide sequence specific interactions. Access to A37 is achieved through an induced‐fit strategy that completely unfolds the tRNA anticodon stem loop. This binding strategy likely facilitates recognition of the diverse sequences found in the tRNA and rRNA substrates. Mutagenesis of interacting residues suggests key interactions that are necessary for the modification of the tRNA substrates. The observation of reaction products in the active site suggests the order of substrate binding and product release, providing previously unknown mechanistic details. This structure represents the first structure of a dual specificity methylase in complex with the entire tRNA substrate.Support or Funding InformationThis work has been supported by NIH grants GM100011 (A.K.B.) and GM101957 (S.J.B.), the Searle Scholars Program (A.K.B.), and Tobacco Settlement Funds (TSF13/14 SAP_4100062216) to S.J.B.
A Spatiotemporal‐Controlled Targeting Tool for Understanding the Roles of Redox Signals on Bacterial Growth
Authors: Pham Vanha Nhat 
Published: November 2023

Redox‐mediated modifications are becoming increasingly appreciated as finely‐tuned signaling events that affect organism fitness. Greater understanding of these redox signals and cellular communication is imperative to being able to influence specific cellular functions and growth. However, precise spatiotemporal control to investigate these redox‐mediated inter‐ and intracellular communications has thus far been impossible. A recently developed method, targetable reactive electrophiles and oxidants (T‐REX), has achieved the precision necessary to explore the impacts of discrete redox signals in live mammalian cells. T‐REX employs an inert photo‐caged precursor of a bioactive redox signal and is targeted to a protein of interest (POI) through a HaloTag. Upon UV light, the redox signal is released onto the POI. This project adapts the T‐REX approach to bacteria to better resolve the roles of redox signals in bacteria stress responses and growth pathways. We have shown that T‐REX in bacteria—using E. coli as the model organism—yields comparable specificity as in mammalian cells, highlighting the generality of the methodology. Using this platform, we also demonstrate the capability of delivering redox signals to specific subpopulations of cells expressing different fluorescent proteins. With this approach, we are making headway toward identification of novel bacterial stress response pathways. This project highlights a novel chemical biology tool used to address the longstanding challenges in precise targeting of small‐molecule redox signals to specific protein targets in living cells.Support or Funding InformationWe acknowledge Frank L. and Lynnet Douglas Fellowship for Undergraduate Summer Research for Vanha N. Pham and NIH Director's New Innovator Award (1DP2GM114850‐01) for Yimon Aye.
Stabilin‐mediated cellular internalization of phosphorothioate‐modified Antisense Oligonucleotides (ASOs)
Authors: Miller Colton M. , Donner Aaron J. , Blank Emma K. , Eggar Andrew , Kellar Brianna , Seth Punit P. , Harris Edward N. 
Published: November 2023

IntroductionAntisense oligonucleotides (ASOs) are short chemically modified oligonucleotides (5–7.4 kDa) that can produce a pharmacological effect by binding to RNA and affecting intermediary metabolism. Over 35 phosphorothioate (PS) ASOs are at various stages of clinical development for use as therapeutic agents and pharmacological tools. Antisense therapy is a progressing area of research, as these small strands of nucleotide oligomers can be produced to silence genes that aggravate chronic disorders or infections. An important distinction for ASOs compared to DNA is the substitution of the phosphodiester (PO) backbone with the PS modification. This sulfur substitution allows for these polar polyanionic molecules to have high stability in biological fluids and selective binding to cell surfaces. Although there is a premium for clinical development of these short chain molecules, the current understanding of the pathways for their ability to traverse plasma membranes remains unresolved. Injected gymnotic ASOs have been shown to accumulate in the liver via the organ's functional scavenging mechanism in highly endocytically active sinusoidal endothelial cells (SECs) and Kupffer cells (KC) compared to hepatocytes. Our work outlines how a non‐DNA binding class of scavenger receptors known as Stabilins binds to, and internalizes these small PS ASOs.MethodsPrimary cells from rat and mouse were isolated and cultured with 125I‐ASOs. Stable cell lines expressing Stabilin‐1 and two Stabilin‐2 isoforms (315‐HARE and 190‐HARE), were used to analyze binding affinity, endocytosis and degradation of 125I‐ASOs in the cell. Co‐localization was also done to analyze trafficking of ASOs once internalized within the cell by use of fluorescent ASO and lysotracker.ResultsIt was determined that PS ASOs bind with high affinity to the Stabilin class receptors with the majority of internalization performed by clathrin‐mediated endocytosis. Binding was determined to be dependent on proper folding of the receptor, along with relying on salt‐bridge formation. Once inside the cell via the Stabilin receptors, co‐localization analysis showed ASOs being trafficked for degradation in the lysosome. Increased internalization rates of an ASO targeting the non‐coding RNA of malat‐1, in the Stabilin‐expressing cell lines reduced malat‐1 expression more efficiently, indicating not all ASOs are trafficked to the lysosome after internalization.ConclusionOur work shows that ASOs are internalized into the cells of the liver through clathrin‐mediated endocytosis. The understanding of the pathway(s) for chemically modified ASO internalization and trafficking with cell surface receptors will aid in future clinical design of ASOs as therapeutic agents.Support or Funding InformationNational Institutes of Health – NHLBI R01 5R01HL094463‐07University of Nebraska Undergraduate Creative Activities and Research Experience
Introducing Glycans to Molecular Visualization Software
Authors: Thieker David F. , Hadden Jodi , Stone John , Woods Robert 
Published: November 2023

Despite the essential role of glycoconjugates in biological processes, molecular visualization software fail to recognize carbohydrates as a unified class of macromolecules. Although proteins and DNA are detected by default, carbohydrate residues are treated as ‘non‐standard’ (HETATM) and grouped together with solvent and ions. As a result, carbohydrates currently lack a simplistic representation for 3D structure, and are predominantly visualized with all atoms displayed. Discerning the glycan sequence from an atomic view is challenging for short fragments and prohibitive for oligosaccharides. The following work describes the implementation of algorithms for the automatic identification and representation of carbohydrate moieties within the highly‐cited Visual Molecular Dynamics (VMD) program. Carbohydrate residues are now detected with a robust algorithm that distinguishes carbohydrate moieties from other ring‐based structures based upon atomic coordinates, rather than residue names. A 3D adaptation of the symbolic nomenclature developed by the Consortium for Functional Glycomics (CFG) has been implemented as a cartoon representation of carbohydrate residues, analogous to the canonical depiction of protein backbones with ribbons. Incorporation of a standard representation scheme familiar to the glycobiology community should improve adoption and allow researchers to more efficiently convey complex structural concepts pertaining to carbohydrate‐containing biomolecules (i.e. glycoforms of proteins, ligand sequences, etc.). Alternatives to the 3D‐CFG representation have also been included to enhance the atomic view of carbohydrate moieties. These additions to VMD finally create a program capable of treating carbohydrates as a unified class of molecules, complete with custom representations.Support or Funding InformationThe authors thank the NIH for support (R01 GM100058).
Glycosylation Alters Oligomerization Status of the Human CEACAM1‐IgV Domain
Authors: Prestegard James H , Moremen Kelley , Zhuo You , Yang Jeong‐Yeh 
Published: November 2023

Human carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) is a cell‐surface signaling molecule involved in cell adhesion, proliferation and immune response. It is also implicated in cancer angiogenesis, progression and metastasis. This diverse set of roles likely arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other members of the CEACAM superfamily. Its N‐terminal Ig‐like variable (IgV) domain has been suggested to be a principal player in these interactions, facilitating cis and trans homo‐dimer formation, as well as complex formation with other molecules. The IgV domain crystal structure, which used E. coli expressed material that lacks glycosylation, shows a dimer interface that obscures a glycosylation site, while other studies using mutagenesis and electron microscopy suggest dimer formation may also be mediated by a non‐glycosylated surface on the opposite side of the molecule. Here, we use NMR cross correlation measurements and residual dipolar coupling measurements to examine the effect of glycosylation on CEACAM1‐IgV domain oligomerization status. Both a non‐glycosylated form expressed in E. coli and a glycosylated form expressed in HEK293 GnT1‐cells and trimmed to a single GlcNAc residue, were examined. Our findings demonstrate that IgV with a single N‐linked GlcNAc at all glycosylation sites inhibits dimer formation of any type, whereas formation of a dimer similar in structure to that found in the crystal structure occurs in solution when glycosylation is absent. Future studies will be directed at the role additional domains and more complex glycosylation may play in native dimer formation.Support or Funding InformationFinancial support provided by the National Institutes of Health, R01GM033225
Common Jamaican Herbal Medicine, Bizzy Nut, Inhibits Cell Cycle in Prostate Cancer Cells
Authors: Gray Wesley , Chumbow Anne , Phillips Orville 
Published: November 2023

Prostate cancer development is dependent on androgens, and the majority of patients respond to androgen ablation. However, virtually every patient will develop hormone‐resistant prostate cancer and no longer respond to androgen deprivation therapy (ADT). Biz‐2 (the common Jamaican herbal medicine) Kola acuminate elicited anti‐proliferative effects in both hormone‐responsive (LNCaP cells) and hormone‐resistant (DU145 cells) prostate cancer cells. In this study we determined the cytotoxic efficacy of Biz‐2 in hormone‐responsive (LNCaP cells), the hormone‐resistant (DU145 cells) and a normal transformed (RWPE‐1) prostate cells. We determined growth inhibitory parameters (GI50, TGI and LC50) over 24hrs to 96 hrs using the MTT assay and cell count. Biz‐2 was found to inhibit the proliferation of all tested cell lines in a concentration‐dependent and time‐dependent manner. Both cancer cells showed significantly higher cytostatic and cytocidal (TGI 230ppm in DU145 verses 860ppm in RWPE‐1 and 156ppm in LNCaP verses 860ppm in WRPE‐1) as compared to the normal non‐transformed RWPE‐1. To begin to understand the mechanism of Biz‐2 anti‐proliferative activity, we used flow cytometry to determine where in the cell‐cycle Biz‐2 induced proliferation blockage. Analysis of untreated synchronized LNCaP cells by flow cytometry shows that 63% of the cells were in the Gi/Go, and 36% in the G2/M phases of the cell cycle. Upon treatment with Biz‐2, over 95% of the cells were in the Gi/Go phases. When these experiments were performed in DU‐145 we observed an accumulation of cells in the G2/M phases of the cell cycle. Both western blot and RT‐PCR showed a down regulation in the levels of the cell cycle protein. Our results suggest that the mechanism of action of Biz‐2 in androgen‐dependent and androgen‐independent prostate cancer cells is different.Support or Funding InformationNIH grant, Grant No. NIH R15AT006821
Novel Role for Lysocardiolipin Acyltransferase (LYCAT) in Non‐Small Cell Lung Cancer
Authors: Kotha Sainath R , Huang Longshuang , Van Scoyk Michelle Mai , Avasarala Sreedevi , Winn Robert A , Bikkavilli Rama Kamesh , Natarajan Viswanathan 
Published: November 2023

Lung cancer is the leading cause of deaths in United States and non‐small cell lung cancer (NSCLC) accounts for ~85% of all lung cancers with a 5‐year survival rate of approximately ~16%. Therefore there is an immediate need to develop new strategies for early detection and more effective treatments options. Mitochondrial dysfunction including but not limited to defects in mitochondrial genomics and dynamics has long been implicated to play a role in human health and disease particularly in cancer initiation, progression and treatment options since it plays a pivotal role in cell death and survival. Lysocardiolipin acyltransferase (LYCAT), a Cardiolipin remodeling enzyme regulating the 18:2 linoleic acid pattern of mammalian mitochondrial cardiolipin, plays a crucial role in maintaining normal mitochondrial function and vascular development. LYCAT was shown to be up‐regulated in cancers; however, the role of LYCAT in lung cancer is yet unclear. Probing the protein expression of LYCAT in lung cancer specimens, non‐transformed bronchial epithelial cell lines and 5 lung cancer cell lines revealed increased LYCAT expression and activity in all the lung cancer samples and cell lines tested in comparison to the control lung tissues and non‐transformed epithelial cell lines. To determine the role of LYCAT in lung cancer, NSCLC cell lines H2122 and H23 were transfected with either scrambled or LYCAT shRNA and differences in serum‐induced cell proliferation, migration, clonogenecity and mitochondrial dynamics were determined. Our results demonstrated that down‐regulation of LYCAT by shRNA significantly attenuated cell migration, proliferation, and invasion in NSCLC cell lines compared to control cell lines. Furthermore knockdown of LYCAT expression in NSCLC cell lines inhibited mitochondrial fragmentation and enhanced mitochondrial fusion. Taken together, these data demonstrate a strong association between increased LYCAT expression and cell proliferation, motility, invasion and mitochondrial dynamics in NSCLC cells. Thus, development of targeted therapies to reduce LYCAT expression in NSCLC should be beneficial.Support or Funding InformationThis work in part was supported by funds from the College of Medicine, UIC and NIH HL98050 to VN.