bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of proper Type 3 Secretion System Translocon Assembly using a split Luciferase Assay
Authors: Guo Hanling , Heuck Alejandro P.
Published: May 2022

Type 3 secretion system (T3SS) is used by many Gram‐negative bacteria to colonize the host and inject effector proteins into target cells. Inhibitors targeting T3SS are important to combat the appearance of multidrug‐resistant pathogens. In Pseudomonas aeruginosa, the T3SS secreted proteins PopB (SctE) and PopD (SctB) are required to inject effector proteins into the host cells. PopB and PopD associate with the target membrane and they are proposed to form a translocon pore by which effectors are translocated. This translocon is a potential target for inhibitors because these molecules do not need to overcome the intrinsic problems associated with cell membrane permeability and the presence of efflux pumps.

In previous studies, we have reported that the PopD N‐terminus is exposed to host cell cytosol (J. Biol. Chem. 2018 293 8982–8993). We reasoned that the PopD N‐terminal exposure could be used as a reporter for properly assembled translocons. Using a split form of the small NanoLuc (NLuc) luciferase, we have developed an assay to detect the exposure of this N‐terminus to the host cytosol. HeLa cells producing the large truncated version of NLuc are used as targets. These cells are incubated with a P. aeruginosa ΔpopD PAK strain that produces a PopD variant fused with the missing portion of NLuc at the N‐terminus (Nluc10‐PopD). Assembly of a functional translocon is detected by the maturation of NLuc enzyme in the HeLa cell cytosol and the production of luminescence. Failure to assemble a functional translocon did not produce luminescence. The assay has a typical signal‐to‐background ratio (S/B) of 6 ‐ 10 and a Z factor above 0.5, constituting an excellent tool for the screening and detection of small molecule inhibitors of T3SS translocon assembly.
Mechanisms of Orthosteric Agonist and Allosteric Modulator Activity at mGlu7
Authors: Lei Xia , Rodriguez Alice , Niswender Colleen
Published: May 2022

Metabotropic glutamate receptor 7 (mGlu7) is a dimeric, group III metabotropic glutamate (mGlu) receptor. It is a G protein‐coupled receptor that acts to modulate neurotransmission across many brain structures. mGlu7 is most highly expressed presynaptically in neurons and is widely distributed in the central nervous system (CNS) and body. Mutations, deletions, or decreases in mGlu7 result in symptoms and phenotypes of neurodevelopmental disorders in humans and mice, including Rett syndrome.

Our group is focused on developing new ligands that interact with mGlu7 to understand the therapeutic potential of the receptor in various CNS disorders. However, in vitromolecular pharmacology studies show that the orthosteric ligand, glutamate, has a low affinity for mGlu7 that limits its ability to be routinely used for drug screening. An alternative agonist, L‐AP4, is a synthetic compound that, while not produced naturally in vivo, activates mGlu7 with higher affinity than glutamate. However, our preliminary research indicates that there are differences in the profiles of positive allosteric modulators (PAMs) when assessed using glutamate versus L‐AP4. This difference creates a challenge for the development of mGlu7‐selective PAMs, and the focus of our current studies is to understand the difference between these two agonists in activating the mGlu7 receptor.

The mGlu receptors function as constitutive dimers. Here, we hypothesize that glutamate and L‐AP4 activate mGlu7 either via a different number of agonist binding sites or via distinct effector interactions; additionally, we hypothesize that mGlu7potentiation by structurally distinct PAMs may differ based on these agonists. We have found that two agonist binding sites are required for mGlu7 activation for both glutamate and L‐AP4, and two G‐protein/effector binding sites are required for any glutamate‐dependent activation. L‐AP4, however, can partially activate the dimer with one functioning G‐protein binding site, and two G‐protein binding sites fully activate mGlu7 with L‐AP4. The PAM VU0422288 requires two agonist binding sites to potentiate mGlu7; in contrast, VU0155094 and VU6005469 show partial potentiation of mGlu7 if one of the agonist binding sites is mutated. VU0422288 induces partial potentiation of mGlu7 with only one functional G‐protein binding site and requires two G‐protein binding sites to induce full potentiation. In contrast, other PAMs (VU0155094 and VU6005469) can fully potentiate mGlu7 activity with one functioning G‐protein binding site. Additionally, VU0422288 also shows elevated efficacy with L‐AP4 compared to glutamate while the other two PAMs do not show differences in efficacy between these two agonists. These studies suggest that mGlu7 can be differentially regulated by various agonists and PAMs and provide insight into considerations for chemical optimization campaigns to generate new allosteric ligands for the receptor.
Phosphatidic Acid Mediates the Nem1‐Spo7/Pah1 Phosphatase Cascade in Saccharomyces cerevisiae
Authors: Kwiatek Joanna M. , Han Gil‐Soo , Carman George M.
Published: May 2022

The Nem1‐Spo7/Pah1 phosphatase cascade plays an essential role in the synthesis of the neutral lipid triacylglycerol in the yeast Saccharomyces cerevisiae. Pah1 is a phosphatidic acid (PA) phosphatase that is phosphorylated and inactive in the cytoplasm. The Nem1‐Spo7 complex is a nuclear/endoplasmic reticulum (ER)‐associated protein phosphatase that recruits, dephosphorylates, and activates Pah1. Upon its dephosphorylation, Pah1 hops onto the membrane surface for its interaction with and dephosphorylation of PA to produce the diacylglycerol that is then converted to triacylglycerol. Pah1 then scoots along the membrane to associate with and dephosphorylate another PA molecule. In this work, the Nem1‐Spo7 complex was reconstituted into unilamellar phospholipid vesicles (proteoliposomes) composed of phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, and PA to mimic the nuclear/ER membrane. The phospholipid composition of the proteoliposomes, which had an average diameter of 60 nm, was confirmed by thin‐layer chromatography. The reconstituted Nem1‐Spo7 complex supported the recruitment and dephosphorylation of Pah1 phosphorylated by the Pho85‐Pho80 protein kinase complex in vitro or the Pah1 phosphorylated by multiple protein kinases in vivo. The Nem1‐Spo7‐mediated dephosphorylation of Pah1 was dependent on the presence of PA in the proteoliposomes. Additionally, the dephosphorylated Pah1 catalyzed the dephosphorylation of PA to produce diacylglycerol in the proteoliposomes. This reconstituted system permits a defined membrane environment to examine the regulation of the Nem1‐Spo7/Pah1 phosphatase cascade.
Regulation of Mitogen‐Activated Protein Kinase Hog1 Phosphorylation Induced by DNA Damage Agent in Saccharomyces cerevisiae
Authors: Weng Fangli , Wang Yuqi
Published: May 2022

DNA damage is a harmful and unavoidable stress frequently encountered by living organisms. It is crucial for organisms to have elaborated mechanisms in response to DNA damage, and a thorough understanding of these mechanisms and their regulation is very important. Hog1 is a stress‐activated protein kinase in Saccharomyces cerevisiae, which responds to osmolarity changes as well as various other stressors. Recently, we found that Hog1 can be activated by DNA damage agent, and the activation is primarily mediated by the Sln1 branch of the HOG pathway. Interestingly, we observed that DNA damage agent‐induced activation of Hog1 is substantially elevated when treated with cycloheximide, a protein synthesis inhibitor, suggesting the existence of a mechanism that keeps the extent of Hog1 activation in check. Notably, the cycloheximide effect is diminished in several phosphatase deletion mutants, suggesting a profound involvement of phosphatase in this process. The detailed characterization of the role of phosphatase in regulating DNA damage‐induced Hog1 activation is currently underway.
Comparative Study of Quantitative TSS Maps among Yeast Strains Demonstrates Key Genetic Sites Required for TSS Selection
Authors: Park William , Lu Zhaolian , Lin Zhenguo
Published: May 2022

Gene transcription is finely regulated to ensure proper cellular functions. Transcription initiation is a critical step of gene regulation as it integrates most regulatory signals, determines the location of transcription start sites (TSS), and establishes the transcript abundance. Recent studies have shown that transcription of most genes in eukaryotes can be initiated from multiple spatially located TSSs. Furthermore, different TSSs were used in various cell types as well as in response to environmental stress. How TSSs are selected during transcription, however, remains largely unknown. We aimed to infer important genetic variations associated with divergence of TSSs to better understand the role of genomic context in TSS selection. Specifically, we used no‐amplification non‐tagging cap analysis of gene expression (nAnT‐iCAGE) sequencing to accurately determine TSSs on a genome‐wide scale for three different populations of budding yeast (Saccharomyces cerevisiae). Comparative studies of these quantitative TSS maps and their genomic sequences showed that transversion substitutions at several positions, such as ‐8, ‐1, and +1 position relative to the TSS, coincided with divergence of the dominant TSS in many promoters. Additionally, a range of transversion substitutions upstream of the TSS at around the ‐150 position showed divergence of the dominant TSS in many promoters as well. However, many divergent TSSs did not have genetic variations nearby, suggesting that transcription factors likely played a major role in TSS selection. This study lays a solid foundation for further functional characterization of these genetic variations, contributing to a better understanding of the genetic mechanisms underlying the regulation of transcription initiation.
Evaluation of the protein cross‐link breaking effect of selected medicinal plants used in the management of diabetes mellitus
Authors: Adeniran Oluwaseyefunmi I. , Gololo Stanley S.
Published: May 2022

One of the strategies proposed for combating advanced glycation end‐products (AGEs) and its devastating effects is the breaking of preformed AGEs‐protein cross‐links. AGEs are linked to the pathogenesis of late onset complications of diabetes mellitus, age‐related and neurodegenerative diseases.

This study evaluated the AGEs‐protein cross‐link breaking effect of some medicinal plants used for managing diabetes mellitus. These plants, selected based on their blood glucose lowering property, include Sclerocarya birrea, Psidium guajava, Murraya koenigiiand Cinnamon cassia.

Of the selected plants, the highest AGEs‐protein cross‐link breaking effect was observed with the hexane and ethyl acetate leaf extracts of Psidium guajava.Methanol extracts of both Psidium guajava leaf and Sclerocarya birrea stem‐bark also showed high cross‐link breaking effect. Chemical screening tests for the presence of phytochemicals and Gas Chromatography Mass Spectrometry (GC‐MS) analysis for the detection and identification of the phytoconstituents in the extracts were also carried out.

Compounds detected in the hexane leaf extract of Psidium guajava, using GC‐MS, include I‐(+)‐Ascorbic acid 2,6‐dihexadecanoate, Octadecanoic acid (Stearic acid), Vitamin E (D‐alpha‐tocopherol), Alpha‐Tocopherol‐beta‐D‐mannoside and 2,6,10,14,18,22‐Tetracosahexaene, 2,6,10,15,19,23‐hexamethyl‐, (all‐E) (Squalene). The hexane leaf extract of Psidium guajava was subjected to column chromatography. Using Nuclear Magnetic Resonance and Infra‐red spectroscopy, the isolated compound from the hexane leaf extract of Psidium guajava was identified as a flavonoid.

The results of the study indicate that present in these extracts are potent agents with the ability to break AGEs‐protein cross‐links which could be exploited for drug discovery.
Fishbowl As Pedagogical Innovation In Anatomy Teaching
Authors: Santos Cerqueira Gilberto , Silva Renata Souza e S. , da Santos Silva José Mariedson , Naconecy de Souza Horst S. , Pimenta Bindá Helder S. , da Silva Ivan do Nascimento , Silva Teixeira Cláudio S. , Maria Silvio Morano Domingos Antonio Clemente S.
Published: May 2022

The Fishbowl is an active teaching methodology whose group discussion format promotes dialogue and exchange of experience among session students and allows everyone to have an equal chance to express their opinions and views during class. So, we use this method to teach endocrine system anatomy to undergraduate students. However, there is little evidence of the use of this methodology to guide teachers on the best way to implement the Fishbowl in teaching anatomy. Based on these premises, the objective of this work was to report our experience based on the use of the Fishbowl technique in the teaching and learning process of the anatomy of the endocrine system. A quasi‐experimental study with a quantitative approach was carried out with students from the discipline of anatomy of the endocrine system. The technique used was the open fishbowl in online mode. For data analysis, we used the relative frequency and the Mann Whitney test with the aid of the Graph Pad Prism version 7.0 program, with the data with a value of p<0.05 being considered significant. It was observed that there was no statistically significant difference between the participants' ages (p>0.05). In our studies, we found that Fishbowl was more effective for teaching undergraduate anatomy than traditional teaching (p<0.05). The Fishbowl proved to be a satisfactory teaching technique for students when compared to the traditional class. We found that students who participated in the Fishbowl got statistically significantly more questions on the test when compared to students in traditional education (p<0.05). It was found that the Fishbowl allowed the educational process to be carried out with greater interaction in a more playful way. In addition, students became protagonists in the teaching and learning process, participating more effectively in classes. Thus, the Fishbowl becomes another active tool in the process of teaching and learning anatomy.
Anti‐CovidMicroRNA Therapy Blocksthe Expression of the SpikeGeneof SARS‐CoV‐2
Authors: DeMarco Victoria , Sine Laura C. , Hintelmann Thomas , Reardon Sean , Hicks Martin J.
Published: May 2022

Emerging viral diseases have increased in recent decades. In December 2019, an epidemic with low respiratory infections emerged in Wuhan, China. The disease, Covid‐19 was found to be caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). As of October 14, 2021, WHO has confirmed greater than 239,000,000 global cases, nearly 5 million deaths worldwide, including greater than 720,000 in the USA. Fortunately, a vaccine has been approved and distributed, yet there are no approved therapeutics for infected individuals, and the threat of emerging vaccine‐resistant strains remain. From advances in biotechnology, the genome and structure of SARS‐CoV‐2 is known. Three proteins are anchored in the viral envelope, Spike (S), Envelope (E), and Membrane (M), which is linked to the Nucleocapsid (N) protein connecting to the viral RNA genome. Our lab is developing an innovative therapy that delivers multiple therapeutic microRNAs to block the expression of distinct Covid viral proteins. The design of the anti‐Covid microRNAs 1) mimics human microRNA cluster 17‐92a structural stability, 2) forms guide‐RNA substrates for the RNA induced silencing complex, and 3) are complementary to specific regions of the SARS‐CoV‐2 RNA genome without off‐targets effects in the human genome. Twenty‐one microRNA sequences were designed to target the S gene, six for N, two for M, and one for E. These were cloned into our microRNA‐17‐92 therapy vector which expresses six distinct anti‐Covid RNA therapeutics simultaneously. We have transfected the S gene into our tissue culture model to measure the efficacy of the anti‐Covid microRNA therapy to down‐regulate the S gene expression. In our preliminary experiments we show a significant 2.5‐fold reduction in the relative abundance of the Spike mRNA in the treated cells (p < 0.05). We are currently testing additional therapies and verifying changes in spike protein levels. Next steps are to examine the secondary structure of our RNA therapy using SHAPE‐MAP to optimize RNA therapeutic stability in comparison to the stable structure based on the human gene, microRNA Cluster 17‐92a.
Anti‐Covid Gene Therapy: Integration of Authentic Research into a Continuously Evolving Undergraduate Laboratory Course
Authors: Hicks Martin J. , DeMarco Victoria , Sine Laura C. , Gawargi Flobater
Published: May 2022

Undergraduate biology students often graduate without exposure to authentic research experiences. Laboratory courses follow a one or two week fail‐proof experiment resembling a cookbook recipe, lacking the uncertainty of genuine research. Techniques in molecular biology cover an array of skills essential to succeed in a biotechnological laboratory today. I have been developing an evolving molecular biology lab course based on the teaching of concepts while imparting the skills and applications of modern techniques, providing students with theoretical concepts and laboratory skills. We prepare students to carry‐out scientific protocols that can be applied to a future workforce setting. Students are immersed in a 10‐week series of labs with the objective to use molecular cloning to make a novel gene therapy vector; therapies are designed to inhibit the expression of genes of the virus that causes Covid‐19, severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). Students are introduced to PubMed and GenBank to research the background of SARS‐CoV‐2 and its target genes; the Spike gene (S), the membrane gene (M) the nucleocapsid gene (N) and the envelope gene (E). Students use the DNA software, Serial Cloner, as a tool to evaluate DNA sequences and mFOLD to analyze RNA structure. Students generate and visualize the design of an antisense gene therapy directed against one of the target viral genes. Using our gene therapy platform, students generate a vector with a unique target. Subsequently student‐generated vectors were transfected into mammalian tissue culture cells that express one of the target genes (S, M, N or E) and RNA and protein was collected to measure the efficacy of the gene therapy vector to reduce the expression of the target viral gene. In our initial experiments, we observed greater than 2.5‐fold reduction in the Spike Covid mRNA. Currently, students are testing additional therapies to maximize efficacy.
Proton‐gated Coincidence Detection is a Common Feature of GPCR Signaling
Authors: Isom Daniel G.
Published: May 2022

The evolutionary expansion of G protein‐coupled receptors (GPCRs) has produced a rich diversity of transmembrane sensors for many physical and chemical signals. In humans alone, over 800 GPCRs detect stimuli such as light, hormones, and metabolites to guide cellular decision‐making primarily using intracellular G protein signaling networks. This diversity is further enriched by GPCRs that function as molecular sensors capable of discerning multiple inputs to transduce cues encoded in complex, context‐dependent signals. Here, we show that many GPCRs are coincidence detectors that couple proton (H+) binding to GPCR signaling. Using a large panel of receptors covering hundreds of individual GPCR‐Gα coupling combinations, we show that H+ gating both positively and negatively modulates GPCR signaling. Notably, these observations extend to all modes of GPCR pharmacology including ligand efficacy, potency, and cooperativity. Additionally, we show that GPCR antagonism and constitutive activity are regulated by H+ gating and report the discovery of new GPCR H+ sensors that can be activated solely by changes in pH. Together, these findings establish a paradigm for GPCR signaling, biology, and pharmacology applicable to acidified microenvironments such as endosomes, synapses, tumors, and ischemic vasculature.
Dishevelled localization and function are differentially regulated by structurally distinct sterols
Authors: Sengupta Sonali , Freel Bethany , Francis Kevin
Published: May 2022

Aberrant cholesterol synthesis, characterized by depletion of cellular cholesterol and accumulation of sterol precursors, is directly linked to a group of rare human diseases exhibiting broad clinical phenotypes and developmental malformations. Despite decreased cholesterol content being shared across these diseases, patients exhibit distinct clinical manifestations associated with specific tissues or systems with varying degrees of phenotypic overlap. This tissue specificity may result from disease‐specific disruption of important critical signaling pathways due to the inability of sterol precursors to structurally compensate for cholesterol. We previously determined that the cholesterol precursor 7‐dehydrocholesterol (7DHC), which accumulates in Smith‐Lemli‐Opitz syndrome, perturbs Dishevelled 2 (DVL2) protein function due to reduced binding affinity of 7DHC to DVL2’s PDZ domain (Postsynaptic density 95, Disc large 1, and Zonular occludens 1 domain) in comparison to cholesterol, resulting in loss of canonical Wnt/β‐catenin signaling (1). We hypothesized that structural differences between cholesterol and alternative sterols will inhibit binding with DVL2 and other PDZ‐domain containing proteins, producing dire cellular consequences and directly affecting Wnt signaling (2). Protein homology modeling and ligand‐protein docking analysis suggested a differential binding affinity and specificity of DVL2 for cholesterol versus alternative sterols. Using PM‐mimetic vesicles incorporating cholesterol versus alternative sterols in liposome/DVL2 pulldowns, we experimentally observed a reduced binding affinity between DVL2 and alternate sterols. Surface plasmon resonance analysis to define the kinetics of protein‐sterol interaction corroborates in silicomodeling and pulldowns. Our data suggests orientation of the beta‐ and alpha‐sterol face within the DVL2‐PDZ domain binding pocket is predictive of DVL2‐sterol interaction. At the cellular level, we have also observed disruption of DVL2‐sterol binding shifts the subcellular localization of DVL2 from the plasma membrane to the nucleus. Increased nuclear DVL2 promotes non‐canonical WNT signaling and DVL2 protein‐protein binding with nuclear factors. Our work suggests that disrupted sterol homeostasis broadly impacts cellular signaling in a sterol‐specific and pathway‐specific manner. Defining the structural and biochemical requirements for sterol interactions with DVL2 and other cholesterol‐binding proteins will help define the critical role sterol homeostasis plays in human health.

(1)Francis KR et al, Nature medicine. 2016;22(4):388‐96.

(2) Sheng R et al, Nature communications. 2012;3:1249.
Altering NAD(P)H cofactor specificity by structure‐guided modification of class II HMG‐CoA reductase
Authors: Ragwan Edwin , Huo Raining , Kung Yan
Published: May 2022

HMG‐CoA reductase (HMGR) is a central enzyme in metabolism and natural product biosynthesis, as it catalyzes the committed and rate‐determining step of the mevalonate pathway, which produces steroids and isoprenoids, the largest and most diverse class of natural products. Here, HMGR converts HMG‐CoA to mevalonate using two equivalents of a redox cofactor, either NADH or NADPH. Class I HMGRs are highly specific for NADPH, including human HMGR, the target of cholesterol‐lowering statin drugs. However, cofactor specificity of microbial class II HMGR varies widely, as some homologs exhibit a strong preference for either NADH or NADPH, while others homologs are less specific. We have recently identified a key structural feature termed the “cofactor helix” that may govern cofactor specificity in class II HMGR, but until now this helix’s role in conferring NAD(P)H preference has not been tested. In this work, we performed structure‐guided enzyme modification with the aim of altering the cofactor preferences of various class II HMGR homologs. Through kinetic and X‐ray crystallographic studies on these enzyme variants, we have found that the cofactor helix plays a critical but not exclusive role in controlling NAD(P)H preference, expanding our understanding of the structural determinants of cofactor specificity in class II HMGR.
Isolation and Characterization of Antimicrobials from Icelandic Aquatic Bacteria
Authors: Corona‐Avila Irene , Lee Jeongho , Hernandez Antonio , Krull Nyssa , Augustinovic Mario , Jones Derick , Ndekwe Tatyana , Murphy Brian T.
Published: May 2022

Since the Golden Age of natural product (NP) antibiotic discovery in the mid‐1950s, NP discovery has declined despite methodological advances. As a result, antibacterial discovery is being outpaced by antibiotic resistance. One significant challenge in antibiotic discovery is the rediscovery of antibiotic compounds previously isolated from bacteria. Generating a diverse microbial library can help overcome the rediscovery of antibiotic compounds, but major challenges to this approach include the inefficiency of commonly used methods to create high‐throughput libraries of bacteria and the difficulty of reducing redundancy within these libraries. To combat these issues, the Murphy lab works with an innovative NP discovery using IDBac, an efficient, rapid process developed to reduce taxonomic and chemical redundancy in microbial libraries. In the current study, we performed liquid chromatographic separations of Icelandic bacterial isolates K391, K765, and K802. We then investigated each isolate's antibacterial ability against pathogens through growth inhibition assays. Bacterial isolate K391 displayed weak antibacterial activity in fractions 3‐5, 14‐15, and 25‐26 and showed presence of a small molecule, which may hold potential as an antibiotic via MS/MS peaks from fractions 14‐15. We are further investigating the K391 fractions through GNPS analysis and running liquid chromatographic separations of K765 and K802 in normal phase.
Journal Club 3.0: An Effective Method of Training High School Students to Understand and Present Relevant Academic Research Papers
Authors: Patel Anya , Ramkumar Akshar , Teter Willow , Lin Lucie , Sostena Juliet , Choi Peter , Hauser Paul , De Luke
Published: May 2022

People are asked to make decisions influencing the world we live in based on peer‐reviewed academic research, a fundamental component of scientific research and development. However, high schoolers are provided with little scaffolding to read and understand relevant scientific papers, limiting their ability to engage with the field on this level. Here, we compare two self‐propagating student‐driven systems, in which experienced students help inexperienced peers learn to read and present on recent academic research papers. In the first system, student presenters undergo a series of meetings, each led by a different team consisting of two students. The teams each specialize in one of four areas: the broad scope of the paper, its novelty, numerical data, and presentational skills. In the second system, every presenter attends meetings led by a consistent team of 3‐5 students who are each trained in one of the following areas: the personal story and hook, the main question and supporting data, and presentational skills. Our data suggests that working with a consistent group of reviewers led to more continuity between the review meetings, eliminating redundancies and increasing preparedness for weekly meetings. This continuity also enabled review teams to tailor the preparation process to fit the presenter’s needs. The improved process increased efficiency, enhanced the presenter experience, and led to a higher quality of presentations. This research contributes to our goal of implementing a peer‐to‐peer training system that effectively heightens students’ scientific literacy skills and allows them to develop a deeper understanding of current issues in different fields that are being solved through a variety of research paradigms and data analyses.
Structures of LIG1 engaging with mutagenic mismatches inserted by polβ in base excision repair
Authors: Caglayan Melike
Published: May 2022

DNA ligase I (LIG1) catalyzes final ligation step following DNA polymerase (pol) β gap filling and an incorrect nucleotide insertion by polβ creates a nick repair intermediate with mismatched end at the downstream steps of base excision repair (BER) pathway. Yet, how LIG1 discriminates against the mutagenic 3′‐mismatches at atomic resolution remains undefined. Here, we determined X‐ray structures of LIG1/nick DNA complexes with G:T and A:C mismatches and uncovered the ligase strategies that favor or deter ligation of base substitution errors. Our structures revealed that LIG1 active site can accommodate G:T mismatch in a similar conformation with A:T base pairing, while it stays in the covalent LIG1‐adenylate intermediate during initial step of ligation reaction in the presence of A:C mismatch at 3′‐strand. Moreover, we showed mutagenic ligation and aberrant nick sealing of the nick DNA substrates with 3′‐preinserted dG:T and dA:C mismatches, respectively. Finally, we demonstrated that AP‐Endonuclease 1 (APE1), as a compensatory proofreading enzyme, interacts and coordinates with LIG1 during mismatch removal and DNA ligation. Our overall findings and ligase/nick DNA structures provide the features of accurate versus mutagenic outcomes at final BER steps where a multi‐protein complex including polβ, LIG1, and APE1 can maintain accurate repair.
Desmoglein‐2 and Desmoplakin modulate Cell Spreading Dynamics via PDZ‐GEF2 and Rap1 Signaling
Authors: Shelton William T. , Thomes Charles E. , Thomas Sarah M. , Conway Daniel , Dubash Adi D.
Published: May 2022

The desmosome is a cell‐cell adhesion complex which facilitates the mechanical stability of tissues and cell‐cell communication. Desmosome function depends upon a tripartite organizational structure wherein transmembrane cadherins (Desmoglein and Desmocollin) link adjacent cells in the extracellular space, armadillo proteins (Plakophilin and Plakoglobin) stabilize the intracellular plaque, and the cytolinker Desmoplakin (DP) connects the plaque to the intermediate filament network. In addition to their central role in maintaining cell‐cell junction integrity, desmosomal cadherins also coordinate biological processes such as proliferation, apoptosis, differentiation and cell migration. In our study, we sought to investigate the signaling mechanisms involved in control of actin architecture via desmosomal cadherins, using A431 cells lacking Desmoglein‐2 (Dsg2 KO), generated via CRISPR‐mediated knock‐out. Compared to control cells, Dsg2 KO cells displayed a significant increase in spreading area on both fibronectin and collagen. As these experiments were performed in singly spreading cells, these experiments have identified a novel cell‐autonomous, cell‐cell adhesion‐independent role for Dsg2 in regulation of cell spreading. Spreading changes in Dsg2 KO cells were dependent on Rap1 GTPase, as siRNA‐mediated knockdown of Rap1 or pharmacological inhibition of Rap1 in Dsg2 KO cells rescued the increase in spreading area. Rap1 activity was significantly increased in Dsg2KO cells, but no change was observed in the activity of other GTPases tested (Rho, Rac and Cdc42). Changes in Dsg2‐mediated cell spreading was also shown to require the Rap GEF PDZ‐GEF2 (but not PDZ‐GEF1 or other Rap GEFs), and PDZ‐GEF2 localization at the cell periphery markedly increases upon loss of Dsg2. Further investigation into signaling pathways known to affect cell spreading identified a role for TGF‐beta signaling, but neither Erk or p38 MAPK were involved. In singly‐spreading cells, the expression of other desmosomal proteins is unchanged in Dsg2 KO cells (mRNA and protein levels), and the localization of Plakophilin‐2, Plakoglobin and Desmoplakin is also not significantly perturbed (specifically in singly‐spreading cells). Nevertheless, siRNA‐mediated knockdown of DP was able to rescue Dg2KO‐dependent increases in cell spreading (while siRNA for Plakophilin‐2, Plakoglobin and Plakophilin‐3 did not). In addition, DP siRNA was also able to rescue changes in localization of PDZ‐GEF2 seen upon loss of Dsg2. Taken together, these data have identified novel cell‐cell adhesion independent role for Dsg2 and DP in mediating cell spreading via Rap1 signaling, which provides significant insight into the signaling mechanisms via which desmosomal cadherins control cell‐matrix attachment and cytoskeletal architecture.
Kinase Domain Autophosphorylation Rewires the Activity and Substrate Specificity of CK1 Enzymes
Authors: Cullati Sierra , Chaikuad Apirat , Chen Jun‐Song , Gebel Jakob , Tesmer Laura , Zhubi Rezart , Navarrete‐Perea Jose , Guillen Rodrigo X. , Gygi Steven P. , Hummer Gerhard , Dotsch Volker , Knapp Stefan , Gould Kathleen L.
Published: May 2022

CK1s are acidophilic serine/threonine kinases with multiple critical cellular functions; their misregulation contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Here, we describe an evolutionarily conserved mechanism of CK1 activity: autophosphorylation of a threonine (T220 in human CK1δ) located at the N‐terminus of helix αG, proximal to the substrate binding cleft. Crystal structures and molecular dynamics simulations uncovered inherent plasticity in αG that increased upon T220 autophosphorylation. The phosphorylation‐induced structural changes significantly altered the conformation of the substrate binding cleft, affecting substrate specificity. In T220 phosphorylated yeast and human CK1s, activity toward many substrates was decreased, but we also identified a high‐affinity substrate that was phosphorylated more rapidly, and quantitative phosphoproteomics revealed that disrupting T220 autophosphorylation rewired CK1 signaling in Schizosaccharomyces pombe. T220 is present exclusively in the CK1 family, thus its autophosphorylation may have evolved as a unique regulatory mechanism for this important family.
An endogenous polyunsaturated fatty acid, dihomo‐gamma‐linoleic acid, induces neurodegeneration in C. elegans via ferroptosis
Authors: Lee Kin Sing Stephen , Sarparast Morteza , Zhang Fan , Seyedehelham Pourmand , Gonzalez Roland E. , Kessler Benjamin , Heasman Benjamin , Alan Jamie
Published: May 2022

Ferroptosis, an iron‐dependent non‐apoptotic programmed cell death, becomes a novel target and mechanism for age‐associated neurodegenerative diseases. Although several ferroptosis regulatory proteins have been identified, the signaling molecules and the pathways of ferroptosis remain largely unknown. Despite membrane lipid peroxidation being one of the molecular hallmarks of ferroptosis, how polyunsaturated fatty acids (PUFAs) involved in the ferroptosis pathway is understudied. Here, we hypothesize that a specific PUFA and/or its downstream metabolites are lipid mediators of ferroptosis. However, it is challenging to identify by which metabolites regulate ferroptosis and to study the associated mechanism due to many different endogenous PUFAs are presented endogenously and each PUFAs can be metabolized to almost a hundred of metabolites.

To take on these challenges, we employed Caenorhabditis elegans (C. elegans) as a novel biological model due to the availability of many genetic and imaging tools, conserved ferroptosis pathways between mammal and C. elegans, and adaptability for high‐throughput whole animal aging study. Using the well‐established fluorescent imaging assay in C. elegans, we showed that treatment of dihomo‐gamma‐linoleic acid (DGLA), but not other PUFAs, induces significant (>40% as compared to control) degeneration in dopaminergic neurons immediately after 1 day of treatment. We also found that treatment of DGLA does not affect other somatic tissues of the C. elegans. Our lipidomic analysis demonstrated that DGLA is well‐absorbed by C. elegans. Besides, our results indicated that DGLA triggers neurodegeneration in dopaminergic neurons, likely through ferroptosis. Using our oxidized lipid metabolites analysis and synthetic chemical probes, we revealed that DGLA likely triggers neurodegeneration viatheir oxidized metabolites. Finally, we will also present the potential ferroptosis signaling pathways triggered by DGLA based on our RNA‐seq experiments.

Our results will help us identify novel lipid mediators for ferroptosis, which will lead to a better understanding of the molecular mechanism of ferroptosis. Our findings regarding the neuronal specific effects triggered by DGLA also revive a novel cell‐specific regulatory mechanism of ferroptosis. Lastly, our study will find novel therapeutic targets for either preventative treatment or cure for neurodegenerative diseases.
The effect of the linker in urea‐based soluble epoxide hydrolase inhibitors’ on their blood‐brain penetration ability and drug‐like properties
Authors: Lee Kin Sing Stephen , Prahalad Keshav , Giletto Matthew , Maghami Katayoon , Dickson Alex , Ellsworth Edmund
Published: May 2022

Soluble epoxide hydrolase (sEH) is a novel therapeutic target for the treatment neurodegenerative diseases, including Parkinson’s disease and Alzheimer’s disease. Although many sEH inhibitors exhibit sub‐nanomolar potency, they suffer from poor blood‐brain barrier penetration and drug‐like properties. As high in vivolevel is required to achieve clinical efficacy for sEH inhibitors for other therapeutic applications, low CNS penetration represents a significant barrier for success for CNS diseases. Therefore, sEH inhibitors with high blood‐brain barrier penetration are needed.

Although significant structure‐activity relationships (SAR) have been reported, little data exist exploring the features that overcome the blood‐brain barrier. Most SAR studies have focused on terminal substitutions on both ends of the reported inhibitors with little focus on the “linker” or core of the inhibitors. In this presentation, we will show how the “linker” of urea‐based sEH inhibitors affects their potency, physical properties, and ability to penetrate the blood‐brain barrier. Based on our computational model and the crystal structure of our lead compound, we have prepared new sEH inhibitors with linker containing various ring sizes, bicyclic structures, and simple methyl or fluorine substituents. Perhaps not unexpectantly, the linker of sEH inhibitors has substantial effects on the inhibitor’s potency, solubility, and blood‐brain barrier penetration. In some cases, we observed an increase in blood‐brain penetration by 4‐fold and improvements in solubility by almost 10‐fold. In addition, unexpectedly, our data provides new insights that the linker of the sEH inhibitor affects the binding kinetics without improving the potency of the inhibitors. Our results will create a new direction for the design of novel sEH inhibitors, and we are in the process of leveraging the existing SAR to create much‐improved sEH inhibitors to treat neurodegenerative diseases.
Role of a Polar c‐Ring Residue in the Fo Motor of E. coli ATP Synthase
Authors: Shepard Sam J. , Shrestha Rashmi K. , Steed Ryan
Published: May 2022

ATP Synthase is a molecular motor that utilizes a rotary mechanism to synthesize adenosine triphosphate (ATP), the fundamental energy currency of life. The torque for this mechanism is generated in the membrane‐embedded FO motor by harnessing the proton motive force, where protons flow down the electrochemical gradient through two half channels in the motor. The exit half channel on the cytoplasmic side is found at the interface of subunit a (stator) and the subunit c ring (rotor). Previous work on the E. coli ATP synthase has suggested that Thr51 in subunit c is involved in the proton translocation process. To investigate the role of this residue and chemical requirements at this position, we generated six substitution mutants and assayed their in vitro ATP synthesis, H+ pumping, and H+ permeability activities as well as the ability of mutants to carry out oxidative phosphorylation in vivo. Polar and hydrophobic mutations were generally tolerated, except for a notable decrease in function when a negative charge is introduced. Unusually, substitution with Cys completely abolished in vitro functions but only mildly inhibited growth on succinate. Overall, our results suggest that cThr51 may play a supporting role in the interaction between the c‐ring and subunit a in the region of the proton exit channel.
Phosphorylation barcode ensembles encoded by biased CXCR3 agonists direct non‐redundant chemokine signaling
Authors: Eiger Dylan S. , Smith Jeffrey , Shi Tujin , Stepniewski Tomasz M. , Honeycutt Christopher , Boldizsar Noelia , Gardner Julia , Tsai Chia‐Feng , Nicora Carrie , Moghieb Ahmed , Kawakami Kouki , Choi Issac , Smith Richard , Inoue Asuka , Selent Jana , Jacobs Jon , Rajagopal Sudarshan
Published: May 2022

Chemokine receptors, a group of G protein‐coupled receptors (GPCRs), interact with transducers such as G proteins, β‐arrestins, and GPCR kinases (GRKs). In the chemokine system, many chemokine agonists act as “biased agonists” that preferentially activate distinct signaling effectors when binding to the same receptor, resulting in distinct physiological effects. Although one third of FDA‐approved drugs target GPCRs, there has been limited success in targeting the chemokine system. Currently, there is little evidence that differential receptor phosphorylation, or “phosphorylation barcodes,” direct these biased responses at chemokine receptors. To address this knowledge gap, we used mass spectrometry to demonstrate that chemokines of CXCR3 promote different ensembles of phosphorylation barcodes that are associated with differential activation of G proteins, β‐arrestins and GRKs. Chemokine stimulation also resulted in distinct changes throughout the kinome in global phosphoproteomic studies. Mutation of specific CXCR3 phosphosites altered β‐arrestin conformation and impacted β‐arrestin activation in molecular dynamics simulations. T‐cells expressing phosphorylation‐deficient CXCR3 mutants resulted in distinct agonist‐ and receptor‐specific chemotactic and signaling profiles that were not completely explained by engagement of G proteins, β‐arrestins, and GRKs alone. Our results directly link distinct GPCR phosphorylation patterns with non‐redundant chemokine signaling (Figure 1).
Middle Cerebral Artery Pulsatility Index is Elevated in Chronic Kidney Disease
Authors: Jones Touré , Sprick Justin , DaCosta Dana , Park Jeanie
Published: May 2022

Introduction

CKD patients exhibit an increased risk of cerebrovascular disease such as stroke, transient ischemic attacks, and cerebral small vessel disease. One mechanism that may contribute to this risk is increased cerebrovascular stiffness and augmented pulsatility of blood flow into the cerebral microcirculation. Middle cerebral artery pulsatility index (MCA PI) provides an indirect measure of cerebrovascular stiffness and can be measured with transcranial Doppler (TCD) ultrasonography. Prior work suggests that MCA PI is elevated in CKD patients following stroke, but it remains unclear if MCA PI is elevated in CKD patients without a history of cerebrovascular disease. Moreover, whether MCA PI correlates with central (aortic) arterial stiffness in CKD remains unknown. We hypothesized that MCA PI would be elevated in CKD patients stages III‐IV compared to controls without CKD matched for age, race, sex, diabetes and hypertension. Furthermore, we hypothesized that MCA PI would correlate with central arterial stiffness and estimated glomerular filtration rate (eGFR) in CKD.

Methods

13 CKD participants stages III‐IV (mean eGFR=38±3 ml/min/1.73m2) and 10 controls without CKD (age=60±2y) underwent measurement of middle cerebral artery blood velocity via TCD during 5 minutes of seated rest. MCA PI was averaged over the 5 minute period and calculated as MCA PI = (peak systolic blood velocity – diastolic blood velocity)/(mean blood velocity). In CKD, central arterial stiffness was measured via carotid‐femoral pulse wave velocity (cfPWV) after 5 minutes of supine rest. MCA PI was compared between CKD vs CON via 2‐tailed, independent sample T‐tests and the relationships between MCA PI, cfPWV, and eGFR CKD were examined via linear regression.

Results

MCA PI was elevated in CKD compared to CON (0.91±0.06 vs 0.73±.04, P=0.04) and was associated with cfPWV (R=0.72, P=0.01). However, we observed no relationship between MCA PI and eGFR in CKD (P=0.83).

Discussion

In support of our hypothesis, MCA was elevated in CKD compared to controls and was associated with central arterial stiffness. These findings suggest that cerebrovascular stiffness is elevated in CKD patients without a history of cerebrovascular disease, and that MCA PI correlates with central arterial stiffness in CKD. Increases in cerebrovascular stiffness and subsequent flow pulsatility may comprise one mechanism of cerebrovascular disease risk in CKD. Future work should clarify the relationship between MCA PI and severity of renal disease in a larger cohort, and examine the prognostic value of MCA PI as a predictor of future cerebrovascular disease burden in CKD.
Understanding Alpha‐synuclein Aggregation Propensity with a Fragment Peptide Library
Authors: Fortin Jessica S. , McCarty Annie M. , Penticoff Hailey B. , Hetak Ashley A.
Published: May 2022

Alpha‐synuclein (α‐syn) is an intrinsically disordered presynaptic protein of the central nervous system, and its aggregation has been described to play a central role in the pathogenicity of Parkinson’s Disease (PD). Although the mechanism is not fully understood, certain mutations in the α‐syn protein can increase its susceptibility to oligomerization, fibrillation, and cell permeabilization that creates the cytotoxic effects defined in PD and other forms of neurodegeneration. The α‐syn amino acid variability could further our understanding of the mechanisms involved in the aggregation process. This study aims to characterize the aggregation propensity of α‐syn via a library of fragment peptides designed based on substitutions (or variability) found in various animal species. We used biophysical assays such as Thioflavin T (ThT) and transmission electron microscopy (TEM) to identify critical amino acid residues for misfolding and aggregation. The α‐syn peptide fragments in non‐mammalian species are less prone to aggregate compare with mammal sequences analyzed. These non‐mammalian peptide fragments included the 1‐25 and 26‐50 regions in the wild turkey, two‐lined caecilian, mainland tiger snake, Tanaka’s snailfish, Greenland sleeper shark, and tiger pufferfish. All 88‐113 peptide fragments from different animal species generated fibrils. Region 1‐25 was least prone to aggregation for most fragment peptides. The understanding of these identified protein motifs with increased risk for amyloidosis may serve as new potential therapeutic targets for PD and neurodegeneration.
Optimizing the production, purification, characterization of E. coli OMVs
Authors: Kasper Anna , Henretta Sarah , Dinu Milena , Smith Callum , Videva Martina , Crawford Jamie , Shanahan Caitlin , Gaborski Thomas , Michel Lea V.
Published: May 2022

Outer membrane vesicles (OMVs) are 20‐250 nm particles released from Gram‐negative bacteria, including commensal and pathogenic Escherichia coli(E. coli). OMVs contain outer membrane and periplasmic proteins, toxins, nucleic acids, and pieces of peptidoglycan. OMV production is enhanced by environmental stressors, and OMVs are thought to facilitate bacterial cell growth and division, to contribute to bacterial communication (quorum sensing), and can also act as decoys during antibiotic or host immune system attacks. We aim to optimize E. coli OMV production and purification using ultracentrifugation and alternative techniques, to characterize OMVs using immunoblotting and nanoparticle tracking analysis (NTA), and to ultimately determine how OMVs can be used as molecular biomarkers for E. coli infections and Gram‐negative sepsis. Preliminary studies have shown that antibodies to peptidoglycan associated lipoprotein (Pal) can be employed to successfully identify E. coli OMVs.