bluuebunny/crossref_metadata_2025 huggingface dataset:
Defining lagging‐strand polymerase dynamics in vivo
Authors: Smith Duncan
Published: November 2023

At the replication fork, the leading strand can be continuously synthesized in the direction of DNA unwinding. However, the lagging strand must be discontinuously synthesized via the repeated priming, extension, processing and ligation of Okazaki fragments. Lagging‐strand synthesis in eukaryotes requires the sequential action of two polymerases – Pol α/primase and Pol δ. We have previously found that the location of Okazaki fragment termini is determined by interaction between Pol δ and newly deposited nucleosomes on the replicated DNA, and that this interaction generates fragments whose size is quantized according to the chromatin repeat (1). However, although the length of Okazaki fragments appears to be broadly similar across diverse eukaryotes and the mechanism that fine‐tunes Okazaki fragment size is known, it is not clear how the overall range of fragment length is determined. Moreover, although it is clear that the association of the lagging‐strand polymerases with DNA must be dynamic, the stoichiometry and stability of lagging‐strand polymerase binding at active replication forks have not been extensively investigated in eukaryotes.

By titrating the levels of wild‐type and mutant replicative polymerases in S. cerevisiae and analyzing newly synthesized DNA on the lagging strand, we investigate the dynamics of DNA polymerase association with the replisome, the mechanisms by which eukaryotic Okazaki fragment length is specified and maintained in vivo, and the consequences for genome integrity of perturbing this length. Data from these investigations, and their mechanistic implications, will be discussed

Support or Funding Information

R01 GM114340 (NIGMS)Searle Scholars Program
The Effect of UVB Irradiation on Histone H4
Authors: Musson Chase Ryker , Zurita‐Lopez Cecilia
Published: November 2023

Histone modifications such as phosphorylation, alter chromatin and accessibility to transcription factors. Consequently, they play an important role in gene expression. During normal cell development, histone H4 serine 1 becomes phosphorylated and arginine 3 becomes methylated. Using different proteins, studies have established a crosstalk relationship between these two modifications whereby methylation will block phosphorylation. In histone H4, serine 1 becomes phosphorylated in response to DNA damage. In addition, there is evidence that exposure to ultraviolet light increases the activity of protein arginine methyltransferase 1 (PRMT1), the enzyme responsible for arginine 3 methylation. However, whether phosphorylation or methylation can directly respond to environmental stresses, such as ultraviolet B (UVB) irradiation, remains unknown. It is also not known whether phosphorylation or neighboring arginine methylation respond synergistically or antagonistically to UVB irradiation. Preliminary in vitro studies have confirmed that histone H4 is methylated by PRMT1 at arginine 3. The effects of UVB irradiation will be assessed by directly measuring the amount of serine 1 phosphorylation and arginine 3 methylation of histone H4 under set conditions. In vitro modification reactions and HeLa cells incubated with kinase and methyltransferase inhibitors, will undergo UVB (280 – 315 nm) irradiation through a homemade beam collimating device. Histone H4 will be purified and the amount of phosphorylation and methylation will be detected by immunoblotting. We hypothesize that methylation of arginine 3 at histone H4 will increase in response to UVB irradiation and block neighboring phosphorylation of serine 1. PRMT1 is implicated (along with p300, a lysine acetyltransferase) in a signal transduction pathway that is responsible for the production and function of p53, a tumor suppressor. If the amount of arginine methylation increases in response to ultraviolet light, it will confirm that PRMT1 plays an indirect role in the suppression of tumors via histone modification. Finally, the effect of this modification on serine 1 phosphorylation will lead to a greater understanding of the histone code.

Support or Funding Information

This research was funded through the Minority Access to Research Careers Undergraduate Student Training in Academic Research Fellowship Program (NIH Grant GM008228)
Elucidating a Putative Enhancer Element for the Human LAT Gene
Authors: Ghanim Ghida , Finco Timothy Scott
Published: November 2023

The LAT gene encodes a transmembrane adaptor protein required for the function of several immune system cell types, including T cells, mast cells, and natural killer cells. In order to better understand transcriptional regulation of the LAT gene, the laboratory sought to identify and characterize distal regulatory regions, specifically enhancer elements. Data available through the University of California Santa Cruz Genome Browser was assessed to identify genomic regions displaying features consistent with enhancer elements such as DNA sequence conservation, DNase hypersensitivity, and specific post‐translational histone modifications. Identified regions were then PCR amplified and tested for enhancer activity using transient transfection assays in Jurkat T cells and HMC‐1 mast cells. Deletion analysis narrowed enhancer activity to a highly conserved 300 bp region that contains a number of putative transcription factor binding sites, including two Ets and two GATA sites. Point mutations within these Ets and GATA sites significantly inhibited enhancer activity.

CRISPR/Cas9‐mediated deletion was then employed in Jurkat T cells to confirm the functional relevance of this region in the regulation of LAT gene expression. Surprisingly, only heterozygous deletion clones were obtained. Ongoing steps focus on attempting once again to obtain homozygous knockout clones and performing RT‐qPCR to analyze LAT gene expression in both heterozygous and homozygous deletion clones. Collectively, these results identified a putative 300 bp enhancer for the LAT gene which appears to function in part through Ets and GATA transcription factor binding sites.

Support or Funding Information

National Institutes of Health grant # 1R15GM107734‐01
Stat3 is a upstream regulator of Granzyme g that triggers the minor zygotic gene activation in mouse preimplantation embryo
Authors: Ou‐Yang Huan , Wu Shinn‐Chih , Sung Li‐Ying , Chen Chuan‐Mu
Published: November 2023

Maternal zygotic transition (MZT) that control from maternal massage to zygotic gene activation (ZGA) promote preimplantation embryo development in mouse 1‐ to 2‐cell stage. Mouse Granzyme g (Gzmg) is an important protease and specificity expressed at 2‐cell stage of mouse embryos. In our previous report, we found that zygotic RNA synthesize is blocked in Gzmg knock‐down 2‐cell embryo. These data indicated that Gzmg is a necessary protein in MZT. Recently, we found the −417 ~ +28 nt upstream sequence of Gzmg gene may contain cis‐elements to regulate Gzmg expression pattern in mouse zygote and 2‐cell stages by promoter assay in mouse preimplantation embryos. Furthermore, we predicted transcription factor binding sites in promoter sequence by JASPAR and expression atlas website. Gabpa and Stat3 were selected by comparing promoter assay and JASPAR website. These two transcription factors were located in zygote and 2‐cell embryo's nuclei by immunofluorescence. These data supposed that Gabpa and Stat3 may have transcriptional activity to regulate genes in zygote and 2‐cell stages. Surprisingly, the Stat3 can be accumulated in mouse zygote and 2‐cell nuclei by injecting −417~+28 nt upstream sequence of Gzmg into zygote and 2‐cell nuclei. These data indicated that Stat3 is a maternal transcription factor and may regulate Gzmg to promote MZT. Thus, we used inhibitor treatment to define timing of embryo development affected by Stat3. After treatment with S3I‐201, Stat3 inhibitor, mouse embryos were arrested at zygote and 2‐cell stages and some of embryos were arrested at 2‐cell when treated with another inhibitor, WP1066. These results suggest that Stat3 may play important roles at zygote and 2‐cell stages. Take together, Stat3 may promote mouse preimplantation embryo development by triggering the minor ZGA and activating Gzmg gene.
Targeting High‐Mobility Group Box2 by miR‐127 Modulates Pluripotency of Mouse Embryonic Stem Cells and Contributes to Aggressiveness of Hepatocellular Carcinoma
Authors: Zhao Yulan , Yang Zhihong , Wang Li
Published: November 2023

Background

Cancer progenitor cells possess some characteristics associated with normal stem cells. MiR‐127 is associated with germ layer differentiation and its promoter is hypermethylated in cancer cells. High‐mobility‐group protein 2 (HMGB2) is highly expressed in embryonic stage and hepatocellular carcinoma (HCC), but low in adult liver. The current study aimed at elucidating a novel regulation of HMGB2 by miR‐127 in mouse stem cells and liver cancer disease.

Methods

Mouse ESCs (CGR8, Sigma) were maintained on 0.1% gelatin coated dish in Knockout DMEM containing 15% ES‐FBS, 2 mM L‐glutamin, 0.1 mM NEAA, 0.1 mM β‐mercaptoethanol 50 μg/ml penicillin‐streptomycin and 10 ng/ml Leukemia inhibitory factor (LIF). For embryonic body (EB) formation, ESCs were trypsinized into single cells and cultured by seeding 900 ESCs in 30 μl hanging droplets of ES medium devoid of LIF. Liver tumor initiating cells (TICs) spheroid assay: 1000 cells were suspended in serum free TIC medium with 1% methylcellulose supplemented with B27, 20 ng/ml EGF and 20 ng/ml FGF in each well of 96‐well ultralow attachment plate for 7 days. Other methods: luciferase reporter assay, qPCR, Western blots, Co‐immunoprecipitation (Co‐IP), cell invasion, shRNA knockdown and adenovirus overexpression.

Results

The level of miR‐127 was markedly elevated in CGR8 cells during EB differentiation, which correlated with the significant down‐regulation of HMGB2, OCT4 and SOX2. A conserved seed region for miR‐127 exists in the 3′UTR of human, mouse and rat HMBG2 genes. Ectopic expression of miR‐127 inhibited HMGB2 3′UTR activity in a dose dependent manner, which was abolished by mutation of the miR‐127 seed region and miR‐127 antagomir (anti‐miR‐127). Ectopic expression of miR‐127 reduced HMGB2 protein levels in a variety of human HCC cells. HMGB2 interacted with OCT4 and increased protein levels of OCT4 and SOX2, which further enhanced their transcriptional activity. On the other hand, HMGB2 inhibitor Inflachromene (ICM) induced differentiation of mouse stem cells and alleviated proliferation of TIC cells. Ectopic expression of miR‐127 in mouse stem cell reduced mRNA levels of Oct4, Nanog and Sox2, but increased mesendoderm markers during EB formation. Ectopic expression of miR‐127 in TIC cells lead to smaller spheroid formation, which was rescued by HMGB2. This was associated with decreased RhoA, CDC42, and Rac1/2/3 expression by miR‐127.

Conclusion

miR‐127 is a novel regulator of mouse stem cell differentiation and liver cancer progenitor cell proliferation by targeting HMGB2. Our work provides a therapeutic perspective for future translational studies to inhibit HCC metastasis by blocking HMGB2.

Support or Funding Information

Grant support: NIH R01DK104656, R01DK080440, R01ES025909, R21AA022482, R21AA024935, VA Merit Award 1I01BX002634.
Alteration of miR‐186 expression modifies inflammatory markers in normal epithelial and prostate cancer cell models
Authors: Suman Suman , Jones‐Reed Dominique Z. Z. , Schmidt M. L. , Clark Geoffrey J. , Klinge Carolyn , Barve Shirish , Kimbro Kevin S. , Kidd La Creis R.
Published: November 2023

Dysregulation of miRNAs and chronic inflammation are strongly implicated in the development of various malignancies, including prostate cancer (PCa). Previously, our lab identified several inflammatory and immune response sequence variants in CCL5 and CCR5 significantly modified PCa risk. Recently, we propose an oncogenic role for miR‐186‐5p based on its up‐regulation in serum from PCa patients and metastatic PCa cell lines. Moreover, miR‐186‐5p inhibition reduced proliferation, anchorage independent growth and invasion in metastatic PC cells (PC3, MDA‐PCA‐2b). We hypothesize dysregulation of inflammatory and immune response markers may enhance immune surveillance leading to a reduced aggressive tumor phenotype. Following modulations in miRNA‐186 levels in normal prostate epithelial (RWPE1) and metastatic PCa (PC3) cell lines, we evaluated alterations in mRNA expression using micro‐array analysis. Ectopic expression of miRNA‐186 in the RWPE1 resulted in a 1.3–2.6‐fold down‐regulation in TLR2, IRAK2, CCL20, IL1RAP, IL1RAP, IL15, and IL17RD (FDR p‐value ≥7.8×10−06). Whereas, inhibition of miR‐186 in PC3 cells corresponded with a 1.4‐fold up‐regulation of IL18R1 (p=0.0095). Notably, these inflammatory markers are involved in cell invasion (TLR2), apoptosis (IL17RD), tumorigenesis (TLR2, IRAK2) and autoimmune diseases (CCL20). Validation of these targets is underway using qRT‐PCR, western blots and luciferase reporter assays. We will assess the mechanism by which these miR‐186 targets suppress aggressive cancer behavior using knock‐out and knock‐in cell and ultimately animal models. Future studies may identify inflammatory targets that may guide immune therapies for the effective treatment of aggressive prostate cancer.

Support or Funding Information

James G. Brown Cancer Center, Our Highest Potential endowment in cancer research
Investigation of PAX3‐FOXO1 Effect on NEAT1 Expression in Alveolar Rhabdomyosarcoma Cells
Authors: George Victoria Agbeke , McDaniel Brianyell , Johanson Kelly E.
Published: November 2023

Rhabdomyosarcoma (RMS) is pediatric sarcoma in the soft muscular tissue. One of the two major types of rhabdomyosarcoma is alveolar rhabdomyosarcoma (ARMS). ARMS generally occurs in muscles in the abdomen, arms, and legs. Due to its tendency to grow more rapidly, it is a more aggressive form of the cancer that has a higher risk factor than other forms of RMS. The expression of PAX3‐FOXO1 in ARMS may contribute to the formation of tumors by inducing tumor growth activities or inhibiting tumor suppressing activities. There are many studies investigating targets of PAX3‐FOXO1 and some indicate that PAX3‐FOXO1 binds to long noncoding RNA (lncRNA), although it is not known if this interaction affects the expression of the lncRNA. Nuclear‐enriched autosomal transcript 1 (NEAT1) is a 4kb lncRNA that potentially is bound by PAX3‐FOXO1. Several putative PAX3‐FOXO1 binding sites are present in the DNA fragment of NEAT1 that was identified in a ChIP assay. We are examining the NEAT1 sequence to narrow down the specific site of PAX3‐FOXO1 interaction as well as determining the effect of PAX3‐FOXO1 on NEAT1 expression. The expression of NEAT1 is necessary for differentiation of muscle cells and a change in this expression due to PAX3‐FOXO1 may be linked to ARMS development. Finding the effect of PAX3‐FOXO1 on expression of NEAT1 will help to uncover more information about the mechanisms involved in the development and progression of ARMS.

Support or Funding Information

NIH BUILD TL4GM118968, NIH BUILD RL5GM118966‐02
Nonstop Decay in C. elegans: Examination of a Possible Role for Small Noncoding RNAs
Authors: Youngman Elaine M
Published: November 2023

In C. elegans, small noncoding RNAs known as 22G RNAs are synthesized by RNA‐dependent RNA polymerases and target thousands of protein‐coding mRNAs, repetitive sequences, and cryptic transcripts throughout the genome. In some cases, 22G RNA synthesis appears to be triggered as a secondary event downstream of targeting by C. elegans piRNAs (known as 21U RNAs) in a pathway that defends against non‐self RNA species. (1, 2) However, the synthesis of the majority of 22G RNAs is unaffected by loss of 21U RNAs, and the triggers for biosynthesis of these 21U‐independent 22Gs remain elusive. Using a comparative genomic approach in geographically distributed wild isolates of C. elegans, we have identified a locus (F43E2.6) at which loss of the stop codon is correlated with increased production of 22G RNAs. The F43 mRNA has a short 3′UTR with no further in frame stop codons, raising the intriguing possibility that 22G RNAs could be involved in a nonstop decay pathway in C. elegans. F43 is also targeted by an unusual Dicer product; however this Dicer product does not appear to act as a primary siRNA to trigger F43 22G RNAs. First, the Dicer product is expressed equally in strains with large differences (more than 10 fold) in 22G RNA levels. Second, this product is expressed only during embryogenesis, whereas F43 22G RNAs are expressed most strongly in adults. We have demonstrated that the 22G RNA expression phenotype at the F43 locus is genetically linked to the locus itself, and are currently assaying worms carrying CRISPR‐edited F43 loci that bear or lack a stop codon to determine whether the loss of a stop codon is indeed causal for increased 22G RNA production. In addition, we are generating CRISPR‐mediated edits that create additional nonstop alleles to ask whether this phenomenon is general, and are integrating reporter constructs that will allow us to ask whether 22G RNAs play a functional role in the nonsense‐mediated and no‐go decay pathways.
The Role of a Flexible loop in Metal transfer between periplasmic zinc proteins
Authors: Fullam Stephanie Hope
Published: November 2023

Zinc is an essential nutrient, and proteins mediating zinc acquisition are important virulence factors among pathogenic bacteria, making them attractive targets for the development of novel antibiotics. Of particular interest are the ATP binding cassette (ABC) transporters, which utilize extracellular solute binding proteins (SBPs) to bind zinc with high affinity and specificity for delivery into the cell via integral membrane permeases. We are studying the zinc import machinery of Paracoccus denitrificans as a model for highly homologous systems in pathogenic bacteria. Using a simple fluorescence assay, we have found that the zinc SBP AztC can acquire zinc from solution or from a metallochaperone, AztD. The crystal structure of AztC shows that, like all known zinc‐specific SBPs, this protein has a flexible loop near the zinc binding site. We hypothesized that this flexible loop is required for the recognition and subsequent transfer of zinc from its chaperone AztD. To test this, we generated a deletion mutant lacking the flexible loop (Δloop AztC) and tested zinc acquisition from solution and from AztD. Consistent with our hypothesis, the affinity of the mutant protein for zinc in solution is unaffected, yet it can no longer acquire zinc from AztD. In order to refine our understanding of those residues required for AztD recognition and zinc transfer, several site directed mutants of conserved loop residues were generated and evaluated for their ability to acquire zinc from AztD. Three His residues appeared to be absolutely required for zinc transfer, while mutation of a conserved Asp had no effect. Tyr121 appeared to be dispensable for zinc transfer, although the process was significantly slower for the Y121A mutant relative to WT. A detailed understanding of the mechanisms governing extracellular zinc management in bacteria may eventually lead to the development of novel antibiotics targeting this important process.

Support or Funding Information

NIH (National Institutes of Health) grant number: 1sc2gm111170‐01
Molecular mechanisms associated with early onset primary dystonia (DYT16) caused by mutations in PACT
Authors: Burnett Samuel B , Vaughn Lauren , Patel Rekha
Published: November 2023

Dystonia is an inherited neuromuscular movement disorder that has been shown to result from mutations in 25 different genes and classified into 21 categories. The resultant phenotype consists of both agonist and antagonist muscles firing simultaneously. Consequently, patients suffer from inability to control their dystonic limb, often sustained repetitive movements, and have a compromised posture. One type of primary dystonia, the early onset dystonia 16 (DYT16), has been shown to be result from various mutations in the PACT (also known as PRKRA) gene that has been well characterized for its role in regulating apoptosis in response to cellular stress.

Under conditions of viral, oxidative, or endoplasmic reticulum stress, the cellular apoptosis has been shown to be regulated by prolonged translation inhibition resulting from kinase activity targeting the eukaryotic translation initiation factor 2 alpha (eIF2a). Events leading to eIF2a phosphorylation in response to cell stress are attributed to interactions between three double stranded RNA binding proteins: PACT, TRBP, and PKR. These three proteins interact with each other forming homo‐ and heterodimers to impact cellular fate in response to stress signals. PACT is a protein activator of PKR, a serine‐threonine kinase involved in innate immunity. PACT‐PKR interaction is essential for facilitating apoptosis; whereas, transactivation RNA binding protein (TRBP) serves as a PKR inhibitor via PACT‐TRBP and TRBP‐PKR heterodimerization. These three proteins share evolutionarily conserved double stranded RNA binding motifs that also facilitate protein‐protein interactions and regulate the cell survival and apoptosis.

We investigated the altered protein‐protein interactions between PACT, PKR, and TRBP that result from various mutations reported in DYT16 patients, and for the first time in a mouse model of DYT16. Using various biochemical techniques, we characterized how these mutations influence PACT's function during stress response to regulate apoptosis. By utilizing DYT16 patient samples as well as an analogous mouse model we are attempting to better understand how PACT mutations may lead to early onset dystonia. Our research highlights the insight obtained by applying biochemical, and molecular analysis to patient cells and mouse models to understand the etiology and pathophysiology of dystonia.

Support or Funding Information

The Dystonia Medical Research Foundation
Bacteriophytochromes in myxobacteria: Implications for light‐control of cell development
Authors: Tracy Gregory Cornell , Gallagher Kevin D , Varela Joseph D , Bizhga Dorina , Duong Phu , Nugent Angela , Stojkovic Emina A
Published: November 2023

Bacteriophytochromes (BphPs) are red‐light photoreceptor proteins found in photosynthetic and non‐photosynthetic bacteria. They are unique among photoreceptors in their ability to undergo reversible photoconversion between spectrally distinct red (Pr, 700nm) and far‐red (Pfr, 750nm) light absorbing states. BphPs are composed of an N‐terminal photosensory module, consisting of PAS, GAF, and PHY domains, covalently linked to an effector domain, usually a histidine kinase. For photoresponse, BphPs require biliverdin (BV), an open‐chain tetrapyrrole covalently attached to an N‐terminal conserved cysteine. BphPs of the non‐photosynthetic myxobacterium Stigmatella aurantiaca are of special interest. Myxobacteria are distinguished among prokaryotes by a multicellular stage in their life cycle known as fruiting bodies, which in Stigmatella aurantiaca is controlled by light. The two BphPs from S. aurantiaca, SaBphP1 and SaBphP2, have distinct photochemistry, although they bind the same bilin chromophore and share a large sequence identity. Unlike classical BphPs, wild‐type SaBphP1 lacks a conserved histidine (His289) that stabilizes the bilin chromophore and undergoes limited Pr/Pfr photoconversion, that can be restored by a single Thr289His mutation in the bilin‐binding domain. The crystal structures of SaBphP1 wild‐type and Thr289His mutant differ in the conformation of the chromophore binding cavity. Furthermore Cystobacter fuscus, from the same suborder of myxobacteria as S. aurantiaca, contains three BphPs, two of which are lacking the conserved His. CfBphP1, like SaBphP1, contains a Thr in place of the conserved His and shows the same photochemistry. In CfBphP3 the conserved His is replaced by a glycine (Gly) which results in a complete lack of Pr/Pfr photoconversion. Therefore, we hypothesized that if we mutate Thr in SaBphP1 and His in SaBphP2 to Gly we can suppress photoconversion of these proteins. Surprisingly, we observed complete recovery of Pr/Pfr photoconversion in SaBphP1 Thr289Gly although reversion to the thermally stable, dark‐adapted Pr state was shorter than the His mutant. Currently, we are in the process of characterizing SaBphP2 mutant where the conserved His is substituted by a Gly. Our structural and sequence analyses of S. aurantiaca's and C. fuscus BphPs highlight critical His289 interactions with the chromophore and suggest the function for BphPs in fruiting myxobacteria.

Support or Funding Information

NSF MCB RUI 1413360
Lactoferrin interacts with SPLUNC1 to amelioate lipopolysaccharide‐induced inflammation of human nasal epithelial cells
Authors: Chen Chuan‐Mu , Tsou Yung‐An , Chen Hsiao‐Ling
Published: November 2023

Chronic rhinosinusitis is a chronic inflammatory disease caused by a chronic bacterial infection that last over 3 months. Innate immune deficiency is considered a crucial factor in the etiology of mucosa disease. The short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein is an important innate material in the upper airway, and lactoferrin (LF) aids the innate functions in humans. In this study, an nasal epithelial model was used to investigate how LF modulates SPLUNC1 to reduce the inflammatory process mediated by lipopolysaccharide (LPS). The inflammation of human RPMI‐2650 cells was induced by LPS to evaluate SPLUNC1 expression after treating the cells with bovine LF (bLF). The interaction pathway between LF and SPLUNC1 in LPS‐induced inflammation was further investigated. Our study reveals that the addition of bLF results in the recovery of SPLUNC1 expression in nasal epithelial cells under LPS‐induced inflammation. MAPK is involved in the main pathway for the SPLUNC1 and bLF interaction. Decreased SPLUNC1 function could be recovered by addition of bLF. The MEK1/2‐MAPK signaling pathway is crucial for the SPLUNC1 and bLF interaction. Therefore, LF could support SPLUNC1 in the innate immunity recovery process.

Support or Funding Information

This research was supported in part by grants MOST‐104‐2313‐B‐005‐043‐MY3 and MOST‐105‐2314‐B‐039‐030 from the Ministry of Science and Technology, Taiwan.
Evaluating the role of Corynebacterium matruchotti MdbA in oral biofilm formation
Authors: Tirgar Reyhaneh , Luong Truc Thanh , Ton‐That Hung
Published: November 2023

Oxidative protein folding via disulfide bond formation is a well‐characterized process in Gram‐negative bacteria; however, this pathway has only recently been revealed in the two Gram‐positive actinobacteria Actinomyces oris and Corynebacterium diphtheriae. In these organisms, post‐translocational folding of the majority of proteins exported by the Sec translocon requires a thiol‐disulfide oxidoreductase named MdbA. MdbA is essential for many cellular processes, as genetic disruption of mdbA causes aberrant cell morphology, growth arrest, and attenuation in bacterial virulence. The focus of my work is to characterize a putative MdbA homolog in Corynebacterium matruchotti, a newly found oral actinobacterium proposed to be a key organism in the development of oral biofilms. By molecular techniques, we generated a recombinant MdbA protein of C. matruchotii in E. coli and purified the protein in homogeneity for antibody production. In collaboration with Argonne National Laboratory, we have determined the crystal structure of C. matruchotii MdbA (1.7 Å resolution) that reveals two conserved features present in actinobacterial MdbA proteins: a thioredoxin‐like domain and an extended α‐helical domain. In addition, I found that C. matruchotii forms a monospecies biofilm as determined by the Congo Red assay and that C. matruchotii interacts with Fusobacterium nucleatum, a key pathogen involved in oral biofilm formation. Future experiments will focus on determining if genetic disruption of C. matruchotii mdbA affects cell growth, morphology, biofilm formation, and interaction with F. nucleatum. This study will contribute to our understanding of oxidative protein folding in actinobacteria and allow for research of MdbA inhibitors in actinobacterial pathogens including A. oris, C. diphtheriae, and Mycobacterium tuberculosis.
Evolutionary fine‐tuning of conformational ensembles in FimH during host‐pathogen interactions
Authors: Kalas Vasilios , Pinkner Jerome S. , Hannan Thomas J. , Hibbing Michael E. , Janetka James W. , Hultgren Scott J.
Published: November 2023

Positive selection in the two‐domain type 1 pilus adhesin FimH enhances Escherichia coli fitness in urinary tract infection (UTI). Here, we report a comprehensive atomic‐level view of FimH in two‐state conformational ensembles in solution, composed of one low‐affinity tense (T) and multiple high‐affinity relaxed (R) conformations. Positively selected residues allosterically modulate the equilibrium between these two conformational states, each of which engage mannose through distinct binding orientations. A FimH variant that only adopts the R state is severely attenuated early in a mouse model of uncomplicated UTI but proficient at colonizing bladder transitional‐like epithelial cells in vitro or catheterized bladders. Thus, the bladder habitat possesses barrier(s) to R state‐mediated colonization possibly conferred by the terminally differentiated bladder epithelium and/or decoy receptors in urine. Together, our studies reveal the conformational landscape in solution, binding mechanisms, and adhesive strength of an allosteric two‐domain adhesin that evolved “moderate” affinity to optimize persistence in the bladder during UTI.
Thermodynamic partitioning forces at the membrane protein interface
Authors: Mahalakshmi Radhakrishnan
Published: November 2023

Transmembrane proteins fold by the orchestrated interplay of membrane chaperones, holdases and the lipid membrane. Upon folding, membrane protein systems attain a thermodynamically stabilized framework through optimized energetics involving protein – membrane – solvent interactions. In particular, the water‐bilayer interface demands selectively positioned amino acids to arbitrate interactions between the hydrophobic interior and polar phospholipid headgroups. A fundamental question that arises is the extent to which the amino acid side chain contributes to membrane protein folding and in maintaining protein‐lipid interactions at the intricate interface environment of membrane proteins. Our goal is to experimentally deduce the partitioning cost of amino acids at the interface, and the associated physical principles governing the overall scaffold stability.

Interface residues are important contributors for membrane protein folding, and additionally serve as post‐folding membrane anchors. In this study, we have quantified the free energy change associated with the chemical nature of the residue at the membrane interface. We achieve this by studying the unassisted folding equilibrium of the 8‐stranded transmembrane β‐barrel enzyme PagP in phosphocholine lipidic micelles and lipid vesicles. We present the first experimentally measured whole‐protein free energy scale for side chain partitioning at the membrane interface, for all naturally abundant amino acids. We obtain differential contributions of the protein‐ and lipid‐facing interface residues to the barrel folding pathway and stability of the folded scaffold. The most favorable transfer at the amphiphilic lipid‐facing interface is for hydrophobic amino acids, whereas we observe the highest energetic cost of transfer for charged and polar groups. On the contrary, we find that small and polar residues are most favorably transferred to the protein‐facing interface, whereas hydrophobic and aromatic residues promote alternate folding pathways in PagP.

Our studies establish that the side chain non‐polar accessible surface area shows a direct correlation with the partitioning free energy change at the lipid‐facing interface residue. We also find that the non‐polar accessible surface correlates inversely with the thermodynamic partitioning free energy change when the interface residue is protein‐facing. We demonstrate how PagP maintains a balance between concerted folding and hydrophobic collapse by evolutionary choice of small polar residues at the protein‐facing interface. We also identify the importance of interface polar residues to the folding pathway of β‐barrel membrane proteins.

Support or Funding Information

R.M. is a recipient of the Wellcome Trust/DBT India Alliance Intermediate Fellowship. This work was supported by the Wellcome Trust/DBT India Alliance award number IA/I/14/1/501305 to R.M.
Pinpointing the divergence of quaternary structure in the lysine biosynthetic pathway
Authors: Pearce Grant , Watkin Serena , Keown Jeremy
Published: November 2023

While quaternary structure is highly conserved among protein families, few studies have investigated the evolutionary history of enzyme complexes. In the work described here, we describe the evolution of quaternary structure in the lysine biosynthetic pathway. We have previously shown that there are significant differences in quaternary structure between the DHDPS (dihydrodipicolinate synthase) and DHDPR (dihydrodipicolinate reductase) enzymes of plants and bacteria. While both plant and bacterial DHDPS enzymes are mostly tetrameric, the arrangement of the subunits differs. Similarly, while bacterial DHDPR enzymes are tetrameric, plant DHDPR enzymes characterised are all dimers. Here, we present the first crystallographic data of dimeric DHDPR enzymes. In an effort to expand the knowledge of when these enzymes evolved to have different quaternary structures, we have characterised the structure and activity of DHDPS and DHDPR enzymes from moss, green algae, and red algae. We have found that while DHDPS from green algae has a similar structure to plants, red algal DHDPS has a similar structure to bacteria. Moss DHDPS has a novel arrangement in which it can form either dimers or tetramers, depending on ligand binding. Conversely, both red algal and green algal DHDPR enzymes appear to have dimeric structures, which are similar to that observed in plants. This work helps provide a better understanding of some of the evolutionary pressures behind self‐assembly.
Cooperative binding of cinnamon polyphenols as activators of Sirtuin‐1 protein in the insulin signaling pathway
Authors: Brennemen Martin , Mahfouz Tarek , Stockert Amy
Published: November 2023

Cinnamon has been shown to decrease blood glucose levels in type‐II diabetics. Analysis of cinnamon extract have determined that it contains a number of polyphenolic compounds and the antidiabetic effect of cinnamon is hypothesized to be due to those phenolic compounds. The protein target of those phenolic compounds has not been identified yet. However, Sirtuin‐1 (Sirt‐1), a deacetylase in the insulin signaling pathway, seems a likely target. To investigate this possibility, we docked several polyphenolic compounds that are commonly found in cinnamon extract to Sirt‐1 in order to determine their binding affinities and compared these to Sirt‐1 affinity for resveratrol which is not in cinnamon extract, but it is a known activator of Sirt‐1. We used Sirt‐1 structure 5BTR for the docking which contained three molecules of resveratrol with a 7‐amino‐4‐methylcoumarin (AMC)‐containing peptide in the binding site. Our results indicate a cooperative binding of resveratrol molecules when docked sequentially in the binding site with the AMC peptide. We further hypothesized that the polyphenolic compounds within cinnamon would have a similar effect. To investigate this, a second set of docking simulations was performed on the polyphenolic compounds within cinnamon extract to investigate potentially similar interactions as shown by resveratrol. These docking simulations were done using AutoDock Vina in two different trials; one using sets of three of the same molecule, and the second using sets of combinations of three molecules. Results from those trials showed similar and sometimes stronger interactions than resveratrol between Sirt‐1 and the molecules docked. Molecules with affinities at least as strong as resveratrol's were used for sequential docking runs. These results strongly suggest that Sirt‐1 is a possible target for cinnamon extract.

Support or Funding Information

Department of Pharmaceutical and Biomedical Sciences, Ohio Northern University
Correlation of fitness landscapes from three orthologous TIM barrels originates from sequence and structure constraints
Authors: Chan Yvonne H. , Venev Sergey V. , Zeldovich Konstantin B. , Matthews C. Robert
Published: November 2023

Sequence divergence of orthologous proteins enables adaptation to environmental stresses and promotes evolution of novel functions. Limits on evolution imposed by constraints on sequence and structure were explored using a model TIM barrel protein, indole‐3‐glycerol phosphate synthase (IGPS). Fitness effects of point mutations in three phylogenetically divergent IGPS proteins during adaptation to temperature stress were probed by auxotrophic complementation of yeast with prokaryotic, thermophilic IGPS. Analysis of beneficial mutations pointed to an unexpected, long‐range allosteric pathway towards the active site of the protein. Significant correlations between the fitness landscapes of distant orthologs implicate both sequence and structure as primary forces in defining the TIM barrel fitness landscape and suggest that fitness landscapes can be translocated in sequence space. Exploration of fitness landscapes in the context of a protein fold provides a strategy for elucidating the sequence‐structure‐fitness relationships in other common motifs.

Support or Funding Information

This research was supported by NIH GM23303 to CRM.
Testing c‐Type Heme Sources for Nontypeable Haemophilus influenzae
Authors: Stanton Sarah , Pierce Jeanetta , Sgheiza Valerie , Bren Kara L , Michel Lea
Published: November 2023

The property that defines c‐type heme proteins is the covalent attachment of the heme group to the polypeptide chain of the protein, such as in cytochrome c. The covalent attachment of the c‐heme (usually by thioether linkages donated by two Cysteine residues) is energetically expensive, and the biological motivation for the attachment has been debated for decades. We propose that one biological motivation for the covalently attached c‐heme is to prevent heme‐dependent bacteria, such as Nontypable Haemophilus influenzae (NTHi), from scavenging heme from c‐heme proteins. To test this hypothesis, we grew NTHi in the presence of cytochrome c variants with zero, one, or two thioether linkages to heme, which we prepared using site‐directed mutagenesis. Our results suggest that one covalent attachment is enough to prevent NTHi from scavenging heme from cytochrome c. Microperoxidase‐11 (MP‐11) is obtained from enzymatic cleavage of cytochrome c, and contains the heme group and residues 11–21 of the protein, including the two covalent attachments to heme and the Histidine axial ligand. Our experiments show that NTHi cannot utilize MP‐11 as a heme source.
Characterizing the Interactions Between Mg2+ and a Periplasmic Lipoprotein Involved in Mg2+ Homeostasis in Salmonella enterica
Authors: Davie Timothy J , May John F
Published: November 2023

The ability to acquire Mg2+ is needed for certain bacteria to cause disease due to the nutrient's importance in physiological processes. Pathogenic bacteria have proteins which help them to adapt to environments where Mg2+ is in low supply. We have identified a periplasmic lipoprotein found in Salmonella enterica, a bacteria that is a major contributor to food‐borne illness, that allows the pathogen to survive in low Mg2+ environments. The Salmonella lipoprotein of 19.7 kDa monomeric molecular weight consists of a signal peptide and a single domain of unknown function that is conserved among enterobacteria. The purpose of this research project is to determine if the protein binds Mg2+ and identify the amino acid residues that are required for the protein to function properly. We have developed a protocol to overproduce and purify the recombinant lipoprotein. Protein variants are created using site directed mutagenesis to replace residues thought to be critical for binding Mg2+. To test for whether a particular residue is required for the ability of this lipoprotein to promote growth in a low Mg2+ environment, growth of Salmonella expressing these protein variants in low Mg2+ is being assayed. This physiological assay will indicate which residues are required for the physiological function of this protein in Mg2+ homeostasis. A fluorescence assay that utilizes Mg2+ sensor 8‐hydroxyquinoline‐5‐sulfonic acid (HQS) has provided evidence that the lipoprotein is sequestering Mg2+ from solution. We observed a change in HQS fluorescence when the protein is present, indicating that HQS and the protein compete for Mg2+. The results from the HQS biochemical assay, combined with our site directed mutagenesis experiments, will provide a model for how the protein functions. The results of the project may allow for a further understanding of how Salmonella and other bacteria adapt to their environment and maintain virulence in harsh conditions.

Support or Funding Information

This research is supported by an UW‐La Crosse Undergraduate Research and Creativity Grant (TD) and a UW‐La Crosse Faculty Research Grant (JM).
Translation of heat shock proteins is regulated by poly(A)‐binding protein assembly
Authors: Katanski Christopher D , Riback Joshua , Pilipenko Evgeny , Drummond David Allan
Published: November 2023

How do cells sense and respond to changes in the environment? During heat stress, numerous eukaryotic RNA and protein species assemble into macromolecular bodies ‐ stress granules ‐ the function of which is largely unknown. Simultaneously, cells increase transcription of genes encoding heat shock proteins, many of which are molecular chaperones. In addition to increased transcription, the mRNA for these genes is also translated more efficiently. Thus, heat shock protein production is regulated by both transcription and translation. These heat‐induced mRNAs have exceptionally A‐rich 5′ untranslated regions (UTRs), which have long been known to mediate cap‐independent translation. We observe that poly(A)‐binding protein (PABP, Pab1 in S. cerevisiae) is bound to these A‐rich UTRs during exponential growth. Pab1 is able to repress translation of its own mRNA by binding a similar A‐rich UTR. We find that Pab1 represses translation of mRNA by binding A‐rich UTRs of heat shock proteins. Further, we propose that during heat‐shock, as Pab1 assembles into heat‐induced RNA‐protein granules, Pab1 releases bound 5′ UTRs, and relieves repression. We have recapitulated Pab1‐mediated repression and heat‐induced relief with an in vitro translation system. Moreover, yeast expressing assembly‐impaired Pab1 are not able to grow at elevated temperature; this result is consistent with disruption of an assembly‐mediated regulatory mechanism. Viewed through this lens, formation of RNA‐protein granules is a regulatory response, whose functions include translational regulation.

Support or Funding Information

The molecular and cellular biology training grant T32 GM007183
Tau and α‐synuclein protein expression, purification, and their amyloid inhibitor discovery
Authors: Marcus Madeleine , Henderson Frances , Kelly Katherine , Umana Ariana , Velander Paul , Wu Ling , Xu Bin
Published: November 2023

Alzheimer's and Parkinson's diseases (AD/PD) are two major neurodegenerative diseases. AD is characterized by the accumulation in brain of two types of abnormal structures, extracellular amyloid plaques and intraneuronal neurofibrillary tangles. Prevalent evidence now indicates that the respective building blocks of plaques and tangles – oligomers of Aβ peptide and the neuron‐specific, axon‐enriched microtubule‐associated protein, tau, represent the most toxic molecular species contributing to AD pathogenesis. Similarly, alpha‐synuclein protein amyloid is a major constitute of Lewy body, protein aggregates that are the pathological hallmark of Parkinson's disease.

Currently there are no effective drugs to prevent or cure AD and PD. Because of protein aggregates, including oligomers, mediate neuronal toxicity in AD/PD, small molecule aggregation inhibitors are attractive drug candidates. Many drug trials aimed at preventing or clearing Aβ pathology have failed in recent years. Investigations on tau yielded innovative targets backed by recent findings about the central role of tau in AD pathogenesis (1,2). Preliminary data from a phase 2 trial with the small molecule, methylene blue chloride, have shown some promise and thereby validate the potential of tau aggregation inhibitors (TAIs), but superior TAIs are needed.

In this poster we will present our work on expression and purification of human tau and α‐synuclein proteins and their amyloid formation and inhibitor discovery. Tau and α‐synuclein are expressed as recombinant proteins in E. coli and were purified to homogeneity using multiple chromatographic steps. They are further validated by western blot analysis and mass spectrometry. We have developed multiple biochemical, biophysical, and cell‐based assays to monitor oligomer/amyloid formation and amyloid‐induced cytotoxicity assays in characterizing amyloidogenic proteins (3). We use ThT fluorescence‐based assay for semi‐high throughput library screens. Upon identification of “hit” compounds, we validate them with multiple secondary assays including transmission electron microscopy (TEM), photo‐induced crosslinking of unmodified proteins (PICUP), IC50 measurements, and cell‐based cytotoxicity assays. We will further perform mechanistic studies for these validated inhibitors.

Support or Funding Information

This work is in part supported by Virginia Tech new faculty start‐up funds, Commonwealth Health Research Board (CHRB), Alzheimer's and Related Diseases Research Award Fund (ARDRAF) from Virginia Center on Aging, and Diabetes Action Research and Education Foundation (DAREF) to B.X.