bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the E. coli SOS response protein YbfE
Authors: Kramer Caitlin , Muenter Mark , Leifer Becky , Travers Meghan , Judge Richard , Beuning Penny
Published: November 2023

The transcription of DNA damage response genes in prokaryotes is largely regulated by LexA and controlled through the SOS mechanism. Over 50 genes have been identified as part of the LexA regulon by their upstream LexA binding sequences. Prior work by our lab has shown that loss of the uncharacterized LexA‐regulated gene ybfE is associated with poor survival in E. coli exposed to alkylating agents. In order to elucidate a mechanism, the structure and function of the ybfE gene product were examined. A homology model was built that indicates that YbfE is a DNA‐binding protein that contains a C‐terminal ribbon‐helix‐helix motif and a domain of unknown function at its N‐terminus. The in vivo transcription start site of YbfE is not known, therefore two open reading frames downstream of the SOS operator were examined in vitro for sequence‐specific DNA binding. The first open reading frame contains an additional 23 amino acids at the N‐terminus. Both open reading frames were cloned, expressed, and purified. Electrophoretic mobility shift assays (EMSAs) were performed using the YbfE protein and DNA sequences selected to test binding specificity. Our observations support that YbfE binds DNA nonspecifically in vitro at concentrations above 500 nM. Characterization of sequence specific DNA binding at lower concentrations and protein‐protein interactions is ongoing.

Support or Funding Information

Supported by American Cancer Society Research Scholar Grant RSG‐12‐161‐01‐DMC.
Investigating the Metabolism of a Major Peroxidation DNA adduct, M1dG, in the Genome
Authors: Wauchope Orrette R , Beavers William N , Galligan James J , Mitchener Michelle M , Kingsley Philip J , Marnett Lawrence J
Published: November 2023

Chronic inflammation results in increased production of reactive oxygen species (ROS), which can oxidize cellular molecules including lipids and DNA. Our laboratory has shown that 3‐(2‐deoxy‐b‐D‐erythro‐pentofuranosyl)pyrimido[1,2‐a]purin‐10(3H)‐one (M1dG) is the most abundant DNA adduct formed from the lipid peroxidation product, malondialdehyde, or the DNA peroxidation product, base propenal. M1dG is mutagenic in bacterial and mammalian cells and is repaired via the nucleotide excision repair system. Here, we report that M1dG levels in intact nuclear DNA were increased from basal levels of 1 adduct per 108 nucleotides to 2 adducts per 106 nucleotides following adenine propenal treatment of RKO, HEK293 or HepG2 cells. We also found that M1dG in genomic DNA was oxidized in a time‐dependent fashion to a single product, 6‐oxo‐M1dG, (to ~ 5 adducts per 107 nucleotides) and that this oxidation correlated with a decline in M1dG levels. Investigations in RAW264.7 macrophages indicate the presence of high basal levels of M1dG (1 adduct per 106 nucleotides) and the endogenous formation of 6‐oxo‐M1dG. Interestingly, in all cell lines investigated, 6‐oxo‐M1dG was not observed in mitochondrial DNA. Basal levels of M1dG in the mitochondria were found to be approximately two orders of magnitude higher (~ 1 adduct per 106 nucleotides) than those observed in nuclear DNA and were increased with electrophilic stimulation. Further studies, in all the investigated cell lines, indicated a correlation between M1dG levels and oxidative stress in the mitochondrion. This is the first report of M1dG in mitochondrial DNA in intact cells and it has significant implications for understanding the role of inflammation in DNA damage, mutagenesis and repair.

Support or Funding Information

This work was supported by a research grant from the National Institutes of Health, R37 CA0878819 (L.J.M.)
Mutations in The RQC Domain of Human WRN Helicase Compromise It's Ability To Unwind G‐Quadruplex DNA
Authors: Ketkar Amit , Voehler Markus , Mukiza Tresor , Eoff Robert Lawton
Published: November 2023

G‐quadruplex (G4) DNA is a specialized DNA structure that is now well established to occur in vivo. G4‐DNA structures present a significant block for replication, and must be overcome to complete the process efficiently. G4 motifs have been shown to occur at functional regions of prokaryotic and eukaryotic genomes, like promoters of oncogenes, replication origins and telomeres. G4 sequences are also enriched at chromosomal breakpoints in multiple types of cancer. The Werner's syndrome protein (WRN) is a RecQ‐family helicase that has been implicated in efficiently unwinding G4‐DNA structures during replication in humans. The aim of our study was to identify the residues of WRN that were involved in this role. Using a c‐Myc G4‐DNA model sequence and recombinant WRN, we determined that the RecQ‐C‐terminal (RQC) domain of WRN imparted a 2‐fold preference for binding to G4‐DNA relative to non‐G4 DNA substrates. NMR spectroscopic studies were performed using 15N‐labeled RQC‐domain in DNA titration experiments where both non‐structured and G4‐DNA substrates were used to identify residues that are involved specifically in interacting with G4‐DNA. Residues that showed the largest G4‐specific changes in NMR signal were chosen for further validation. Point mutations were made in these RQC‐domain residues in the WRN construct 500–1092, to generate single‐mutant proteins WRNT1024G, WRNL1063G, WRNT1086G, and a double‐mutant WRNT1024G/T1086G. The mutant proteins were studied in detail for their ATPase activity, ability to unwind G4‐DNA and their relative affinities towards non‐structured or G4‐DNA substrates. ATPase activity of all mutant proteins was reduced compared to wild‐type. The effect was moderate when nonG4‐DNA was used, and most pronounced for the double mutant when G4‐ssDNA was used to stimulate ATPase activity (activity was only 40% of wild‐type). The helicase/DNA‐unwinding activity of all mutant proteins was relatively unaffected on a non‐G4‐DNA substrate. However, for the WRNT1024G mutant, this activity was reduced by ~50% on a G4‐DNA substrate, while for the double mutant, it was drastically reduced (by 90%) on G4‐DNA as compared to wild‐type. DNA‐binding affinity of all mutant proteins except WRNT1024G/T1086G was relatively unaffected for both control and G4‐DNA substrates. Binding preference towards G4‐DNA was reduced only moderately for the single mutants. In the case of the double mutant, the preference for G4‐DNA was reduced from ~2‐fold (in wild‐type) to ~1.1‐fold. Our work has, thus, for the first time, helped identify the residues of WRN that are involved specifically in its interaction with G4‐DNA, and has laid the groundwork for future studies on properties of RecQ‐helicases, which enable several of them to unwind G‐quadruplex DNA.

Support or Funding Information

Grants GM084460, CA183895
IDENTIFICATION AND CHARACTERIZATION OF HISTONE VARIANT H2A.Z IN THE LONG TERMINAL REPEAT RETROTRANSPOSON INTEGRATION
Authors: Rai Sudhir K. , Guo Yabin , Levin Henry L.
Published: November 2023

Unique integration patterns of Long Terminal Repeat (LTR)‐retrotransposons and the use of retroviruses in gene therapy have generated great interest in target‐site selection. Initially, the RNA transcripts of retroviruses and retrovirus‐like elements are reverse transcribed into full‐length cDNA copies that are subsequently inserted into the host cell genome. Although integrase (IN), encoded by retroviruses and LTR‐retrotransposons, is responsible for catalyzing the insertion of cDNA into the host genome, it is now clear that a variety of host factors are required for targeting and selection of the integration sites. To understand how retroviruses and LTR‐retrotransposons position their sites of integration in the host genome, we study the LTR‐retrotransposon Tf1 of Schizosaccharomyces pombe. Tf1 integrates with a strong preference for the promoters of RNA pol II transcribed genes. A genetic assay that measures cDNA present in the nucleus allows us to determine whether mutations that reduce transposition frequencies have a direct impact on the process of integration. We utilized these assays to screen a collection of 3,004 S. pombe deletion strains to identify genes that contribute to integration. We found that 181 deletion strains had reduced transposition and 98 of these likely have specific defects in integration. All 98 deletion strains were tested for Tf1 IN and Gag expression using immunoblots. A total of 73 strains had normal levels of IN and GAG proteins, suggesting the deleted genes played a direct role in various steps of Tf1 integration. Candidates that may contribute to integration include factors responsible for DNA repair, transcription activation, signal transduction, and nucleosome structure. We characterized the role of candidate protein H2A.Z (pht1) because this histone variant assembles into the nucleosomes at the promoters of RNA pol II transcribed genes. We found that the reduced transposition of the strain lacking pht1 could be complemented with a plasmid encoding a wild type copy of pht1. In addition, bioinformatics analysis of 73,125 unique Tf1 integration sites demonstrated that the nucleosome‐free regions (NFRs) of promoters were the preferred targets of integration. Nucleosomes with H2A.Z are adjacent the NFR suggesting the possibility that H2A.Z may contribute directly to targeting integration.

Support or Funding Information

NICHD/NIH Intramural Research Funding
5‐Azacytidine Increases Class I HLA ABC Expression in Tumor Cell Lines
Authors: Long Patrick L , Louis Cassandre L , Nicholas David W. , O'Donnell Robert W.
Published: November 2023

MHC‐I expression is commonly down regulated during carcinogenesis due to the proliferative advantage acquired. Without class I MHC surface proteins, neoplastic cells are able to avoid surveillance of the adaptive immune system by preventing the presentation of endogenous antigens, providing a proliferative advantage. 5‐azacytidine is a novel epigenetic therapeutic drug that inhibits DNA methyltransferase through methyltransferase trapping. Tumor cells display global hypomethylation of their genome but specific hypermethylation of tumor suppressor CpG islands. We hypothesized that tumor cell lines treated with 5‐Azacytidine would display an increased amount of Class I HLA expression on their surface. To test this model we have begun to treat MCF‐7, MDA‐MB‐435, MDA‐MB‐231, A431 and HL‐60 tumor cell lines with relatively high and low doses of 5‐Azacytidine over a 48 or 72‐hour period. For the MDA‐MB 435 cell line we also compared long‐term exposure. Class I HLA‐ABC proteins were detected with a PE‐conjugated anti‐HLA‐ABC monoclonal antibody and fluorescence measured by flow cytometry. Preliminary results have shown an increase in expression in 5‐Azacytidine treated cells compared to controls but the magnitude of change varied among the cell lines tested. MDA‐MB‐435 cells treated with 5‐Azacytidine for more than six months showed the greatest change (mean fluorescence values of 785 in the untreated cells and 1180 for the 5‐azacytidine treated ). HL‐60 treated with 5‐Azacytidine for three weeks showed little to no change in fluorescence. Future experiments will optimize dose and length of exposure and attempt to identify methylated promoter regions in MHC via bisulfate conversion. With further experimentation, 5‐Azacytidine may prove to assist cancer immunotherapies where low MHC expression in tumor cells is encountered.
Transcriptional Activity of TEAD4 Ensures Self‐Renewal of Trophoblast Progenitors during Post‐Implantation Placental Development
Authors: Saha Biswarup , Home Pratik , Gunewardena Sumedha , Paul Soumen
Published: November 2023

In placental mammals, trophoblast cells are essential for embryo implantation leading to successful progression of pregnancy. During placental development, distinct trophoblast cell types are specified from trophoblast stem cells (TSCs) or TSC‐like progenitors. However, the molecular mechanisms that regulate self‐renewal or differentiation of trophoblast stem/progenitor cells are poorly understood. In this study, we show that transcriptional activity of TEAD4, a TEA domain containing transcription factor, plays a crucial role in promoting cell growth and proliferation of the trophoblast progenitors in post‐implantation developing placenta. TEAD4 is expressed in trophoblast progenitors throughout the placental development and conditional depletion of TEAD4 in post‐implantation mouse embryos result in impaired placental development causing embryonic lethality by embryonic day (E) 9.5. Our global gene expression (RNA‐Seq) analysis with TEAD4‐depleted murine trophoblast stem cells (mTSCs) along with ChIP‐Seq data revealed that TEAD4 directly regulates different cell cycle regulators including multiple Cyclins/CDKs. Our ex‐vivo analysis with mouse ectoplacental cone (EPC) explant cultures confirmed direct TEAD4‐mediated regulation of Cyclins/CDKs in primary trophoblast progenitors and found to be essential for their self‐renewal and expansion. Collectively, our data indicate that TEAD4 plays an essential role in trophoblast cells homeostasis during placental development to prevent placental insufficiency leading to successful pregnancy in mammals.

Support or Funding Information

This project was supported by grants from National Institute of Child Health and Human Development to S.P., HD079363, HD062546 and HD075233.
Investigating the Structural Dynamics of Oncogenic Histone Methyltransferases
Authors: Teichert Kristian Eric , Iacob Roxana E. , Wales Thomas , Paulk Joshiawa , St. Pierre Roodolph , Engen John , Bradner James
Published: November 2023

Aberrant expression or activity of histone‐modifying enzymes has been linked to a number of cancers, such as multiple myeloma, diffuse‐intrinsic pontine glioma (DIPG), B‐cell lymphomas and others. Drug discovery for inhibitors of histone‐modifying enzymes, such as histone methyltransferases (HMTs) is critical for the well‐being of patients with malignancies involving these enzymes. The structures of several of the histone‐modifying enzymes have been reported, however information regarding their structural dynamics is lacking. We have previously reported that binding of a peptide substrate accelerates the conformational dynamics of a small region of histone methyltransferase NSD2. This insight directed us to investigate the flexibility of several other histone methyltransferases, such as G9A, SUV39H2 and SETD2 with and without their peptide substrates. These data help us to further understand the conformational dynamics of histone methyltransferases and their mechanisms of substrate engagement. Through this understanding, critical regions and movements required for substrate binding can be identified and compared across several HMTs. This insight can be utilized in drug discovery efforts to develop a substrate‐competitive inhibitor to specific histone methyltransferases such as NSD2.

Support or Funding Information

NIH R21 CA179159‐01A1
RNA Multifunctional Antisense Gene Transfer Strategy to Alter Hepatocyte Growth Factor Receptor Expression in Glioblastoma Multiforme
Authors: Patel Priyal
Published: November 2023

The most common central nervous system malignancy is glioblastoma multiforme (GBM). The aggressive tumor spreads rapidly migrating through the white matter of the brain. Current therapy includes surgical removal, radiation and chemotherapy extending survival 14 months. Furthermore, the blood‐brain barrier is a challenge to targeted therapy. Our research is to develop a novel gene transfer vector to deliver the DNA encoding an antisense RNA (ASR) to alter the splicing pattern of the hepatocyte growth factor receptor (HGFR). In this approach, the ASR blocks recognition of splice sites and splicing enhancers. The current strategy targets the splicing of exons 11 to 12 of the pre‐mRNA transcript. Blockage of this splicing event leads to retention of intron 11. Because this intron includes 14 intronic alternative polyadenylation (IPA) signals, there is great potential to generate a shortened transcript and its subsequent soluble therapeutic HGFR decoy. A 1st generation construct containing an antisense with a human U7 snRNA was transfected into human glioblastoma cell lines, A172 and U87MG. The 1st generation therapy was successfully expressed in transfected cell lines. To optimize therapeutic efficiency, we have designed a 2nd generation gene transfer construct with multiple elements with distinct functions. These include the ASR with an hnRNPA1 splicing inhibitory element and an sm‐Opt U7snRNA (sm‐Opt/U7) localization signal with the novel tertiary ‘kiss domain’. The ASR targets the 5′ splice site of Exon 11, the sm‐Opt/U7 signal stabilizes and directs the ASR into the spliceosome of the nucleus, the hnRNPA1 tail blocks the splicing machinery and the ‘kiss domain’ stabilizes tertiary structure of the therapy vector. The new design makes use of the U7 promoter and 3′ transcription termination element to allow more efficient transcription. Using the 1st generation therapy vector we have developed assays to measure efficiency of therapy vector delivery, as well as levels of pre‐mRNA and spliced HGFR transcript. In the next steps, we are delivering the 2nd generation therapy vector to measure the efficacy to increase the abundance of shortened transcript and reduction of full length HGFR in the GBM cells. Real‐ time polymerase chain reaction (PCR) and ELISA will be used to determine the ratios of the pre‐mRNA, mRNA and decoy in the cell and in the media.
Structural and Mechanistic Basis for Self versus Non‐self RNA discrimination by the Innate Immune Receptor RIG‐I
Authors: Devarkar Swapnil , Wang Chen , Ramanathan Anand , Jiang Fuguo , Miller Matthew , Khan Abdul , Marcotrigiano Joseph , Patel Smita
Published: November 2023

RNAs with 5′‐triphosphate (ppp) are detected in the cytoplasm principally by the innate immune receptor RIG‐I, whose activation triggers a Type I interferon response. Cellular RNAs undergo post‐transcriptional modifications which ensures that self RNAs do not carry 5′ppp and/or blunt‐ends. We carried out a thorough biochemical characterization of RIG‐I complexes with RNAs carrying various end‐modifications as well as measured the effect of these end‐modifications on their signaling activity. We find that discrimination of RNA ligands occurs at multiple stages and signaling RNAs have high affinity and ATPase turnover rate and thus a high katpase/Kd. Our studies reveal that the autoinhibitory interface between CARD2 (second CARD) and Hel2i (Helicase 2 insertion domain) plays a critical role in RNA selectivity, wherein this interface acts as a “gate” that prevents cellular RNAs from generating productive complexes that can signal.

It is thought that self RNAs like mRNAs are not recognized by RIG‐I because 5′ppp is capped by the addition of a 7‐methyl guanosine (m7G) (Cap‐0) and the 5′‐end nucleotide ribose is 2′‐O‐methylated (Cap‐1). We provide a structural and mechanistic basis for the exact roles of capping and 2′‐O‐methylation in evading RIG‐I recognition using enzymatic assays, X‐ray crystallography and cell‐based signaling studies. Surprisingly, we find that Cap‐0 and 5′ppp dsRNA have similar binding affinities, activate RIG‐I's ATPase to the same extent, and shows the same cellular signaling response. Three new crystal structures of RIG‐I complexes with dsRNA bearing 5′OH, 5′ppp, and 5′Cap‐0 reveal that RIG‐I accommodates m7G in a cavity created through conformational changes in helicase‐motif IVa without perturbing the triphosphate interactions. In contrast, Cap‐1 modifications abrogate RIG‐I signaling through a mechanism involving the H830 residue, wherein 2′‐O‐methylation works synergistically with m7G capping to weaken RNA affinity and ATPase activity. Our work provides new structural insights into the host and viral immune evasive mechanisms from RIG‐I explaining the complexity of cap structures over evolution.

Support or Funding Information

This work was supported by NIH grant GM111959 to Joseph Marcotrigiano and Smita S Patel.
Reprogramming Msl5 to recognize mutant branch point sequences during spliceosome assembly
Authors: Turner Brexton , Rodgers Margaret , Hoskins Aaron
Published: November 2023

Saccharomyces cerevisiae Msl5 is an essential protein responsible for the assembly of a catalytically active spliceosome. Msl5 mediates spliceosome assembly through the recognition of the intronic branchpoint sequence 5′‐UACUAAC. Upon binding, Msl5 establishes an interaction with U1 bound to the 5′ splice site before recruiting the U2 snRNP to the intronic branchpoint sequence. We have generated and expressed mutant Msl5 proteins containing L176N and L167A/Q255A mutations, designed to bind specifically to mutant branch point sequences 5′‐UACUACC and 5′‐UACAAAC, respectively. Electrophoretic mobility shift assays will be performed to determine if reprogrammed Msl5 can specifically recognize and bind to a mutant branch point sequence. The reprogramming of Msl5 can be used to generate an orthogonal Msl5 system in vivo, and to analyze the effectiveness of intron splicing containing different branchpoint sequences.
In Vivo Studies of Function of RRF (Ribosome Recycling Factor) with Translationally Coupled Two ORFs Revealed Its Major Function: Release of mRNA from the Post‐Termination Complex
Authors: Kaji Akira , Inokuchi Yoshio , Hirashima Akikazu , Kaji Hideko
Published: November 2023

Objective

Study the RRF reaction in vivo to correct wrong prevalent concept that RRF's major function is to split ribosomes of post‐termination complexes.

Methods

Use translationally coupled two ORFs and determine how much of the upstream ribosomes is reading the downstream ORF using the reporter gene. RRF is known to be essential for the translational coupling. We control RRF activity using tsRRF.

Results

In the translationally coupled 2 ORFs, the downstream ORF is exclusively read by the ribosomes reading the upstream ORF. No free ribosomes participate in the reading of the downstream ORF. In this system, the border region of the two ORFs is such that termination codon of the upstream ORF and the initiation codon of the downstream ORF overlap as in UAAUG. At this junction, ribosomes just completed reading the upstream are released by RRF and about 25% of the released ribosomes re‐bind to the AUG and start translating the downstream gene. For this reason, the system offers unique opportunity to study the fate of ribosome finishing one round of translation which is under the control of ribosome recycling Factor (RRF). We placed this system in a strain (LJ14) which contains the temperature sensitive RRF so that one can study what happens in the absence and presence of RRF. Using this system, we showed 1) The major function of RRF is to release the ribosomes from mRNA but not splitting the ribosome. 2) When Ribosomes are not released due to the inactivation of RRF, ribosomes at UAA undergoes thermal frame shift and read all three frames. 3) For the ribosome released by RRF from UAA, AUG but not other triplet functions to read the downstream ORF. 4) This binding of released ribosomes occurs only if the AUG is present near UAA. 5) Introduction of short sequence hybridizable with ribosomal RNA helps ribosomes to rebind to the AUG. 6) When the upstream sequence is extremely short (4 codon or less), the ribosomes released from UAA by RRF rebinds to the upstream AUG with Shine Dalgarno (SD) sequence and the reading of the downstream ORF does not occur.

Conclusions

Our results show that the early workers were led to a wrong concept that RRF does not release mRNA from the ribosome because they studied the action of RRF using short ORF with SD. Therefore, our original concept for which RRF was discovered as “ribosome releasing factor” was correct. The current wrong concept of the RRF action or ribosome recycling factor in general including the eukaryotes stems from the use of short ORF in in vitro studies in the early days.

Support or Funding Information

Funded by Creative Bio‐medical Research Institute, Phildelphia Pa 19147
Structural Characterization of a Mutant form of the Chlamydial Protein Pgp3
Authors: Trinh Timothy , Waninger‐Saroni Jessica , Galaleldeen Ahmad
Published: November 2023

Chlamydia trachomatis infection is the most common sexually transmitted bacterial infection worldwide. Untreated infection of the human ocular or urogenital tract can lead to blindness, ectopic pregnancy, pelvic inflammation, and infertility. The C. trachomatis protein Pgp3 is a homotrimer believed to play a prominent role in chlamydial pathogenesis. Pgp3 is one of the most immunodominant antigens found in infected people, and it stimulates macrophages into releasing inflammatory cytokines.

We have recently determined the structure of Pgp3. The protein is comprised of three domains: a C‐terminal domain that resembles the TNF family of cytokines, an N‐terminal domain with structural motifs similar to those found in trimeric viral proteins, and a central triple‐helical coiled‐coil (THCC) that forms a rare right‐handed superhelical twist.

The structure determination of Pgp3 was challenging due to the strong diffuse scattering observed in the diffraction pattern of the crystals. The diffuse scatter is believed to be caused by the flexibility of the THCC domain as a result of a sharp turn at glycine 85.

To improve the model and produce better diffracting crystals, we mutated glycine 85 into alanine to remove the sharp turn, thus producing better diffracting crystals to build a better model of the THCC domain.

The G85A protein was expressed, purified, and, crystallized, and the structure was determined. The crystal shares the same space group with WT Pgp3 and the diffuse scattering persisted. The G85A mutation did not alter the twist in the THCC domain.
Calcineurin Homologous Protein Isoforms 1 and 2 Binding to the Sodium Hydrogen Exchanger 1
Authors: Marshall Chelsea , Wallert Mark , Provost Joseph John
Published: November 2023

The Sodium Hydrogen Exchanger (NHE1) is a ubiquitously expressed protein, which regulates cell volume, intracellular pH, and motility. This antiporter exchanges an intracellular proton for an extracellular sodium and is regulated by a range of protein or lipid interactions and phosphorylations. Calcineurin Homologous Protein isoform 1 (CHP1) and isoform 2 (CHP2) both bind to essentially the same site on the carboxyl terminus of NHE1. However, while CHP1 and CHP2 share a 61% amino residue homology, they have different binding affinities for NHE1. Understanding how one isoform can bind NHE with greater affinity than another is of interest because CHP1 is required for basic NHE1 function and a physiological role of CHP2 has not been identified. To study the interaction of each CHP isoform with NHE1, we have generated a GST‐NHE1 fusion protein (aa 803–846) and His tagged CHP1 and 2. After expression and reconstitution of a thrombin cleaved NHE1 peptide with CHP we determined the interaction using pull‐down and thermal melt. To identify possible binding determinates on NHE1 for the each_CHP isoforms we mutated_eight residues of NHE1 thought to play a role binding CHP. These key residues, include: Asn519Ala, Asn519Asp, Ile518Gln/Ile522Gln, Ile534Lys, Ile537Lys, His523Gly, His523Ile, and Asp536Gly. Protein interactions with recombinant CHP1 or CHP2 with each mutant NHE1 peptide was determined using thermal denaturation, circular dichroism and other biophysical methods. This work will illuminate potential interaction sites on NHE1 for CHP, and demonstrate sites common and unique for both CHP isoforms.
Frustrational Analysis of Na+/H+ Exchanger 1 (NHE1) and Calcineurin B Homologous Protein 1 & 2
Authors: Silva Daniela , Latzer Joachim , Wallert Mark , Provost Joseph John
Published: November 2023

The sodium hydrogen exchanger isoform 1 (NHE1) is involved in tumor cell proliferation, metastasis, invasion and morbidity in several cancers including non‐small cell lung cancer. While several proteins, lipids and kinases are responsible for regulating NHE, two proteins, calcineurin homologous protein isoform 1 (CHP1) and 2 (CHP2) are of particular interest in lung cancer cells. While CHP1 binds NHE1 and is responsible for basal activation of NHE1, CHP2 expression is nearly limited to cancer cells and has been reported to replace CHP1‐NHE1 interactions with unknown consequences. Thus an important target for anti‐lung cancer drug design is the binding of a drug which would block CHP2 but not CHP1 from NHE1 activation. To determine unique and common binding sites for CHP isoforms with NHE, we performed molecular dynamics simulations with a coarse‐grained funneled energy function that provided an atomic level picture of the association (and hence activation) of NHE1 with CHP1 and CHP2. In these simulations NHE1 collaboratively folds and binds correctly to both CHP proteins lowering the overall binding free energy. The molecular detail of the simulations helped identify “hot‐spot” residues involved in the encounter complex and in the binding interface that can guide the design of non‐binding mutants. To understand the association of NHE1 on a residue level, frustrated (residues unlikely to bind) and minimally frustrated (residues that bind strongly) contacts of NHE1 residues were computed for bound structures and also the encounter complexes. Based on circular dichroism studies and computational analysis the binding energies were determined for the bound complexes of NHE1 and CHP1/CHP2. In order to assess the optimal storage conditions and establish a time frame for CHP1 and CHP2 structural stability a validation assay was conducted. The nature of the interactions probed served to understand, in molecular detail, the activation of NHE1 and could serve as input for pharmacomodeling to design a therapeutic drugs that fights lung cancer growth.
Blocking the Protein‐Protein Interaction between Gαh and PLCδ1 Inhibits Metastatic Progression in Triple‐Negative Breast Cancer
Authors: Lin Yuan‐Feng , Liu Pei‐Yao , Hsiao Michael
Published: November 2023

Breast cancer is thought to be a leading cause of poor survival rate in woman diseases. Especially triple‐negative breast cancer (TNBC) severely risks patient's life, usually owing to its metastatic progression. However, the molecular mechanism for cancer metastasis is still unclear in TNBC.

Here find that Gαh which is originally identified as tissue transglutaminase acts as a poor prognostic marker in clinical cohort with breast cancer and exerts its G protein function to promote the invasiveness of TNBC cells in vitro and in vivo. Our data showed that the expression of Gαh causally associates with the in vitro invasion ability of TNBC cell lines. Moreover, the knockdown of Gαh obviously suppressed the in vitro invasion ability and the in vivo lung colonization capacity of highly metastatic TNBC cell lines MDA‐MB231 and HCC38 that express Gαh protein abundantly. Conversely, the enforced expression of exogenous Gah markedly fostered the metastatic potentials in vitro and in vivo of TNBC cell lines MDA‐MB157 and HCC70 that express Gαh protein poorly. Certainly, the GTP‐binding activity, but not transamidating activity, of Gαh is crucial for promoting the metastatic evolution in TNBC cells. Furthermore, interrupting the protein‐protein interaction (PPI) of phospholipase C‐δ1 (PLCδ1) with Gαh by a synthetic peptide corresponding to PLCδ1 amino acid sequence TIPLNSLKQGYRHVHLM robustly suppressed the metastatic abilities of MDA‐MB231 cells in vitro and in vivo.

These findings not only elucidate the critical role of Gαh/PLCδ1 signaling pathway in promoting the metastatic progression of TNBC but also provide a new strategy to combat TNBC metastasis via targeting the PPI of Gαh/PLCδ1 complex.
Using ANS to Probe Ligand Induced Conformational States of Malate Dehydrogenase
Authors: Schwabe Michael , Bell Ellis , Bell Jessica
Published: November 2023

Malate Dehydrogenase catalyzes the oxidation/reduction of malate/oxaloacetate coupled with the conversion of NAD and NADH and plays a critical role in energy metabolism in the cell. The enzyme is a dimer and has been suggested to involve subunit interactions in both its normal function and in regulation by citrate. 8‐Anilinonaphthalene‐1‐sulfonic acid(ANS) is frequently used as a conformational probe of protein structure since it often shows a dramatic increase in fluorescence and spectral shifts on binding to exposed regions of a protein and has been used to follow protein folding, ligand induced conformational changes, and aggregation. However little is known about the specifics of changes in fluorescence resulting from different modes of binding. To address this issue, and to characterize ligand induced conformational states in Malate Dehydrogenase we have used a combination of spectral studies combined with ANS titrations to characterize the different modes of binding of ANS to Malate Dehydrogenase in the presence or absence of a variety of ligands. Titrations reveal both high and low affinity sites on the protein. Computational docking studies with both “open” and “closed” conformations shown to exist by x ray crystallography, have been used to identify potential regions of the protein which may constitute these sites. The spectral properties of the various sites are resolved using “Peakfit” to deconvolute the experimental data. Using a titration and spectral deconvolution approach we have examined mitochondrial, cytosolic and glyoxysomal forms of the enzyme to elucidate the effects of addition of varying concentrations of the substrates, Malate and Oxaloacetate, as well as several inhibitors of the enzyme, succinate and malonate on the conformational states of the enzyme. The results suggest a complex array of conformational states associated with possible spatial asymmetry and ligand induced changes in the protein.
Serum Proteins Binding to Tamm‐Horsfall Protein
Authors: Araujo Hugo C. , Rhodes Diana C.J. , Rhodes James A.
Published: November 2023

Tamm‐Horsfall protein (THP), also called uromodulin, is an approximately 86kD, heavily glycosylated, single‐chain acidic protein (estimated pI=3). THP is produced in the kidney within the thick ascending limb of the loop of Henle and is excreted in milligram quantities daily into the urine. Despite its abundance, the normal physiologic function of THP remains enigmatic. One theory is that THP is an immunomodulatory molecule, due to its ability to bind various immune proteins such as IL‐1, TNF‐α, IgG, complement 1q (C1q), and factor H (fH). The purpose of the present study was to determine if other serum proteins could be shown to bind to immobilized THP. Multiple attempts to couple THP (purified from the urine of one healthy female) to a commercially available N‐hydroxysuccinimide (NHS)‐activated agarose resin (Thermo ScientificTM Pierce®) failed, at least partially because of solubility issues with THP, a protein that readily forms large aggregates in solution. As an alternative approach, purified THP was coated to microtiter plate wells and normal human serum diluted 1:5 with 20 mM Tris/20 mM NaCl/0.05% Tween 20, pH 7.5 was incubated in THP‐coated or non‐THP‐coated wells. After rinsing to remove unbound protein, the proteins bound to the wells were extracted by addition of 1X sample buffer containing 5% β‐mercaptoethanol, and the recovered samples were analyzed by SDS‐PAGE. IgG in serum did not appear to bind THP, even though binding of purified IgG to THP could be detected by this method. Among the proteins that specifically bound to the THP‐coated wells was a prominent protein with an estimated molecular weight of about 78 kD. Based on its molecular weight and abundance, transferrin seemed to be a likely candidate for this protein. Purified human transferrin (Sigma) and the protein recovered from THP‐coated microtiter plates had identical mobilities on SDS‐PAGE gels. Direct binding of THP to purified transferrin was analyzed by ELISA, incubating human transferrin (from 4 mg/ml to 100 μg/ml) in THP‐coated and non‐THP‐coated microtiter plate wells and then detecting bound transferrin with goat anti‐transferrin antiserum and rabbit anti‐goat IgG‐alkaline phosphatase. This ELISA demonstrated that transferrin bound to immobilized THP in a dose‐dependent manner. In summary, these studies indicated that immobilized THP does not bind to IgG within diluted serum, even though THP binds to purified IgG (in the absence of diluted serum), suggesting that a component of serum inhibits the THP/IgG interaction. These studies also provided evidence for an interaction between THP and transferrin, the significance of which remains to be elucidated.

Support or Funding Information

Funded by the PNWU Research Seed Program.
Binding of Tamm‐Horsfall Protein to Properdin in Various Ionic Environments
Authors: Morgan Jarrad , Rhodes Diana
Published: November 2023

Tamm‐Horsfall protein (THP), also known as uromodulin, is produced in the kidney and is the most abundant protein excreted into urine. It is an ~86kD acidic glycoprotein (pI≈3) with complex N‐linked carbohydrates often capped with sialic acid. THP may act as an immunomodulatory protein since it binds to IgG, complement 1q (C1q), complement 3b (C3b), and factor H (fH); inhibits the classical complement pathway; and acts as a cofactor for factor I in C3b degradation. THP also binds properdin which is a basic protein (pI > 9.5) consisting of multiple, identical 53 kD subunits. Properdin is the only known positive regulator of complement, stabilizing C3b. The purpose of this study was to investigate the effect of different ionic conditions on the interaction between THP and properdin, which are oppositely charged proteins. Previous studies of the interaction between THP and properdin had utilized Falcon Pro Bind microtiter plates that are no longer available. Hence, the first studies compared the remaining Falcon plates to three other microtiter plates. For these and all subsequent ELISAs, wells were coated with THP purified from the urine of one healthy male. Multiple concentrations of properdin (Complement Technologies) were added to THP‐coated wells, and the bound properdin was detected with goat anti‐properdin serum and rabbit anti‐goat IgG‐alkaline phosphatase. Of the plates tested, the Corning Costar plates acted in a manner most similar to Falcon plates and were used in the remaining studies. The binding buffer used in all studies was 20 mM Tris/1 mM MgCl2/1 mM CaCl2/0.05% Tween 20 (pH ~7.6) with concentrations of NaCl varying from 20 mM to 154 mM. These studies showed that the optimal NaCl concentration was dependent on the properdin concentration, with maximal binding of properdin to THP occurring at 70–80 mM NaCl for low properdin concentrations (0.25 and 0.5 μg/ml) and maximal binding of properdin to THP occurring at 20 mM NaCl with high properdin concentrations (5 μg/ml). The lowest amount of THP/properdin binding consistently was seen in the 154 mM NaCl buffer. These results differ somewhat from our very early, much more limited experiments with the Falcon microtiter plates, where THP binding to properdin was higher at all concentrations of properdin in a 90 mM ionic strength buffer. It is noteworthy that if the interaction between THP and properdin was purely electrostatic in nature, binding would be consistently higher in low ionic strength buffers, which was not what the present study showed. Further studies, not only on the effect of ionic strength but also on the effect of pH on the THP/properdin interaction are warranted since urine, which is where THP and properdin most likely could interact, varies considerably in these parameters.

Support or Funding Information

Supported by the PNWU Research Funds.
A group of novel serum diagnostic biomarkers for multidrug‐resistant tuberculosis by iTRAQ‐2D LC‐MS/MS and Solexa sequencing
Authors: WANG CHONG , LIU CHANG‐MING , WEI LI‐LIANG , SHI LI‐YING , PAN ZHI‐FEN , MAO LIAN‐GEN , WAN XIAO‐CHEN , PING ZE‐PENG , JIANG TING‐TING , CHEN ZHONG‐LIANG , LI ZHONG‐JIE , LI JI‐CHENG
Published: November 2023

The epidemic of pulmonary tuberculosis (TB), especially multidrug‐resistance tuberculosis (MDR‐TB) presented a major challenge for TB treatment today. We performed iTRAQ labeling coupled with two‐dimensional liquid chromatography‐tandem mass spectrometry (2D LC‐MS/MS) and Solexa sequencing among MDR‐TB patients, drug‐sensitive tuberculosis (DS‐TB) patients, and healthy controls. A total of 50 differentially expressed proteins and 43 differentially expressed miRNAs (fold change >1.50 or <0.60, P<0.05) were identified in the MDR‐TB patients compared to both DS‐TB patients and healthy controls. We found that 22.00% of differentially expressed proteins and 32.56% of differentially expressed miRNAs were related, and could construct a network mainly in complement and coagulation cascades. Significant differences in CD44 antigen (CD44), coagulation factor XI (F11), kininogen‐1 (KNG1), miR‐4433b‐5p, miR‐424‐5p, and miR‐199b‐5p were found among MDR‐TB patients, DS‐TB patients and healthy controls (P<0.05) by enzyme‐linked immunosorbent assay (ELISA) and SYBR green qRT‐PCR validation. A strong negative correlation, consistent with the target gene prediction, was found between KNG1 and miR‐199b‐5p (r=−0.232, P=0.017). Moreover, we established a diagnostic model for MDR‐TB patients and healthy controls with a sensitivity of 100.00% and a specificity of 88.33% (overall accuracy 93.14%), and also established a diagnostic model for MDR‐TB patients and DS‐TB patients with a sensitivity of 83.33% and a specificity of 88.33% (overall accuracy 86.27%). Our study proposes potential biomarkers for MDR‐TB diagnosis, and also provides a new experimental basis to understand the pathogenesis of MDR‐TB.

Support or Funding Information

This work was supported by grants from the National Natural Science Foundation of China (No.81573709, No.81273882), the National Basic Research Program of China (No.2014CB543002) and the National Special Sci‐Tech Projects (No.2012ZX10005001‐006).
Metal affinity chromatography and protein refolding techniques: some observations and strategies
Authors: Odunuga Odutayo O , Weihe Jamie M , Tovar Carmel N
Published: November 2023

Immobilized metal affinity chromatography (IMAC) is a popular technique used for large‐scale purification of proteins because of its high binding specificity, capacity and relative ease of operation. Despite recent advances in improving the robustness of the technique, several factors still affect column binding capacity, yield and purity of proteins during IMAC. Using (His)6‐tagged proteins with comparably high level of expression, we investigated the effect of protein size on the binding capacities of commercial Ni2+ and Co2+ IMAC columns. Our data suggest that small and medium‐sized proteins (< 50 kDa) bind more tightly to the columns (Ni‐NTA and Co‐TALON) than bigger proteins, resulting in higher yields during purification. In another study, we employed a modified gel filtration technique, gradient gel filtration to successfully refold a spectrum of highly aggregation‐prone enzymatic proteins back to their native states during denaturing purification. Some of the proteins chosen are well‐known to precipitate significantly during refolding. However, using this technique, precipitation was either so minimal or non‐existent. Enzymatic assays and dynamic molecular mass determination confirmed that the proteins were correctly refolded and biologically active. This strategy provides a robust approach to refolding denatured proteins that can also be used simultaneously as buffer exchange and additional purification step.

Support or Funding Information

Welch Foundation Grant # AN‐0008
New insight into the role of ATP in iron sulfur cluster scaffolding by the Mrp/Nbp35 family of cluster scaffolds
Authors: Perlstein Deborah L , Grossman John
Published: November 2023

The yeast enzymes Nbp35 and Cfd1 form the iron sulfur (FeS) cluster scaffold for the assembly of cytosolic and nuclear FeS enzymes. As prototypical members of the Mrp/Nbp35 family of scaffolds, Nbp35 and Cfd1 each possess motifs for FeS cluster binding and a P‐loop ATPase domain of unknown function. Although it has been known for a decade that conserved residues in the ATPase domain are required for cluster biogenesis activity in vivo, it has been difficult to pinpoint the role of ATP because Nbp35 and Cfd1 are essential, limiting the scope of information that can be gained from genetic approaches. Furthermore, it has been challenging to detect nucleotide binding and hydrolysis by Nbp35 and Cfd1. We recently developed the first assays for detecting ATPase activity and demonstrated that the Nbp35 homodimer and the Nbp35‐Cfd1 heterodimer both hydrolyze ATP, whereas the Cfd1 homodimer does not display detectable hydrolysis activity. However, these assays did not reveal any evidence for communication between the cluster scaffolding site and the ATPase site since chemical reconstitution of FeS clusters on the scaffold did not significantly affect the ATP hydrolysis rate. To determine if there is any allosteric communication between the ATPase site and the cluster scaffolding site, we probed nucleotide affinity of the Nbp35‐Cfd1 complex using a bodipy‐labeled ATP analog (BoATP). We demonstrate that the apo‐Nbp35‐Cfd1 complex has a KD for BoATP of 52 μM whereas the holo‐complex has a KD ≥ 135 μM. Since the Nbp35‐Cfd1 complex binds two FeS clusters, one on the Nbp35 N‐terminus and a second bridging the Nbp35‐Cfd1 interface, we created a truncated Nbp35‐Cfd1 heterodimer (trNbp35‐Cfd1) which can only bind the bridging cluster. As observed with the full length constructs, holo‐trNbp35‐Cfd1 bound boATP with weaker affinity (KD ≥ 294 μM) than the apo‐construct (KD= 65 μM). This work demonstrates that the bridging cluster is sufficient to affect ATP affinity, uncovering some of the first direct evidence for allosteric communication between the cluster scaffolding site and the ATPase site. This breakthrough allows us to begin unraveling the cryptic role of ATP in cluster assembly and transfer by Mrp/Nbp35 cluster scaffolds.
Synthesis of Artificial Bacterial Cell Wall Fragments as Tools to Study the Innate Immune System
Authors: Gregorzak Gabriel , Melnyk James , DeMeester Kristen , Grimes Catherine
Published: November 2023

During a bacterial infection, the body's first line of defense is provided by the innate immune system. This system is triggered by pathogen‐associated molecular patterns (PAMPs) that are recognized by different members of the body's innate immune system called pattern recognition receptors (PRRs). One such PRR of interest is nucleotide‐binding oligomerization domain‐containing protein 2 (Nod2). Mammalian Nod2 is a cytosolic protein that binds to and signals in response to carbohydrate containing bacterial cell wall fragments; the smallest of which is muramyl dipeptide (MDP). Mutations in Nod2 increase the likelihood of the development of Crohn's Disease and other chronic inflammatory diseases. Previously, our lab has shown that Nod2 binds directly to MDP, but currently the molecular mechanisms of activation and binding are still unknown1. Currently, we are addressing this issue using synthetic chemistry. This project focuses on the synthesis of an assortment of MDP dimers, which vary in the length and functionality of their connecting linkers. To avoid troublesome late‐stage de‐protection issues, the synthesis diverges in its early stages to result in two differing MDP‐like fragments. These two fragments can then be coupled together and de‐protected, resulting in the MDP dimer. Once synthesized, these dimers will be used in in vitro SPR binding assays, as well as in cell‐based assays to elucidate the binding and activation mechanisms used by Nod2. Ultimately, these synthetic tools will aid in the advancement of our understanding of how are body's sense and respond to bacteria.

Support or Funding Information

Howard Hughes Medical Institute