bluuebunny/crossref_metadata_2025_split huggingface dataset:
Antimicrobial Peptide Turns the Ribosome into a Release Factor Trap
Authors: Florin Tanja , Maracci Cristina , Graf Michael , Karki Prajwal , Klepacki Dorota , Rodnina Marina V , Wilson Daniel N , Vázquez‐Laslop Nora , Mankin Alexander S 
Published: November 2023

Apidaecin (Api) belongs to the group of proline‐rich antimicrobial peptides (PrAMPs), which are produced by insects and mammals and protect the host from bacterial infection. PrAMPs enter the bacterial cell, bind to the ribosome, and inhibit protein synthesis. Biochemical characterization and recent high‐resolution crystal structures have shown that most PrAMPs obstruct the nascent peptide exit tunnel and the peptidyl transferase center in the ribosome and block the initiation step of translation.Despite the lack of the structural information about the Api‐bound ribosome, its similarities with the other PrAMPs suggested that Api also inhibits translation initiation. However, our toeprinting experiments in a cell free translation system revealed that Api does not inhibit initiation but, instead, stalls the ribosome at the stop codon. Isolation of Api‐resistant E. coli mutants with alterations in class‐1 release factors RF1 and RF2, indicated that Api may interfere with the functions of these factors and/or their association with the ribosome. Consistently, our high‐resolution cryo‐EM structures of ribosomes complexed with Api showed that Api establishes interactions with the ribosomal exit tunnel and with the functionally important conserved GGQ motif of RF1. Furthermore, kinetic studies revealed that Api does not prevent the release of the nascent peptide chain but impedes the dissociation of RF from its binding site in the ribosome. By trapping RF1 and RF2 in the ribosome, Api leads to depletion of free class‐1 RFs in the cell, resulting in inhibition of protein synthesis and cell growth arrest.Importantly, the Api‐dependent depletion of RFs facilitates premature stop codon read‐through. We envision the possibility that similar trapping of RFs on eukaryotic ribosomes could aid in relieving the effects of human genetic diseases caused by premature stop‐codons.
Structural differences associated with DNA binding of p53 family member proteins
Authors: Mavodza Grace R , Fang Yayin , Sherif Zaki 
Published: November 2023

Introductionp53 family member proteins, which are made up of p53, p63, and p73, have long been presumed to function as tumor suppressors. They possess significant structural homology, including the presence of a transactivation domain, a DNA‐binding domain, and an oligomerization domain. In spite of their structural similarities there is growing evidence that p63 and p73 initiate unique pathways, which play larger roles in development rather than in tumor suppression. This study seeks to further highlight the structural differences between the DNA‐binding regions of the three proteins and how those structural differences may translate into the functional differences observed for each protein.MethodsThe structure of the DNA binding domain of each protein was obtained from the RCSB Protein Data Bank. The final structures used for protein p53, p63 and p73 were PDB ID 2ADY, 3US0 and 4G82 respectively. MOE 2013 was utilized to visualize the structure of the DNA binding domain of each protein and it was then used to perform a sequence and structural alignment of the three proteins. The three structures were compared at the atomic level, and the amino acids in contact with the DNA were selected for further analysis.ResultsThe six regions of interest, which are all in contact with DNA, have been identified. Five of the areas were verifications of information previously published. The region of greatest interest was a conserved arginine, whose guanidinium group had a noticeably different orientation and an equally unique electrostatic map in all three structures. Arg248, Arg279, and Arg268 correspond to the arginine of interest in p53, p63, and p73 respectively.ConclusionThe presence of a conserved arginine within the p53 family of proteins does not guarantee the proteins will interact with DNA in the same manner. This is highlighted by the unique orientation observed in the arginine of each family member protein and may contribute to the diversity in their ability to initiate the activation of various proteins.Support or Funding InformationThis work was supported by grant #G12 MD007597 from NIMHD, NIH to RCMI program at Howard University, HUMAA (Howard University Medicine Alumni Association) Endowed Founder's Chair in Basic Science, and grant #U01 CA185188‐01A1 from NCI/NIH to Dr. Zaki Sherif.
Solution Structure of the Transmembrane Nogo‐B Receptor and Insight into its Topological Orientation by Small Angle X‐ray Scattering Analysis
Authors: Holcomb Joshua , Spellmon Nicholas , Shang Weifeng , Miao Quing , Yang Zhe 
Published: November 2023

The Nogo‐B Receptor (NgBR) is newly identified class I transmembrane receptor known for its role in binding Nogo‐B and promoting angiogenesis by chemotaxis in vivo. Most recently we discovered that NgBR shows binding specificity for the farnesylated oncoprotein Ras and facilitates Ras activation by its recruitment to the plasma membrane. To understand the potential mode of interaction with farnesylated Ras, we performed Small Angle X‐ray Scattering (SAXS) analysis of the NgBR construct 79–293 which includes the currently accepted cytosolic and transmembrane domains with a portion of the extracellular region. SAXS analysis reveals the radius of gyration (Rg) for our NgBR construct to be 18.2 Å with a maximum end to end distance (Dmax) of 61 Å. Ab initio modeling returns a globular molecular envelope with an estimated molecular weight of 23 kD closely correlated with the calculated molecular weight of our NgBR construct. These data indicate that our NgBR construct is well folded and exists as a globular domain in solution with no apparent intrinsic disordered regions. However, this result is conflicting with the currently accepted topological orientation of NgBR which would separate our NgBR construct into three distinct regions: extracellular, transmembrane and cytosolic domains. However, comparison of the molecular envelope structure of our NgBR construct with the structure of Undecaprenyl Pyrophosphate Synthase (UPPs) which shares similar sequence homology to NgBR, further supports our conclusion that the regions included in our construct are folded into a single globular domain, as the two structures are highly superimposed. This led us to propose the idea that NgBR may exist in more than one topological orientation and that the currently accepted transmembrane domain may internalize and contribute to the overall fold of NgBR cytosolically, thereby a complete binding site for farnesylated Ras. Altogether, our SAXS analysis provides the first molecular envelope structure of the Nogo‐B Receptor and provides evidence for a topological orientation of NgBR not previously identified.Support or Funding InformationDetroit Cardiovascular Training Program, NIH: National Heart, Lung, and Blood Institute
Inter‐Domain Interactions In Nascent Polypeptides Interfere With Productive Protein Folding
Authors: Liu Kaixian , Maciuba Kevin , Kaiser Christian 
Published: November 2023

How large, multi‐domain proteins robustly fold into their native structures is not well understood. It is often assumed that individual domains fold independently, but experimental studies have largely been limited to the special case of tandem repeat proteins. We have used single‐molecule force spectroscopy to study the folding of elongation factor G (EF‐G), which is composed of five domains. Optical tweezers experiments show that full‐length EF‐G refolds inefficiently along highly complex folding pathways. To determine whether vectorial protein synthesis, by reducing complexity, enables efficient domain‐wise folding, we studied successively longer ribosome‐bound nascent polypeptides. The N‐terminal G‐domain forms a stable structure by itself and enables the otherwise unstable subsequent domain II to fold. When both domains fold simultaneously, they form a misfolded species that is more stable than the productive folding intermediates. Interestingly, interactions with the unfolded domain II destabilize the native G‐domain. Together, our data indicate that vectorial protein synthesis, perhaps with rates optimized for folding, helps to avoid intra‐molecular misfolding within nascent multi‐domain polypeptides, allowing the G‐domain to complete folding before domain II is produced. Interactions with molecular chaperones might be required to protect folded domains against interactions with yet unstructured regions in the nascent polypeptide.Support or Funding InformationJohns Hopkins University, The Pew Charitable Trusts
Development of a Microscopic Method for the Exposure of Hemoglobin C
Authors: Schmidt Kayla Leanne , Randolph Tim R 
Published: November 2023

Hemoglobin C is the second most prevalent hemoglobinopathy worldwide behind sickle cell anemia (HbS). HbC disease (HbCC) produces only mild symptoms, but is a life‐threatening disorder if inherited with HbS (HbSC). HbS and HbC are most prevalent in countries underequipped to diagnose its presence through the Gold Standard of electrophoresis. This study aims to create a simple, inexpensive method to identify the presence of Hemoglobin C using limited resources. The hypothesis is that when blood is incubated in a hypertonic salt solution, Hemoglobin C will crystalize intracellularly and become visible microscopically when stained with New Methylene Blue. The method was optimized by modifying the salt type, salt concentration, and incubation time. The optimized method uses a 5× Dulbecco's phosphate buffered saline at 37°C for 4 hours. Blood samples with the HbSC genotype yielded 702 for every 1000 RBCs counted. Blood samples of AC, CC, SC, and AA genotypes will be tested to determine if the number of crystals that form can be used to differentiate genotypes. If so, this inexpensive, simple, and relatively rapid method can be used to identify patients with HbS and potentially determine genotype.Support or Funding InformationKS is supported by the DeNardo Education and Research Foundation Fellowship.
HLA‐DQ1 Alpha and Beta Genotypes Associated with Non‐Celiac Gluten Sensitivity
Authors: Maki Michael , Caporale Diane 
Published: November 2023

Non‐Celiac Gluten Sensitivity (NCGS) is a gastrointestinal disease occurring in 3–6% of the population, while celiac disease (CD) and wheat allergy (WA) are only found in 2–4%. This indicates that 17 million Americans may have NCGS. Only by eliminating a diagnosis of CD and WA through medical testing and the elimination of foods from their diet can a patient be diagnosed with NCGS. Some reports have shown that proteins embedded in the membranes of leukocytes known as human leukocyte antigens (HLAs) are linked to comparable gastric diseases like CD and WA. It is possible that certain HLA genotypes may also be associated with NCGS. To test this hypothesis, we screened a cohort of 50 NCGS and 50 non‐NCGS participants for their HLA DQ alpha and beta genotypes, using AllSet+ Gold DQ alpha 1 and beta 1 high‐resolution allele‐specific PCR kits. DNA fingerprints were generated by electrophoresing the PCR products on large 3% agarose gels and photo‐documented for allele identification. Since the HLADQ surface protein consists of one alpha polypeptide and one beta polypeptide and both subunits are in association with the antigen molecule, it seemed most appropriate to combine the alpha and beta genotypes for each individual and observe allele combinations within and between each cohort. For simplicity, alpha and beta allele types were numbered α1 through 5 and β1 through 6, respectively. So far, the most common genotype combinations within the gluten‐sensitive cohort were A1‐3/B2‐6 and A1‐5/B2‐6. The most common genotype combinations for the control group were A1‐3/B3‐5 and A1‐3/B3‐6; both were also unique to the control group. The genotype combination A1‐3/B2‐6 was only found within the gluten‐sensitive cohort. Interestingly, allele A5 (DQA1:05) and allele B2 (DQB1:02) are both higher in frequency within the gluten‐sensitive group than within the control group. Previous studies have shown high association between these two allele types and Celiac disease. We are continuing to screen more participants within each cohort, to test the statistical significance of our findings. If the A5 and B2 allele frequencies continue to be significantly higher in the gluten‐sensitive cohort, then screening for these alleles may be used as a more efficient diagnosis tool in the future.Support or Funding InformationThis project was partly funded by URSCA student grant awarded to M. Maki and CL&S UEI grant awarded to D. Caporale.
Ras1 Phosphorylation and its Role in Nutrient Signaling
Authors: Sethupathi Sheetal , Jin Xin , Starke Samuel , Li Yang , Kung George , Wang Yuqi 
Published: November 2023

Ras1 is a small GTPase found in Saccharomyces cerevisiae that regulates nutrient signaling and is known to undergo phosphorylation. Studies were conducted to gain a better understanding of the functional consequences and the regulation of Ras1 phosphorylation. Site‐directed mutagenesis was used to mutate critical serine residues to alanine residues. A serine residue at the C‐terminal region of Ras1 was identified as an important residue for Ras1 phosphorylation as mutating this serine residue to alanine drastically diminished the level of Ras1 phosphorylation. Additionally, phosphorylated Ras1 accumulates as cells approach the stationary phase of growth. Similarly, cells exposed to nitrogen starvation exhibit increased levels of phosphorylated Ras1. By blocking the phosphorylation of Ras1, we find that the level of autophagy decreases. Together, these findings reveal phosphorylation of Ras1 as a mechanism in regulating cellular responses to nitrogen starvation.Support or Funding InformationThis work was funded by a research grant from the National Institutes of Health and the Knoedler Undergraduate Research Fund.
Nanoparticles mimic exosomes and attenuate growth factor‐induced cell migration in melanoma cells
Authors: Gallant Benjamin Paul , Clark Matthew , Madigan Audrey , Wan Yinsheng , Zhang Wei , Lu Hongguang 
Published: November 2023

Melanoma is notoriously aggressive and still considered untreatable, in spite of some of the most recent breakthroughs related to immunotherapy. Our previous studies have shown that melanoma cells, compared to untransformed normal human melanocytes, exhibit constitutive mTOR activity and are resistant to oxidative stress. Exosomes, which are membranous vesicles of various sizes between 30–100 nm, are recently being intensively studied. These exosomes are involved in cell‐cell communications and aggression of cancer cells. We hypothesized that constitutive mTOR activity in melanoma cells is attributable to biogenesis and release of exosomes, resulting in the aggression and metastatic potential of melanoma. Externally applied nanoparticles with similar sizes to exosomes may confuse melanoma cells and thus have biological effects or therapeutic benefits. Using phagokinetic motility assay, we observed that gold and iron nanoparticles (50 nm.) attenuated serum‐induced cell migration in cultured melanoma cells (WM 266‐4 cells), similarly, inhibitors of PI3K/AKT, MEK/ERK, Na+/Ca++ Channels and mTOR showed inhibitory effects. Confocal microscopy results showed that while above inhibitors decreased mTOR activity in melanoma cells, gold and iron nanoparticles also minimally inhibit serum‐induced mTOR activity (expressed in phosphorylation of ribosomal protein S6), and MEK/ERK activity, which are critical for cell migration. Western blot analysis demonstrated that serum induced biogenesis and release of exosomes, as expressed by two of the markers, CD63 and Mart1, which were minimally inhibited by inhibitors of PI3K/AKT, MEK/ERK, and mTOR. Furthermore, when WM 266‐4 were treated with known inhibitors of exosomes, Monensin and Brefeldin A, serum‐induced mTOR activity (expressed in phosphorylation of ribosomal protein S6) and cell migration (phagokinetic motility track assay) were markedly reduced. Together, our data indicate that nanoparticles, mimicking exosomes, inhibit mTOR, resulting in the attenuation of cell migration of melanoma cells. Additionally, our data also suggests that inhibitors of these exosomes greatly reduce cell migration and mTOR activity in these melanoma cells. Our results suggest that nanoparticles with similar sizes to exosomes, and inhibitors of these exosomes, may provide therapeutic benefits to melanoma treatment.Support or Funding InformationRI‐INBRE
Determining the Structure of Oncogenic NRas Mutants
Authors: Ojeda Monica , Reid Derion , Mattos Carla 
Published: November 2023

Ras proteins are small GTPases that are involved in signal transduction pathways that regulates cell proliferation, differentiation, and survival. These proteins act as molecular switches where they are activated by exchanging guanosine diphosphate (GDP) to guanosine triphosphate (GTP), and become inactivated when GTP is hydrolyzed to GDP. Ras GTPases are mutated in 20% of human cancers. Within the Ras family, there are three isoforms: H‐Ras, K‐Ras, and N‐Ras. Due to the high structural similarities of the three isoforms, much of the structural efforts have focused on H‐ and K‐Ras while neglecting N‐Ras. However, it is known that N‐Ras oncogenes are predominantly mutated in melanoma. Two common sites of oncogenic mutations that affect N‐Ras regulation of cancerous pathways are Gly12 and Gln61. Mutants at codons 12 and 61 result in constitutive activation. Therefore, we are interested in using x‐ray crystallography to solve the structure of N‐Ras oncogenic mutants: G12D and G12S. Both mutants are highly expressed in E. coli and behave well through our established purification protocol involving ion exchange and gel filtration chromatography. Both were purified in sufficiently high yield for structural biology studies and are currently undergoing crystallization trials. We expect that mutating glycine 12 will interfere with nucleotide binding and inhibit hydrolysis of N‐Ras leading to constitutive activation. By determining the structure of these oncogenic N‐Ras mutants, better therapeutic targets can potentially be developed.Support or Funding InformationNational Science Foundation, Northeastern University
Probiotic Escherichia coli Nissle 1917 Uses Zinc Transporters and the Siderophore Yersiniabactin to Acquire Zinc in the Inflamed Gut and Outcompete Salmonella Typhimurium
Authors: Behnsen Judith , Liu Janet , Valeri Maria , Hoover Evelyn , Tjokrosurjo Joshua , Montaldo Nicola P. , Treacy‐Abarca Sean , Garibay Odila , Gilston Benjamin A. , Edwards Robert A. , Chazin Walter , Skaar Eric P. , Raffatellu Manuela 
Published: November 2023

ObjectiveSalmonella enterica serovar Typhimurium is a leading cause of foodborne illness worldwide. The pathogen elicits an inflammatory environment in the gut in which it can thrive. S. Typhimurium infection is ameliorated by the probiotic Escherichia coli Nissle 1917 (E. coli Nissle), partly because E. coli Nissle expresses multiple iron transporters and siderophores, and outcompetes S. Typhimurium for iron. Here we sought to determine whether acquisition of the essential metal nutrient zinc also contributes to the probiotic activity of E. coli Nissle during S. Typhimurium infection and in a non‐infectious colitis model.MethodsWe generated S. Typhimurium and E. coli Nissle strains deficient in zinc uptake systems and compared their growth in zinc‐limited media. To test the importance of zinc transport systems in vivo, inflammation was elicited by dextran sodium sulfate and competitive infections with E. coli Nissle wild type and E. coli Nissle mutant strains were carried out. Furthermore, we infected streptomycin pre‐treated mice with S. Typhimurium and co‐administered wild type or mutant E. coli Nissle strains. To further investigate the mechanism by which E. coli Nissle reduces S. Typhimurium colonization, we used mice lacking the zinc‐sequestering protein calprotectin.ResultsWe found that both S. Typhimurium and E. coli Nissle use the transporters ZnuABC and ZupT to acquire zinc in zinc‐limiting conditions. E. coli Nissle, but not S. Typhimurium, could additionally use the siderophore yersiniabactin for zinc acquisition. Yersiniabactin‐mediated zinc uptake rendered E. coli Nissle more resistant to zinc sequestration by the host protein calprotectin in vitro. Similarly, yersiniabactin was required for efficient colonization of E. coli Nissle in an inflamed environment in vivo. In competition with S. Typhimurium, E. coli Nissle required zinc transport systems and the presence of calprotectin to reduce S. Typhimurium colonization levels. In calprotectin‐deficient mice, yersiniabactin was dispensable for E. coli Nissle colonization, and E. coli Nissle was unable to reduce S. Typhimurium colonization.ConclusionsOur findings demonstrate that zinc acquisition plays an important role in the competition between E. coli Nissle and S. Typhimurium. E. coli Nissle can use three mechanisms to acquire zinc: the zinc transporters ZnuABC and ZupT, as well as yersiniabactin, a siderophore that has been shown previously to bind other transition metals beside iron. Zinc acquisition systems and the host protein calprotectin contribute to E. coli Nissle's beneficial effects and the reduction of S. Typhimurium colonization of the inflamed gut.
Linking the Cytosolic Phenylalanine Pathway, and the Auxin Biosynthetic Pathway
Authors: Garcia Alekzander , Qian Yichun , Lynch Joseph , Dudareva Natalia 
Published: November 2023

The shikimate pathway is a biosynthetic pathway that leads to the production chorismate, the precursor of the aromatic amino acids. In production of phenylalanine (Phe) and tyrosine (Tyr), chorismate is first converted to prephenate by the enzyme chorismate mutase. The predominantly expressed isoform of chorismate mutase (CM1) is located in the plastids; however, a second isoform (CM2) is present in the cytosol, but the role of this cytosolic isoform in plants is still unknown. Preliminary results from Petunia hybrida, cv Mitchell flowers that overexpress CM2 suggest a possible decrease in plastid content. As auxin is known to inhibit the formation of leucoplasts and amyloplasts, we hypothesize a metabolic link between cytosolic Phe production via CM2 and the tryptophan‐dependent auxin biosynthetic pathway. In this study, CM2 expression in the previously developed overexpression lines was analyzed by qRT‐PCR. Despite confirmed overexpression of the gene, downstream products Phe and total anthocyanins were decreased. In contrast, there was a significant increase of phenylpyruvate, the immediate precursor of Phe and an amino‐acceptor in auxin biosynthesis. Plastid content was quantified by starch assays and found to be decreased in flowers of CM2 overexpression lines. Finally, the auxin indole‐3‐pyruvic acid (IPA) and indole‐3‐acetic acid (IAA) were increased as compared to the wildtype. Our results demonstrate a connection between the tryptophan‐dependent auxin biosynthetic pathway and the CM2‐dependent cytosolic Phe biosynthetic pathway.Support or Funding InformationNSF‐REU for Molecular and Biochemical Analysis of Proteins at Purdue University 2016