bluuebunny/crossref_metadata_2025 huggingface dataset:
Understanding conformational changes during translesion synthesis: in silico studies of DinB
Authors: Perez Elevit , Benitez Benedetta Sampoli
Published: November 2023

Y‐family DNA polymerases are a class of error‐prone polymerases able to perform DNA translesion synthesis. E. coli DNA Polymerase IV (also called DinB from the name of the gene) is a Y‐family DNA polymerase implicated in stress‐induced mutagenesis. As other DNA polymerases, DinB has the general architecture of a right hand, with palm, thumb and fingers subdomains. In addition, Y‐family polymerases have an extra domain, namely the “little fingers” and have a more solvent accessible active site than their high‐fidelity replicative counterparts. Kinetics studies have shown that DinB is particularly efficient at bypassing damaged guanosines, such as the N2‐furfuryl‐dG, and even more efficient in lesion bypass on damaged DNA than in replication of undamaged DNA. Recent hydrogen exchange experiments have suggested that the protein undergoes a conformational change in the presence of certain DNA damages on the template strand, but not others, implying a possible mechanism of bypass discrimination. However, the exact nature of such movement is not well characterized. Using molecular dynamics (MD) simulations we explored different DNA/protein/incoming nucleotides combinations to dissect the protein conformational change. The CHARMM‐gui generator was used to generate a total of 3 systems: a binary system (with DNA and protein only) a ternary system (with DNA and correct incoming nucleotide), and a ternary system with a O6‐methylated guanine on the templating strand), a damage which DinB cannot bypass. Each system was run for a total of 10 ns using the NAMD software package. Visualization and analysis of the systems was performed using the Visual Molecular Dynamics (VMD) application. As predicted, there was little movement in the ternary system with the correct incoming nucleotide, suggesting the polymerase is in a closed conformation therefore active. While we observed some large‐scale movement in the binary system, suggesting that DinB prefers an open, non‐active conformation in the absence of the substrate, we did not see significant subdomain movements the O6‐methylated system, which was predicted to be similar to the binary system. The results point us toward a new direction, implying that instead large conformational changes, this polymerase achieves a catalytically competent state by small local movement occurring at specific sites.

Support or Funding Information

This work was funded by the Rose M. Badgeley Residuary Charitable Trust and the Sister Colette Mahoney Science Research Grant.
DNA Cytosine Methyltransferase Promotes Stationary Phase Fitness in Escherichia coli
Authors: Kambhampati Ooha , Finnerty‐Haggerty Lara , Huss Rebecca , Knapp Rachel , Militello Kevin
Published: November 2023

In Escherichia coli, DNA cytosine methyltransferase (Dcm) methylates the second cytosine in the sequence 5′CCWGG3, where W is A or T. Dcm is in an operon with the very short patch repair (vsr) gene, an endonuclease that repairs T‐G mismatches caused by the deamination of 5‐methylcytosine. The role of cytosine DNA methylation and the dcm operon in E. coli is unknown and remains an enigma. Growth curve analyses of wild‐type, dcm knockout and vsr knockout strains at both 37°C and 20°C indicated no effect of the dcm or vsr genes on growth rate. Our laboratory's microarray data comparing expression patterns between a wild‐type and dcm knockout strain indicated numerous gene expression changes occur at stationary phase. Similar results were obtained from microarray experiments comparing gene expression differences between untreated cells and cells treated with the cytosine DNA methylation inhibitor 5‐azacytidine. In summary, the microarray experiments suggest a role for Dcm in stationary phase but not logarithmic phase. Then, we tested whether there was an advantage of either the dcm or vsr genes during stationary phase. We performed competition experiments between the wild‐type strain and the dcm knockout strain or vsr knockout strain to determine which strain would persist during long term stationary phase. In the wild‐type/dcm knockout strain competition at 20°C, the wild‐type strain outcompeted the dcm knockout strain; thus the cells lacking the dcm gene have a disadvantage over wild‐type cells. The addition of a plasmid containing a functional copy of the dcm gene to the dcm knockout strain reversed the loss of fitness in the dcm knockout strain. In the wild‐type/vsr knockout strain competition experiment at 20°C, loss of the vsr gene resulted in a modest loss of fitness. Overall, our data indicate that dcm and the dcm operon promote stationary phase fitness. We aim to determine the mechanism for Dcm‐dependent increases in stationary phase fitness which may include changes in the composition of the transcriptome and/or mutation rate.

Support or Funding Information

Geneseo Foundation
Mechanism of PWI Domain Binding to Nucleic Acids
Authors: Szymczyna Blair R , Chanzu Harry , Jandaghijafari Nafiseh , Lopez Elizabeth I , Flores Denise
Published: November 2023

PWI and PWI‐like motifs are four‐helix bundles that are found in several proteins predicted or known to be involved in the regulation of pre‐messenger RNA processing, such as constitutive and alternative splicing and the 3′‐end modification of transcripts. Examples include SR‐related nuclear matrix protein at 160 kDa (SRm160) and RNA Binding Protein 25 (RBM25). The spliceosome also contains several proteins that contain PWI motifs, including Pre‐messenger RNA Processing proteins PRP2, PRP3 and PRP22. The PWI motif is named for a nearly invariant Proline(P)‐Tryptophan(W)‐Isoleucine(I) sequence, although PWI‐like motifs lack this tripeptide sequence and are homologous only in structure. Sub‐families of canonical PWI motifs can be defined by sequence homology, the position of the motif within the protein and the sequence of an adjacent region rich in positively charged (basic) amino acid residues. The PWI motif and the basic region constitute the PWI domain, and both elements are essential for optimal binding to all nucleic acid types. Although RNA is believed to be the natural target, PWI domains non‐specifically bind single‐ and double‐stranded DNA and RNA. The individual roles of the PWI motif and the basic region in the nucleic acid binding mechanism are still not clear. Several biophysical techniques, including electrophoretic mobility shift assays, gel filtration chromatography and fluorescence and nuclear magnetic resonance spectroscopies, are being employed to investigate the mechanism and stoichiometry of PWI domain binding to nucleic acid targets. Our investigation of several PWI domains reveal that both protein‐protein and protein‐nucleic acid interactions participate in the cooperative nucleic acid binding mechanism.

Support or Funding Information

Start‐up funds from Western Michigan University to BRS.
Unique Structures in the 3′ UTR of Blackcurrant Reversion Nepovirus Genomic RNA 1 Promote Translation Initiation
Authors: Baquero‐Galvis Laura D. , Shields Elizabeth , Filbin‐Wong Megan E.
Published: November 2023

Canonical translation initiation is promoted and regulated by an interaction between proteins bound to a cap structure (a methylated guanosine triphosphate, m7G, as the 5′ nucleotide) at the 5′ end and those bound to a polyadenylate [poly(A)] tail at the 3′ end of a messenger RNA (mRNA). This end‐to‐end interaction contributes to the recruitment, positioning, and activation of the translation machinery. Blackcurrant reversion nepovirus (BRV) has two bipartite genomic RNAs (RNA 1 and RNA 2) that do not contain 5′ cap structures, but do have 3′ poly(A) tails. Hence, the mechanism of end‐to‐end interaction in BRV has not been clearly defined. In other cap‐less, positive‐sense RNA plant viruses, structured RNA in the 5′ and 3′ untranslated regions (UTRs) bind via proposed RNA‐RNA kissing‐loop interactions between a 5′ hairpin loop and a 3′ cap independent translation enhancer (CITE). Likewise, the mechanism by which the 5′ and 3′ UTRs recruit protein synthesis machinery is directly related to their primary, secondary and higher‐order structure. Considering that the end‐to‐end communication and recruitment of translation factors is dependent upon RNA structure, our main goal focuses on determining the secondary and possible tertiary structures of the 3′ CITE structure in the genomic RNAs found in the BRV. Toward that goal, we have in vitro transcribed the minimal BRV RNA 1 CITE sequence, and probed our purified RNA with a series of covalent nucleobase, sugar and backbone modifiers. Combining our results, we have found a unique secondary structure for the BRV RNA 1 CITE that differs from a previously proposed structure based upon in silico thermodynamic folding predictions. This structure likely plays an important role regulating protein synthesis of viral proteins encoded by the RNA 1 message by either facilitating end‐to‐end interaction with the 5′ UTR and/or directly recruiting important translation protein machinery.

Support or Funding Information

MSU Denver College of Letters, Arts & Sciences Mini‐grant, MSU Denver Undergraduate Research Grant, MSU Denver Center for Advanced STEM Education Undergraduate Research Grant
Analysis of Eukaryotic Translation Initiation Factor (eIF) Phosphorylation by Mass Spectrometry
Authors: Beglinger Katherine , Villa Nancy , Andaya Armann , Leary Julie , Fraser Christopher
Published: November 2023

Translation initiation is the rate‐limiting step of protein synthesis and is regulated by eukaryotic initiation factors (eIFs), which work together to recruit a mRNA to the ribosome and locate the initiation codon. An additional layer of regulation of this pathway is likely influenced by post‐translational modifications of initiation factors, specifically phosphorylation. While several global studies have identified extensive numbers of phosphorylation sites, it is still unclear which sites are important in regulating translation. The objective of this study to to identify which phosphorylation sites are key in regulating translation initiation and to determine how phosphorylation regulates the function and structure of translation initiation factors.

We have started to determine which phosphorylation sites on the cap binding complex, eIF4F, are important in regulating translation. To build on our previous work, we are recombinantly expressing and purifying this complex from HeLa cells that have been treated with various kinase inhibitors. We are quantifying site‐specific phosphorylation stoichiometries using a novel cerium oxide dephosphorylation and tandem mass tagging (TMT) method followed by nanoLC‐MS/MS. Identified phosphorylation sites that change in response to kinase inhibitors will be characterized using functional and structural assays.

The translation initiation factor eIF4B has been shown to have an important role in translation initiation by stimulation the unwinding of mRNA in preparation of recruitment to the ribosome. Prior studies have identified a phosphorylation site at Serine 422 of eIF4B. This site is known to be important in stimulating translation initiation as well as increasing its affinity to eIF3, however the mechanism in which this site works is poorly understood. To study the role of this phosphorylation site, we expressed and purified recombinant phosphomimetic (Ser422Glu) and null phosphorylation (Ser422Ala) mutants from SF9 cells. We are employing ion mobility mass spectrometry to study the structural changes that occur when eIF4B is phosphorylated. In addition, we are using a reconstituted translation initiation system to understand the effect of eIF4B phosphorylation on eIF4A‐dependent helicase activity and mRNA recruitment.

Support or Funding Information

NIH T32 Training Grant (T32CM113770) in Chemical Biology, NIGMS (NIH) RO1 GM092927, The Floyd and Mary Schwall Fellowship in Medical Research
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.
Molecular Dynamics Simulations Support Multiple Binding Sites for Phospholamban on SERCA
Authors: Smolin Nikolai , Robia Seth L
Published: November 2023

Ion‐motive ATPases use the energy of ATP hydrolysis to transport ions across membrane bilayer against a concentration gradient. Here we investigate the functional regulation of the sarco(endo)plasmic reticulum calcium ATPase (SERCA), which plays a key role in cellular Ca handling. In the heart, the activity of the SERCA is regulated by its inhibitory partner phospholamban (PLB). PLB has been shown to inhibit the transporter by binding to the SERCA M6 helix, but this site is not available when SERCA is bound to Ca. Our previous fluorescence resonance energy transfer (FRET) experiments suggested that SERCA Ca binding causes PLB to translocate from the known binding site to a putative secondary site. Based on an analogous interaction of the sodium/potassium ATPase (NKA) with a regulatory partner (FXYD10), we hypothesized that PLB could interact with the M9 helix on SERCA after leaving the canonical binding site. In the present study we performed molecular dynamics simulations to compare the energetics of binding of PLB to candidate sites on SERCA. We created models of the regulatory complex using structural information from available crystal structures of the PLB‐SERCA and FXYD10‐NKA complexes. Quantification of the interaction energies of these models suggested PLB can form a stable complex with both the canonical (−856 kJ/mol) and the proposed secondary site (−510 kJ/mol). In contrast, docking of PLB to the M3 helix on the opposite side of SERCA shows a much less favorable interaction energy (−108 kJ/mol). We also used umbrella sampling to quantify the free energy of PLB bound to several candidate binding sites on SERCA transmembrane domain. The results provide insight into the relative affinity of PLB for plausible sites of interaction on SERCA. Overall, the molecular dynamics simulations support a 2‐site model in which PLB interacts first with the high affinity conventional site and then relocates to a nearby secondary site when SERCA binds calcium.
Structural differences associated with DNA binding of p53 family member proteins
Authors: Mavodza Grace R , Fang Yayin , Sherif Zaki
Published: November 2023

Introduction

p53 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.

Methods

The 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.

Results

The 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.

Conclusion

The 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 Information

This 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 Information

Detroit 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 Information

Johns 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 Information

KS 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 Information

This 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 Information

This 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 Information

RI‐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 Information

National 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

Objective

Salmonella 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.

Methods

We 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.

Results

We 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.

Conclusions

Our 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.
Hepatic Fat Accumulation Regulates Carnitine Palmitoyltransferase 1 (Cpt1a) Expression through Coordinated Epigenetic Mechanisms
Authors: Moody Laura , Jung Paul Mark , Kriska Adam , Chen Hong , Pan Yuan‐Xiang
Published: November 2023

Carnitine palmitoyltransferase 1 (Cpt1a) is a rate‐limiting enzyme that mediates the transport of fatty acids into the mitochondria for subsequent beta oxidation. Cpt1a is highly regulated at the transcriptional, translational, and substrate level. Epigenetic mechanisms regulating Cpt1a expression were investigated in the liver of rats fed a high fat diet. Hepatic Cpt1a mRNA was increased in response to high fat. High fat diet reduced DNA methylation and increased histone 3 lysine residue 4 dimethylation (H3K4Me2) both upstream of and within the promoter region of Cpt1a. This was accompanied by increased binding of C/EBPβ directly downstream of the Cpt1a transcription start site within the first intron. In addition to the transcriptional regulation of Cpt1a, expression of miR‐499, a predicted repressor of both Cpt1a and C/EBPβ, was also reduced. Treatment of rat hepatoma H4IIEC3 cells with non‐esterified fatty acid (NEFA) confirmed the correlation between the upregulation of Cpt1a gene expression and cellular fat accumulation. While H4IIEC3 cells transfected with non‐specific microRNA upregulated Cpt1a expression at high NEFA concentrations, exogenous overexpression of miR‐499 cells abolished this increase, resulting in suppressed beta oxidation and elevated lipid accumulation. We conclude that hepatic fat increases Cpt1a expression through a highly coordinated epigenetic mechanism including histone tail modifications and reduction of DNA methylation at specific chromatin locations, C/EBPβ transcription factor recruitment, as well as post‐transcriptional regulation by miR‐499. The spatial and temporal coordination of these epigenetic events at the Cpt1a locus will be further investigated to characterize gene regulation in fat metabolism.
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 Information

NSF‐REU for Molecular and Biochemical Analysis of Proteins at Purdue University 2016
Engineering biochemical bypass to photorespiration to improve photosynthesis and crop production
Authors: South Paul , Ort Donald R
Published: November 2023

Worldwide nearly 1 billion people are affected by hunger every day. As climate changes globally and human population increases, traditional methods of crop improvement have become less effective in adapting and improving agricultural production. In C3 crops such as wheat and rice approximately 25% of the fixed carbon dioxide is lost to photorespiration. Photorespiration is an energy expensive metabolic pathway that recycles toxic compounds produced by RubisCO oxygenation reactions. Reducing photorespiratory yield losses by 5% (i.e., to 31% for soybean and 15% for wheat) would be worth ~$500 million annually in the United States. Although photorespiration is tied to other important metabolic functions, the benefit of improving its efficiency appears to outweigh any potential secondary disadvantages. Synthetic biology has provided new opportunities in altering photorespiratory metabolism to improve photosynthetic efficiency. Indeed metabolic bypasses to photorespiration have been generated and have demonstrated improvements in growth. Using a synthetic biology approach we have assembled a series of multigene constructs that contain alternate biochemical pathways to bypass photorespiration. In addition, we designed a screen based approach to test a range of standardized parts (promoters, terminators) in the model plant Nicotiana tabacum. We have successfully transformed in large multigene constructs and have demonstrated detectable gene expression and improvements in photosynthesis and biomass in replicated greenhouse and field trails. Determining robust photorespiratory bypass constructs can provide insight into next generation crops and our utilization of standard parts provide a new tool kit for plant synthetic biology to engineer improvements in photosynthetic efficiency.

Support or Funding Information

Bill and Melinda Gates Foundation (OPP1060461) titled “RIPE‐Realizing Increased Photosynthetic Efficiency for Sustainable Increases in Crop Yield.” United States Department of Agriculture‐Agriculture Research Service.
Liver X Receptor (LXR) Activation Decreases Chronic Myelogenous Leukemia Cell Viability and Alters the Expression of Antiapoptotic and Cholesterol Genes
Authors: Andersen Catherine J. , Dupree Lydia , Doerr Adam , Cintron‐Rivera Layra , Murray Kristina , Ragonesi Nicholas
Published: November 2023

Liver X receptors (LXRs) are oxysterol‐activated nuclear receptors involved in the regulation of cholesterol transport, glucose metabolism, and inflammatory responses. Recently, LXRs have been shown to possess anti‐cancer effects in a variety of cancer types, in part due its capacity to deplete essential lipids required for cell proliferation. However, the role of LXRs in chronic myelogenous leukemia (CML) has yet to be elucidated. In this study, we investigated the effects of LXR activation on the human erythroleukemia K562 cell line – a model for CML. K562 cells were treated with the LXR agonist TO901317 (0μM, 1μM, 5μM, 10μM) to determine dose‐ and time‐dependent effects on cell viability, cell number and size, and the mRNA expression of genes related to cholesterol metabolism (ATP‐binding cassette transporter A1 (ABCA1), low‐density lipoprotein receptor (LDLR), and HMG‐CoA reductase (HMGCR)) and anti‐apoptosis (B cell leukemia‐xL (Bcl‐xL)). TO901317 treatment significantly increased ABCA1 mRNA expression at 24hr and 72hr, with greater expression at the lowest treatment concentration (1μM). HMGCR and Bcl‐xL mRNA expression was dose‐dependently reduced by LXR activation after 72hr, as were decreases in total cell number. Average cell size similarly decreased from 24hr to 72hr with 10μM TO901317 treatment. Further, K562 viability was decreased by TO901317 treatment at all concentrations at 24hr, 48hr, and 72hr, with dose‐dependent effects observed at 48hr only. Together, these findings suggest that LXR agonism decreases CML cell viability and number, and that these changes may be related to modulation of cholesterol metabolism and apoptotic pathways. Current studies are underway to further investigate these pathways, in addition to the capacity of TO901317‐induced LXR activation to modulate cellular differentiation.

Support or Funding Information

The Science Institute of the College of Arts and Sciences and Faculty Research Committee, Fairfield University
Structural Mechanism of Viral Tethering by the Anti‐Viral Protein BST‐2
Authors: Ozcan Kadir , Berndsen Christopher
Published: November 2023

BST‐2/tetherin is a human extracellular transmembrane protein that serves as a host defense factor against HIV‐1, HIV‐2, SIV, and other viruses by inhibiting viral budding. Structurally, BST‐2 is a homo‐dimeric coiled‐coil that is connected to the host cell membrane by N and C terminal transmembrane anchors. The coiled‐coil structure of BST‐2 is strengthened by three disulfide bonds between cysteine residues of the monomers. The structural mechanism of viral budding and tethering as mediated by BST‐2 is not clear. In order to describe the mechanism of viral tethering, we created a physiologically relevant model of BST‐2 embedded in a membrane. We also post‐translationally modified the BST‐2 with glycosylation at the known sites and equilibrated the model to create a stable structure of membrane associated BST‐2. Through steered molecular dynamics, viral tethering was simulated to illustrate the structural transitions in BST‐2 between the host cell membrane associated BST‐2 and the cell‐virus membrane bridging form. We find that the membrane and disulfides did not affect the structural transitions of BST‐2. Regardless of the disulfides, glycosylation, or presence of the membrane, BST‐2 consistently unwound at non‐ideal heptads. Therefore, sequence encoded weaknesses in the BST‐2 coiled‐coil region allow for a smooth transition between the host cell membrane associated BST‐2 and cell‐virus membrane bridging form.

Support or Funding Information

NSF‐REU award CHE‐1461175 the 4‐VA organization
Small Molecule and Lipid Binding Mechanisms of Sortilin and Vps10 Indicated in Altered Trafficking Patterns and Membrane Fusion
Authors: Sparks Robert , Fratti Rutilio A.
Published: November 2023

This study focuses on the regulation of sortilin/Vps10p‐directed protein trafficking. Human sortilin is a Vps10 domain‐containing transmembrane receptor that interacts with Apo B100‐containing VLDL, PCSK9 and LDL in the lumen of hepatic secretory vesicles. The Vps10 domain name comes from the yeast orthologue Vps10p, a carboxypeptidase Y (CPY) sorting receptor responsible for trafficking proteins to the vacuole in yeast. We have recently shown that vesicular VLDL contains phosphatidylinositol (3,4,5)‐trisphosphate (PIP3). Furthermore, we found that sortilin directly binds PIP3 using liposome binding assays as well as surface plasmon resonance using nanodiscs. We found that small molecules designed to bind human sortilin were also effective in yeast, leading to altered protein sorting to the yeast vacuole and secretion of CPY. Additional efforts using liposome floatation assays and microscale thermophoresis show that Vps10p and sortilin bind to additional membrane lipids. We hypothesize that differential binding different types of membrane lipids regulates Vps10‐controlled protein sorting. Further characterization of sortilin probes in yeast identified a variety of potential protein targets including the Rab7 orthologue Ypt7. This late endosomal GTPase is required for retrograde trafficking to the Golgi, anterograde traffic to the endolysosomal pathway, and vacuole homotypic fusion. Both deletion of VPS10 or binding of the receptor with sortilin‐targeting compounds led to defective Ypt7 trafficking to vacuoles and inhibited homotypic vacuole fusion. To complement direct binding studies, yeast Vps10p homologues have been identified and structural research performed to better understand binding features of Vps10‐like luminal domains. Elucidation of the relationship between specific luminal binding interactions of sorting receptors and vesicle trafficking predestination should allow better understanding of mechanistic interactions of yeast Vps10p with cytosolic trafficking factors and components.

Support or Funding Information

NIGMS R01‐GM101132