bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of Gravin in Compartmentalization of cAMP Signaling
Authors: Thompson Gonowolo Faith , Grove Bryon
Published: June 2021

A‐kinase anchoring proteins (AKAPs) play a critical role in signaling by targeting cAMP dependent signaling complexes to specific cellular sites. Gravin, a multivalent AKAP that targets PKA and possibly PDE4 to the cell membrane, undergoes redistribution to the cytosol in response to elevated [Ca2+]i suggesting it may play a role in cross‐talk between Ca2+ and cAMP dependent pathways. We have shown previously that gravin redistribution alters PKA activity in cytosolic and membrane regions, but the effect of gravin redistribution on cAMP levels in these compartments is unknown. To begin assessing this, we used a FRET based cAMP biosensor (lck‐EPAC2‐camps) targeted to the membrane to determine the effect of gravin expression on cAMP levels. Treatment of cells expressing the biosensor and gravin with IBMX showed a greater increase in cAMP levels compared to cells lacking gravin. In addition, cells expressing gravin and the biosensor, showed a significantly higher increase in cAMP when treated with rolipram (PDE4)/forskolin compared to cilostamide (PDE3)/forskolin. These experiments indicate that gravin expression affects cAMP levels at the membrane following PDE inhibition and sets the stage for determining the role of gravin redistribution in regulating cytosolic and membrane cAMP levels. EPAC2‐camps was a gift from Drs. V. Nikolaev and M. Lohse. Supported by NIH grants 1P30GM103329 and 1S100D016250‐01.
Toll‐like receptor 10 Expression in Chicken, Cattle, Pig, Dog, and Rat Lungs
Authors: Balachandran Yadu , Knaus Steven , Singh Baljit
Published: June 2021

Toll‐like receptors (TLRs) are conserved immune receptors that play critical roles in innate immunity and are known as “gate keepers” of immune system. TLR10 is identified as type 1 plasma membrane protein but the identity of its ligand remains unclear. Till date, no data available on tissue and cell specific expression of TLR10 in normal and inflamed lungs of domestic animal species, and rat, which is commonly used as a model to study human diseases. First, we used homologous sequence alignment of published sequences of TLR10 from cattle, pig, dog, chicken and rat and the peptide alignment with antibody sequence to determine that a commercially available TLR10 antibody may be suitable for detection of TLR10. Western blotting of total lung protein extracts from cattle, dog, pig and chicken showed a band in 85‐ 100kDa region indicating the TLR10 protein. The immunohistochemistry, immunoelectron microscopy and confocal microscopy data show TLR10 expression in vascular endothelium and smooth muscle actin of control and inflamed animals. Further, we detected that basal expression of TLR10 in bovine neutrophil is altered upon treatment with E. coli lipopolysaccharide. These data show TLR10 expression in the lungs of these mammalian species and that activation of bovine neutrophils alters the expression of TLR10
Cross‐Course Laboratory Collaboration in Cell Biology and Organic Chemistry II Leads to Increased Knowledge of Science Content and Process
Authors: Carle Sigrid , Miller Justin , Mowery Patricia , MaKinster James
Published: June 2021

To expose students to interdisciplinary research, provide them with a greater sense of ownership over their work, and model a real research laboratory experience, we linked students in Organic Chemistry II and Cell Biology by focusing on the synthesis and testing of anticancer compounds. To this end Organic Chemistry II students synthesized potential anticancer compounds, known as histone deacetylase inhibitors (HDACi), which were tested for cell cytotoxocity by Cell Biology students.

Organic Chemistry II students used standard organic techniques to synthesize novel depsipeptides: peptide bond formation, acyl transfer mechanism, esterification, protecting groups, spectroscopic characterization, and purification techniques. Cell Biology students determined each depsipeptide's IC50 value (or the concentration which kills 50% of the cells) using an MTT assay. Depsipeptides that were cytotoxic were investigated for their ability to induce apoptosis using a CaspACETM Assay.

Students in both courses showed statistically significant increases in self‐perceived learning gains in the following subscales: conceptual understanding, integration, and collaborative scientific process understanding. Increases in learning gains were similar between the two courses and were similar for both years of implementation. Organic Chemistry II students also showed gains in individual science process skills while the Cell Biology students did not. Interestingly, there was no significant change in student interest during the semester, in either course, for the first two years of implementation.
Interdisciplinary Learning Communities at Linfield College Increase Interactions Among Students and Participation in Science
Authors: Kruchten Anne
Published: June 2021

At Linfield College, faculty from multiple disciplines have developed novel STEM programing that includes a week‐long pre‐orientation “boot camp” experience for incoming students, an ongoing learning community program that engages peer mentors in communities working on interdisciplinary problems, and an interdisciplinary seminar series that incorporates both a traditional invited speaker series and preparatory experiences that help undergraduates engage with the advanced material presented by invited speakers.

We report here our progress towards our three initial goals. First, we have examined our student population demographics to develop a baseline understanding of the gender, race and ethnicity, first generation status, and transfer status of our students. We use this information to develop programs to retain all students, but especially those who are at risk of leaking from the pipeline. Second, we encourage students and faculty to engage in interdisciplinary research and problem solving in our boot camp, our learning communities, and our seminar series. Third, we have scaled up from ten students per year in a boot camp setting to fifty plus students in learning communities engaging first year students, transfer students, and upper division peer mentors in research projects and discussions. Through pre‐ and post‐ surveys and conversations, we find that students identify more strongly with the experience of “being a scientist” and develop more interdisciplinary connections and communications between first year and upper division students. We believe that the development of a science community through these programs will lead to increased retention of students in our programs.

Supported by Linfield College, the Rose E. Tucker Charitable Trust, and the Hearst Foundations Inc.
Exploring ethanol: Engaging first‐year undergraduates in the biochemistry of ethyl alcohol
Authors: Joyce Glenna , Howard Rebecca
Published: June 2021

We sought to expose incoming Skidmore College students to principles and applications of biochemistry through a series of experiment‐based activities in an interdisciplinary first‐year seminar course, “Under the Influence: Alcohol in Science and Society.” To provide the diverse student population with first‐hand experience of ethanol's biochemical origins, we developed simple fermentation and distillation protocols using grocery store supplies and standard lab glassware. In subsequent weeks, students used agar plates, spirit lamps, baking supplies, and fruit flies to characterize ethanol as an antiseptic, fuel, solvent, and intoxicant. Activities were deliberately open‐ended to engage students in experimental design as well as laboratory materials and skills. All protocols were developed and validated during a summer faculty/student collaborative research project, supporting pedagogical as well as career development goals. Sample data and outcomes indicated appreciable student engagement in the process as well as content of the activities, and supported new models for accessible biochemistry active‐learning opportunities for a broad range of undergraduates.
Checkpoint Regulation of Replication Forks at Sites of DNA Damage
Authors: Iyer Divya , Rhind Nick
Published: June 2021

Statement of methods:

We have used single‐molecule DNA combing to study how the checkpoint regulates individual origins and forks in response to DNA damage.

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Summary of results

The S‐phase DNA damage checkpoint slows replication and allows cells to replicate damaged DNA, but how slowing relates to damage tolerance remains unclear. The standard explanation — that checkpoint delays cell‐cycle progression until the damage can be repaired — does not seem to apply in the case of DNA replication. Instead, replication proceeds in the presence of the damage, but at a slower rate. Replication slowing can be achieved by inhibition of origin firing, slowing of fork progression or a combination of both. In bulk assays, 1uM 4NQO and 0.03% MMS slow S phase to a similar extent. However the combing data shows that 4NQO slows replication by reducing origin firing rate, while MMS slows S phase by stalling forks and reducing the fork rate. While reduction in origin firing rate is checkpoint dependent, reduction in fork rate has checkpoint dependent and independent components. We are currently investigating the mechanistic difference in response to different damaging agents and how the checkpoint regulates fork components to achieve replication slowing

Another key question is how forks replicate quickly through damaged DNA in the absence of the checkpoint. The MMS‐induced lesions we use are believed to stop replicative polymerases. Nonetheless, in the absence of the checkpoint, replication forks pass them without any apparent delay. This bypass seems to be mainly dependent on recombination and translesion polymerase based synthesis. We are working on how forks bypass polymerase‐stalling lesions and what effect this bypass has on genomic stability.

Funding Source:

NIHGMS
Processing of 3′ Phosphate DNA Termini by the Herpes Simplex Virus‐1 DNA Polymerase: a Novel 3′ Phosphatase Activity
Authors: Boehmer Paul , Rupesh Kanchi Ravi
Published: June 2021

3′ phosphate terminated DNA ends may arise directly following breakage of the phosphodiester bond or by enzymatic processing of DNA damage including βδ elimination catalyzed by NEIL 1 and NEIL 2 during base excision repair. We have previously shown that HSV‐1 has the capacity to perform uracil‐initiated base excision repair. Specifically, we demonstrated that abasic sites generated by the HSV‐1 UL2 glycosylase are incised by human AP endonuclease and a novel 5′ dRP lyase activity inherent in the UL30 DNA polymerase to generate a 1 nt gap that is subsequently filled by UL30 and ligated by human DNA ligase IIIα/XRCC1 complex. We have explored the ability of UL30 to directly process 3′ phosphate terminated DNA. Our data show that UL30 is able to degrade 3′ phosphate terminated ends of both ssDNA and primer‐templates albeit at lower rates and to a lesser extent than 3′ hydroxyl ends. To determine whether this is due to 3′‐5′ exonuclease or direct 3′ phosphatase, we designed experiments that utilize incorporation of chain terminating dideoxynucleotides to distinguish between exonuclease activity and phosphatase activity. Our data show that UL30 has the ability to extend 3′ phosphate terminated DNA without removal of the terminal nucleotide, indicating an inherent phosphatase activity. Moreover, we show that UL30 is able to directly remove 32Pi from 3′ phosphate terminated DNA, directly demonstrating 3′ phosphatase activity. This allows the enzyme to process 3′ phosphate terminated DNA by direct removal of the phosphate group to permit subsequent nucleotide polymerization. This activity may provide a critical function for the processing of damaged DNA that involves the generation of 3′ phosphate ends to ensure completion of viral DNA replication.
Two potential sites of protein‐protein interaction within the Mycobacterium tuberculosis DNA gyrase complex
Authors: Wright Nicholas , Peavey Jeremy , Borg Devin , Raab Ronald , Johann Timothy
Published: June 2021

Recent World Health Organization studies have shown that one‐third of the global population are carriers of latent or active Mycobacterium tuberculosis (TB) infection. With the emergence of antibiotic resistant TB, it is crucial to understand the current drug targets in the treatment of this disease. One of these targets is DNA gyrase, the target of fluoroquinolones and a protein vital to cell function and replication. The goal of this project is to characterize binding interactions between the GyrA and GyrB subunits of M. tuberculosis DNA gyrase. Molecular modeling of the DNA gyrase complex was performed by superimposition of solved GyrA and GyrB M. tuberculosis structures (PDB ID's 3IFZ, 3M4I) over a solved structure of the highly homologous S. pneumoniae Topoisomerase IV protein/DNA complex (PDB ID 3K9F). Structural overlay was performed by the Magic Fit tool in SwissPdb Viewer. The resulting model was used to identify potential sites of charge‐charge interaction between the GyrA and GyrB subunits. Molecular cloning, site‐directed mutagenesis, functional, and structural assays have been utilized to investigate the two identified potential sites of interaction. Initial results from supercoiling assays suggest that the GyrB K658 residue is important for function, as well as a possible interaction occurring between the GyrA R26 residue and the GyrB residues D684 and E685. Gel shift and pull down assays investigating the strength of interaction between wild type and mutant subunits are ongoing.
Cell Death Mechanisms Induced by Altered Telomerase RNA Template Sequence
Authors: Hernandez‐Sanchez Wilnelly , Taylor Derek
Published: June 2021

Due to an inability of DNA polymerases to replicate the extreme end of chromosomes, the telomeres of most somatic cells shorten with each cell division, leading to a state of senescence induced by critically short telomeres. To avoid this, an enzyme called telomerase is required for the synthesis of telomere DNA. In cancer cells, telomerase is upregulated allowing the cancer cells to escape senescence. Due to this unique property of cancer cells, targeting telomerase is a viable therapeutic approach. Telomerase uses an internal RNA template to reverse transcribe telomere DNA with a specific repeating hexameric sequence. Telomere DNA is bound and protected by the shelterin complex. Binding of this complex to telomere DNA is very specific, such that a single nucleotide alteration in telomere DNA sequence results in reduced recognition and binding efficiency. Abrogation of shelterin protein binding induces an immediate DNA damage response. We aim to exploit this mechanism as a putative chemotherapeutic approach by investigating the effects of mutating the template region of telomerase RNA on its overall activity and efficiency. To this end, we created 12 different single nucleotide mutants within the template region of the telomerase RNA that will synthesize non‐native telomere sequences. Using a direct in vitro telomerase assay we identified which sequences affect telomerase function directly. Surprisingly, many of the point mutations resulted in abolished telomerase activity, presumably due to misfolding of the RNA or inhibition of telomerase holoenzyme assembly. Future efforts will include elucidation of the most critical nucleotide within telomere sequence for shelterin binding events by creating stable cell lines with desired mutations at telomerase RNA template.
Differential CYCLOIDEA Expression across Developing Compound Floral Heads
Authors: Thompson Veronica , Lim Aedric , Berger Brent , Ricigliano Vincent , Howarth Dianella
Published: June 2021

Gene duplication has been implicated in the evolution of novel features across flowering plants. Of great interest has been the shift from radial to bilateral symmetry in flowers. Previous studies have demonstrated that the transcription factor CYCLOIDEA (CYC) plays an intricate role in establishing dorsal, lateral, and ventral petals, as well as in the evolution of compound floral heads (i.e., inner and outer flowers differ in symmetry). In this study, we investigated whether multiple copies of CYC differ in their expression patterns between inner and outer flowers of the compound floral heads in Knautia macedonica (Dipsacaceae). qPCR was used to determine relative expression patterns for six copies of CYC in dorsal, lateral, and ventral petals of developing inner and outer flowers. Our data show that different copies of CYC are differentially expressed in different petal types of both inner and outer flowers, with CYC2A likely playing a role in dorsal identity, and CYC2B copies involved in lateral identity. Similar patterns of gene expression were found in inner and outer flowers suggesting a dosage effect in floral head development.
CYCLOIDEA Gene Expression across Dipsacales in Relation to Floral Symmetry
Authors: Lim Aedric , Thompson Veronica , Berger Brent , Ricigliano Vincent , Howarth Dianella
Published: June 2021

The Dipsacales is an order of plants that exhibits a striking shift from radial symmetry in basal lineages like viburnum to strong bilateral symmetry in species like honeysuckle. We are interested in determining how multiple copies of the transcription factor CYCLOIDEA (CYC) may be linked to floral symmetry shifts across Dipsacales. We use quantitative reverse transcriptase PCR (qPCR) to compare relative expression levels of the major copies of CYC (CYC1, CYC2, CYC3) across Dipsacales in seven different species exhibiting radial symmetry, bilateral symmetry, or asymmetry. Our data suggest that radially symmetric species do not exhibit wide variation in expression among petals, whereas bilateral and asymmetric flowers have pronounced differences, especially in CYC2 copies

Research supported by NSF IOS grant 1121301.
Structure and Assembly of Functional Amyloids
Authors: Perrett Sarah
Published: June 2021

The ability of proteins to self‐assemble into beta‐sheet‐rich aggregates called amyloid fibrils is considered to be universal, although certain polypeptide sequences have a particularly high propensity to adopt these conformations. In many cases the formation of amyloid fibrils is deleterious and associated with the progression of disease, but there are also examples of proteins for which the cross‐beta structure represents the functional conformation. Ure2 is the protein determinant of the yeast prion [URE3]. Ure2 consists of an N‐terminal prion‐inducing domain that is disordered in the native state, whereas the C‐terminal functional domain has a globular fold with structural similarity to glutathione transferase enzymes. The C‐terminal domain shows enzymatic activity in both the soluble and fibrillar forms of Ure2. We have used a variety of biophysical approaches to investigate the structure of Ure2 fibrils and their mechanism of assembly. We have also created chimeric constructs where the prion domain is genetically fused to other enzymes of different sizes and architectures. These chimeric polypeptide constructs spontaneously self‐assemble into nanofibrils with fused active enzyme subunits displayed on the amyloid fibril surface. We can measure steady‐state kinetic parameters for the appended enzymes in situ within fibrils, and compare these for the identical protein constructs in solution. We are currently investigating the use of microfluidic techniques to form enzymatically‐active microgel particles from the chimeric self‐assembling protein nanofibrils. These approaches allow amyloid fibrils to be used as a scaffold for enzyme immobilization, facilitating recycling and reuse of the appended enzymes in functional assays, both in batch processes and for microflow chemistry.
Mechanisms of rapid ataxin‐1 degradation in Spinocerebellar Ataxia Type 1
Authors: Kohiyama Mayumi , Toker Nicholas , Hearing Casey , Lagalwar Sara
Published: June 2021

Spinocerebellar Ataxia Type 1 (SCA1) is an autosomal dominant neurodegenerative disease caused by an expansion in the polyglutamine region of the ataxin‐1 (ATXN1) gene. Mutant expanded ATXN1 can be stabilized by phosphorylation at amino acid residue Ser776. In its stabilized form, ATXN1 aggregates in the nucleus and resists degradation, ultimately leading to cell death. However, in the presence of kinase inhibitors, mutant ATXN1 is rapidly degraded. This study examines the mechanism by which ATXN1 is readily degraded, using a Daoy cell model with expanded (82Q) and normal (30Q) ATXN1. Through manipulation of the proteasome, it was found that nuclear ATXN1, though ubiquitinated, is not degraded by the proteasome. Follow up studies will examine the effects of autophagy, a putative mechanism for rapid ATXN1 degradation, on aggregate clearance. Targeting cellular degradation mechanisms in SCA1 will provide a better understanding of the function of ATXN1 in its normal state and a starting point for future therapeutic studies.
Inhibition of Toxic IAPP Aggregation by Extracts of Common Fruits
Authors: Moffet David , Kao Pei‐Yu , Green Evangeline , pereira Catalina , Ekimura Shauna , Juarez Dennis , Whyte Travis , Arhar Taylor , Malaspina Bianca , Nogaj Luiza
Published: June 2021

Aggregation of the short polypeptide, Islet Amyloid Polypeptide (IAPP, amylin), appears to play a direct role in the death of pancreatic β‐islet cells in type 2 diabetes. Inhibiting the aggregation of IAPP has the potential to slow, if not prevent entirely, the progression of this disease. Extracts of thirteen different common fruits were analyzed for their ability to prevent the aggregation of amyloidogenic IAPP. Thioflavin T binding, immuno‐detection and circular dichroism assays were performed to test the in vitro inhibitory potential of each extract. Atomic force microscopy was used to visualize the formation of amyloid fibers with and without each fruit extract. Finally, extracts were tested for their ability to protect living mammalian cells from the toxic effects of amyloid IAPP. Several fruits were identified for having substantial ability to inhibit IAPP aggregation and protect living cells from toxic IAPP amyloid.
Investigation of Aggregative Properties of the Antimicrobial Peptide LL‐37 by Dynamic Light Scattering (DLS) and Electrophoretic Shift Mobility Assay (EMSA)
Authors: Kelly Kaela , Zachary R , Craig Maria
Published: June 2021

The antimicrobial peptide LL‐37 is a member the cathelicidin family of antimicrobial peptides that play a role in mammalian innate immune defense and have potential for combating bacterial infection and antibiotic resistance. LL‐37 is known to induce cell death by disrupting the cell membrane, but the mechanism for membrane disruption is still unknown. By studying peptide properties, such as aggregation, we may better understand how LL‐37 induces cell death. We have used dynamic light scattering (DLS) and electrophoretic mobility shift assays (EMSA) to investigate the aggregative properties of LL‐37. DLS monitors diffusion and thus the hydrodynamic radius of scattering centers by analyzing temporal fluctuations in scattered light intensity using the autocorrelation technique. In the concentration range of 150 to 37 μM, we observe by DLS that LL‐37 shows decreasing mobility as concentration increases, indicative of particle size transition from a hydrodynamic radius on the order of 1 to 1,000 nm. EMSA with peptide detection by western blotting provides information about aggregate size at lower concentrations, ranging from 0.8 to 50 μM. As a result, the particle size distribution shifts decisively in favor of relatively small aggregates as the concentration falls below 50 μM, indicated by the decrease in hydrodynamic radius in DLS and increased protein migration in EMSA.
OASIS is a Novel Cofactor of HIF1α to Promote the Transcription Dependence on Hypoxia Response Element under the Hypoxic Condition
Authors: Cui Min , Cui Xiang , Kanemoto Shoshi , Tanimoto Keiji , Imaizumi Kazunori
Published: June 2021

OASIS/CREB3L1 (Old astrocyte specifically induced substance), an endoplasmic reticulum (ER)–resident transcription factor, plays important roles in differentiation of osteoblasts and astrocytes. OASIS is transported from ER to Golgi in response to ER stress or differential stimuli, and is subjected to be cleaved by Site‐1 protease and Site‐2 protease. The cleaved N‐terminal fragment forms homo/hetero dimers with the other OASIS family members (such as BBF2H7, LUMAN, AIbZIP/CREB3L4 or CREBH). Then the dimerized OASIS complex functions as a transcription factor to induce the expression of various target genes in nucleus. In this study, Co‐immunoprecipitation assay and GST pulldown assay revealed that OASIS can bind to HIF1α (Hypoxia‐inducible factor 1‐alpha) through the bZIP (Basic Leucine Zipper Domain) domain of OASIS. In addition, Luciferase assay showed OASIS and HIF1α synergistically promotes the Luciferase activities through HRE (Hypoxia Responsive Element). On the contrary, a deletion mutant of OASIS without the bZIP domain could not bind to HIF1α, and also not promote the activities via the HRE. These results suggest that OASIS could promote the expression of HIF1α‐targets through the formation of heterodimer with HIF1α under the hypoxic condition.
Mechanistic Basis of Residue Specificity in Targeted Electrophilic Modification
Authors: Lee Dustin , Parvez Saba , Fu Yuan , Le Henry , Aye Yimon
Published: June 2021

Our group recently developed the T‐REX (Targeted Reactive Electrophiles and Oxidants) technology that enables selective delivery of reactive electrophilic signals to a protein of interest (POI) in cells. T‐REX exploits photo‐uncaging chemistry to liberate bioactive electrophiles such as 4‐hydroxynonenal (HNE) at a precise time from the photocaged HNE‐precursor that is covalently bound to the genetically encodable HaloTag (HT) fused to POI. The liberated HNE is captured by the reactive cysteine(s) (Cys) on POI fused to HT. With T‐REX, we have shown the activation of antioxidant response (AR) pathway by selectively HNEylating Keap1—a redox sensitive protein and a key player in mammalian AR, against a backdrop of otherwise unperturbed cell. My project seeks to answer residue specificity in Keap1 HNEylation enabled by T‐REX. Specifically, I aim to elucidate whether residue specificity is dependent on the position of the HT. T‐REX on HT at the N‐terminus of Keap1 in cells resulted in HNEylation of two specific Cys's. I now aim to test the extent to which residue specificity is altered when HT is at the C‐terminus. Toward this goal, I have recently cloned, expressed and purified recombinant Keap1‐HT. I have begun to test residue specificity in T‐REX targeting using Keap1‐HT in vitro and that expressed in human cells. I also aim to characterize residue specificity in vitro when Keap1 is in complex with two known binding partners—Nrf2 transcription factor, and CUL3 ubiquitin E3 ligase protein. Ultimately, these studies will shed light on the Cys residue specificity manifested by the unique T‐REX system and how this property changes upon association with Nrf2 and CUL3 as well as changes in Keap1 conformation in vitro and in cells.
Synthesis and Application of Small Cationic Peptides as Potential Antibiotics
Authors: Ferrante Christopher , Brandt John , Wolyniak Michael
Published: June 2021

Small cationic peptides have been recently found to have potential use as antibiotics. Wenzel et al tested the effectiveness of a peptide consisting of arginine and tryptophan repeats, RWRWRW‐NH2, with a terminal amine group. The arginine and tryptophan residues were chosen because of their highly reactive nature and for the size of the side chain. They found that the peptide would embed itself in the cell wall of a bacterium. The objective of this project is to expand upon the work done by Wenzel et al. Peptide synthesis is being accomplished via the Merrifield Synthesis method. This method utilizes a resin bead with the first amino acid attached to it; through a series of additions and washes the peptide is synthesized in one reaction vessel. Following synthesis, the peptide is cleaved from the bead and eluted from the vessel. The peptide synthesized by Wenzel is being used as a control and seven other scrambled peptides are being used as test groups. Three of the peptides consist of arginine and tryptophan, with the sequences being, RWWRWW‐NH2, RRWRRW‐NH2, and RRRWWW‐NH2. The other four peptides consist of lysine and tryptophan, with the sequences being, KWKWKW‐NH2, KWWKWW‐NH2, KKWKKW‐NH2, and KKKWWW‐NH2. The peptides being synthesized vary in the order of amino acids. We are interested to observe the effect the conformation of the peptide has on the effectiveness as an antibiotic. Moreover, lysine is substituted for arginine in order to determine if one amino acid is more effective than others. Once synthesized, the peptides are being introduced to lawns of eight species of gram positive bacteria in order to test effectiveness as an antibiotic. The mechanism of interaction is unknown for gram negative bacterial cell walls, and our laboratory does not have the capabilities to determine this.
An Yeast‐Based Screening for Signal Motifs That Regulate Membrane Protein Trafficking
Authors: Shikano Sojin , Bernstein Joshua , Okamoto Yukari
Published: June 2021

Cell surface density of membrane proteins are regulated by the interactions that often occur between distinct signal motifs on the cargo proteins and cellular transport machineries. The majority of the trafficking signals have been discovered by studies on individual protein molecules. While effective, a more systematic approach to identify trafficking signals would further facilitate our understanding of membrane trafficking. Here we report development of a new yeast‐based screening system that potentially enables identification of the sorting motifs that down‐regulate surface expression of membrane proteins. B31, a S. cerevisiae strain that lacks potassium (K+) efflux transporters, is unable to grow in high K+ media when expressing the mammalian inward rectifying K+ channel Kir2.1 due to accumulation of excessive intracellular K+. However, B31 cells expressing the disease mutant Kir2.1 deficient in post‐Golgi trafficking or the Kir2.1 that is C‐terminally fused with ER retrieval motif survive in high K+ media due to reduced K+ uptake. This B31 growth complementation in high K+ media allowed a screen for the peptide motifs that would down‐regulate cell surface expression of the reporter Kir2.1 channel. We performed a pilot screening of a C‐terminally‐fused peptide library and identified sequence motifs that reduced surface expression of the channel. Our study provides a technical basis for developing the unique gain‐of‐function screening that can potentially identify novel signal motifs that regulate surface expression of membrane proteins.
Molecular insights into the multispecific recognition of dipeptides of deep‐sea Gram‐negative bacteria Pseudoalteromonas sp. SM9913
Authors: Zhang Yu‐Zhong
Published: June 2021

Peptide uptake is important for nutrition supply for marine bacteria. However, how marine bacteria absorb peptides is still not fully understood. DppA is the periplasmic dipeptide‐binding protein of dipeptide permease (Dpp), an important bacterial peptide transporter. DppA exclusively controls the substrate specificity of Dpp. Here, the substrate recognition mechanism of deep‐sea pseudoalteromonas sp. SM9913 DppA (PsDppA) was studied. The substrate specificity of PsDppA was analyzed for 25 dipeptides with varying properties. PsDppA showed binding affinities for 8 of the peptides. To explain the multispecific substrate recognition mechanism of PsDppA, we solved the crystal structures of unliganded PsDppA and of PsDppA in complex with 4 different types of dipeptides. PsDppA alternates between an “open” and a “closed” form during substrate binding. Structural analyses of 4 PsDppA‐substrate complexes indicate that dipeptides are bound in the same position and the same orientation in PsDppA. Residues facilitating the interactions with the dipeptides show no clear conformational changes when binding to different ligands. Based on structural and mutational analyses, the multispecific recognition mechanism of PsDppA is explained. Additionally, sequence alignment suggests that the substrate recognition mechanism of PsDppA may be common in Gram‐negative bacteria. Our results provide a better understanding of marine bacterial peptide uptake.
An Ionic Lock Between Intracellular Loop 1 and a C‐terminal Amphipathic Helix Stabilizes the Serotonin Transporter Along its Folding Trajectory
Authors: Sucic Sonja , El‐Kasaby Ali , Koban Florian , Freissmuth Michael
Published: June 2021

The serotonin transporter (SERT) terminates neurotransmission via rapid reuptake of serotonin from the synaptic cleft. ER export of solute carrier‐6 (SLC6) family members relies on the recruitment of the COPII coat. The COPII component SEC24C binds to an ER export motif located on SERT C‐terminus. We here describe a C‐terminal amphipathic helix that plays a key role in SERT folding. Non‐conservative mutations in the hydrophobic rim of the helix lead to ER retention. Defective ER export of mutant SERTs is not caused by loss of SEC24C binding, revealed by siRNA‐induced SEC24 depletion. ER export of SERT favors inward‐facing transporter conformations. Treatment with the pharmacochaperone noribogaine, which preferentially binds to and stabilizes inward‐facing conformations, recovered the surface expression of C‐terminal helix mutants. Introducing second site suppressor mutations that promote inward‐facing states also repaired their targeting to the membrane. We also probed ionic interactions between the C‐terminus and the first intracellular loop of SERT. Charge‐reversal mutations revealed an ionic lock between E615 in the C‐terminus and R152 in the first intracellular loop. Reversing the charge of each residue alone (R152E or E615K) dramatically reduced SERT surface expression. Conversely, a tandem charge‐reversal mutant (R152E‐E615K) fully restored SERT cell surface expression. We here propose a mechanistic model where the C‐terminal amphipathic helix is stabilized by an intramolecular ionic lock with the first intracellular loop, to support the folding trajectory of SERT and other SLC6 transporters

This work is supported by the Austrian Science Fund Grant P27518‐B27.
Distributed computing approach to in silico protein function prediction using ProMOL
Authors: Bryan Michael , Kovuri Venkata , Craig Paul , Bernstein Herbert
Published: June 2021

ProMOL is extensible, distributed computing software for implementing computational biochemical workflows. Developed by members of RIT and Dowling College as a plug‐in for PyMOL, ProMOL delivers in silico screening of protein structures by comparison to a library of active‐site motif templates. As a software tool for biochemists, ProMOL has been used to predict the function of uncharacterized protein structures in the Protein Data Bank, identifying candidates for in vitro function determination. ProMOL's motif library has been expanded to include over 33,000 active‐site motifs defined by the Catalytic Site Atlas (http://www.ebi.ac.uk/thornton‐srv/databases/CSA/). The latest version of ProMOL supports high‐throughput in silico screening of novel protein structures for desired catalytic activities. This work has been funded in part by National Institute of General Medical Sciences 2R15GM078077‐02, 3R15GM078077‐02S1 and 3R15GM078077‐02S2.
Urinary Candidate Biomarker Discovery in a Rat Unilateral Ureteral Obstruction Model
Authors: Yuan Yuan , Gao Youhe
Published: June 2021

Urine has the potential to become a better source of biomarkers. Urinary proteins are affected by many factors; therefore, differentiating between the variables associated with any particular pathophysiological condition in clinical samples is challenging. To circumvent these problems, simpler systems such as animal models should be used to establish a direct relationship between disease progression and urine changes. Obstructive nephropathy is one of the most significant causes of chronic kidney disease, and its predominant end‐stage change is irreversible interstitial fibrosis. To better detect a set of obstructive nephropathy candidate urinary biomarkers, a unilateral ureteral obstruction (UUO) model was used to observe tubular injury and the eventual development of renal fibrosis, as well as to identify differential urinary proteins in this process. In this study, urine samples were collected from the residuary ureter linked to the kidney 1 and 3 weeks after UUO to mimic urine from patients with obstructive nephropathy. Compared with the sham operation group, 7 and 19 significantly changed proteins were identified by LC‐MS/MS in the UUO 1 and 3 week groups, respectively. These proteins identified in rat models of UUO may serve as candidate biomarkers of renal tubular injury and interstitial fibrosis in obstructive nephropathy.
Proteomics Study of Human Breast Milk for Breast Cancer Biomarkers Discovery
Authors: Aslebagh Roshanak , Ngounou Armand , Channaveerappa Devika , Arcaro Kathleen , Darie Costel
Published: June 2021

Breast cancer as one of the major causes of mortality in US is a challenging subject to study. Based on recent National Cancer Institute's statistics, it is the second common cancer in US. Detection and treatment of breast cancer at the very early stages and diagnosis of breast cancer risk before it happens is a challenging subject of study. Mammography has greatly increased the early detection of breast cancer, but has known limitations, especially in younger women with dense breasts. Breast milk is an appropriate cancer microenvironment for identifying breast cancer biomarkers that consists of epithelial cells and the proteins which are provided from breast tissue. Mass spectrometry‐based proteomics on human breast milk may offer a novel way to identify young women at increased risk of developing breast cancer. In this study, breast milk samples were separated by SDS‐PAGE and stained by Coomassie for qualitative and quantitative analysis and direct comparison of the samples. The gel lanes of cancerous and healthy samples (as the control) were cut into several bands which were digested with trypsin and then analyzed by nanoliquid chromatography‐tandem mass spectrometry (nanoLC‐MS/MS) using a nanoAcquity UPLC coupled with QTOF Micro mass spectrometer. Data processing and analysis were performed using MassLynx (version 4.1), ProteinLynx Global Served (PLGS 2.4), Mascot and Scaffold 4.0. In the Coomassie‐stained SDS‐PAGE, although the overall protein patterns of most of the major bands are similar, sample specific‐bands are also present which make the differences between cancerous and healthy samples. These outcomes are currently being investigated.
Does PtdIns(4,5)P2 concentrate so it can multitask?
Authors: Hammond Gerald , Balla Tamas
Published: June 2021

PtdIns(4,5)P2 drives many plasma membrane (PM) functions which can be summarized as the delivery of materials and information into, out of and across the membrane, as well as the assembly of structural components. How does the lipid execute so many disparate functions involving a vast array of different protein complexes? A leading hypothesis is that the PM organizes into nanoscopic PtdIns(4,5)P2‐rich platforms that associate with specific molecular complexes. Here, we have employed single molecule, super‐resolution techniques to test this hypothesis. Imaging fixed cells expressing PtdIns(4,5)P2 biosensors (< 40 nm resolution) reveals no evidence for discrete domains. Experiments where the dynamics of biosensor molecules are tracked in living cells reveals rapid diffusion of biosensor/PtdIns(4,5)P2 in the PM, indicating that biosensor:lipid complexes may be released from pre‐existing PtdIns(4,5)P2‐rich platforms. On the other hand, diffusion of biosensor/lipid complexes shows clear signs of anomalous diffusion. To circumvent potential occlusion of PtdIns(4,5)P2‐rich platforms by an expressed biosensor, we have interrogated isolated plasma membrane sheets with exogenous PtdIns(4,5)P2 probes. Again, these results reveal no evidence for PtdIns(4,5)P2‐rich platforms at a resolution levels of < 40 nm. Given the rapid diffusion of the free lipid molecules across such small distances as this, our results are inconsistent with pre‐existing PtdIns(4,5)P2‐rich platforms that recruit protein complexes. Rather, it seems more likely that protein complexes recruit the lipid into functionally dedicated complexes in the PM.
The Biological Actions of 11,12‐Epoxyeicosatrienoic Acid in Endothelial Cells are Specific to the R/S Enantiomer and Require the Gs Protein
Authors: Fleming Ingrid , Ding Yindi , Frömel Timo
Published: June 2021

Cytochrome P450 (CYP)‐derived epoxides of arachidonic acid, i.e., the epoxyeicosatrienoic acids (EETs), are important lipid signaling molecules. Since many actions of 11,12‐EET are dependent on the activation of protein kinase A (PKA), the existence of a cell surface Gs‐coupled receptor has been postulated. To assess whether the responses of endothelial cells to 11,12‐EET are enantiomer specific and linked to a potential G protein‐coupled receptor, we assessed 11,12‐EET‐induced, PKA‐dependent translocation of transient receptor potential (TRP) C6 channels as well as angiogenesis. In primary cultures of human endothelial cells, (±)‐11,12‐EET led to the rapid (30 seconds) translocation a TRPC6V5 fusion protein, an effect reproduced by 11(R),12(S)‐EET, but not 11(S),12(R)‐EET or (±)‐14,15‐EET. Similarly, endothelial cell migration and tube formation were stimulated by (±)‐11,12‐EET and 11(R),12(S)‐EET while 11(S),12(R)‐EET and 11,12‐dihydroxyeicosatrienoic acid were without effect. The effects of (±)‐11,12‐EET on TRP channel translocation and angiogenesis were sensitive to EET antagonists and TRP channel trafficking was also prevented by a PKA inhibitor. The siRNA‐mediated downregulation of Gs in endothelial cells had no significant effect on responses stimulated by VEGF or a PKA activator but abolished responses to (±)‐11,12‐EET. The down regulation of Gq/11 failed to prevent 11,12‐EET‐induced TRPC6 channel translocation or the formation of capillary like structures. Taken together, our results suggest that a Gs‐coupled receptor in the endothelial cell membrane responds to 11(R),12(S)‐EET, mediates the PKA‐dependent translocation and activation of TRPC6 channels, as well as angiogenesis.
Purification and characterization of PldB, an Escherichia coli lysoglycerophospholipase
Authors: Sopeyin Anuoluwapo , Garrett Teresa
Published: June 2021

Headgroup‐acylated glycerophospholipids (GPLs) are an under‐studied class of lipids found in the Escherichia coli cell membrane that are now known to impact cell division by determining potential cell division sites. One such headgroup‐acylated GPL, acyl phosphatidylglycerol (acyl PG), is produced by the enzymatic transfer of the acyl chain from 2‐acyl lysophosphoethanolamine (2‐acyl lysoPE) to the headgroup of phosphatidylglycerol (PG). The enzyme implicated in this trans esterification reaction is the E. coli membrane‐associated protein, PldB. Because PldB is involved in the production of acyl PG, knowledge of the biosynthetic reaction of PldB can provide insight to the mechanism by which other headgroup‐acylated GPLs are formed. To study its biosynthetic reaction, PldB was purified to prevent interference from other enzymes that may metabolize either its reactants or products. A six‐histidine tagged version of PldB was over‐expressed in E. coli. This membrane‐associated protein was removed from the membrane using high salt and then purified using nickel‐affinity chromatography followed by gel‐filtration chromatography. Current research is focused on tracking the activity of PldB at various stages in the purification. The activity being measured is the rate of deacylation of 14C 2‐acyl lysophosphatidylglycerol, which is metabolized the same way that 2‐acyl lysoPE is metabolized. Once the efficiency of the purification is obtained, the substrate specificity with regard to acyl chain length of the acyl donor and acyl acceptor of PldB will be assessed using the purified enzyme.
Lack of Pah1p PA Phosphatase in Yeast Causes Reduced Chronological Life Span through Increased Energy Expenditure for Lipid Synthesis
Authors: Park Yeonhee , Carman George , Han Gil‐Soo
Published: June 2021

In Saccharomyces cerevisiae, Pah1p phosphatidate phosphatase (PAP), which catalyzes the dephosphorylation of PA to yield diacylglycerol, plays a crucial role in the synthesis of the storage lipid triacylglycerol. This evolutionarily conserved enzyme also plays a regulatory control in de novo phospholipid synthesis through its consumption of PA, a precursor of membrane phospholipids. In this work, we found that the pah1Δ mutant is defective in the utilization of non‐fermentable carbon sources and exhibits a great reduction in chronological life span. In line with the phenotypes indicative of mitochondrial dysfunction, the pah1Δ mutant showed a reduction in oxygen consumption and an increase in the level of superoxide. Although sensitive to oxidative stress, the pah1Δ mutant showed no significant decrease in the activities of mitochondrial superoxide dismutase and catalase. Moreover, no significant defect was shown in mitochondrial ATP synthesis, tubulation, and contents of phospholipids and fatty acids. However, in the stationary phase or exponential phase cells transferred to growth medium containing a non‐fermentable carbon source, the pah1Δ mutant showed 2‐fold increase in phospholipid synthesis and a concomitant decrease in ATP. These results indicate that the reduced chronological life span of the pah1Δ mutant was a consequence of ATP depletion due to increased lipid synthesis. Supported by NIH grant GM028140.
Ubiquitin‐Specific Protease 24 Regulates Apoptosis through Deubiquinating Bax and Mediating Ku70 Acetylation
Authors: Wang Shao‐an , Hung Jan‐jong
Published: June 2021

Ubiquitin‐specific protease (USP) 24 is one of deubiquinating enzymes (DUBs), which remove polyubiquitin chains from protein substrates and thereby prevent the substrates from ubiquitin‐mediated proteasomal degradation. However, little is so far known about the possible role of USP24 in control of protein deubiquitination or in physiological function. In this study, we used a time‐lapse technique to observe U2OS cells, and we found that USP24 overexpression caused cell death and increased the detection of caspase‐3 cleavage revealed that apoptosis occurred. Furthermore, we we showed that USP24 specifically interacted with and deubiquitinated Bax. USP24 stabilized Bax protein levels, which required its deubiquitinase activity. Moreover, a yeast two‐hybrid screening of a human cDNA library was performed to identify potential USP24 cellular partners. We identified a nonhomologous end‐joining (NHEJ) factor, Ku70, as a likely USP24‐interacting partner. The Ku70 protein was shown to be involved in multiple cellular pathways including DNA repair, telomere maintenance, V(D)J recombination and Bax mediated apoptosis. Here, we found that USP24 could induce Ku70 acetylation via stabilizing p300 protein levels and decrease the interaction between Ku70 and Bax. Taken together, our findings have uncovered an important role of USP24 in regulating apoptosis via deubiquitinating Bax and enhancing Ku70 acetylation to release Bax.