bluuebunny/crossref_metadata_2025 huggingface dataset:
X‐Ray Single Molecule Tracking Reveal Motions of Peptides in the Binding Groove of MHC II Regulated by DM
Authors: Kozono Haruo , Miyabe Toshihiro , Kozono Yuko , Sasaki Yuji
Published: June 2021

Class II MHC can take loosely bound peptides conformations, which generated in the recycling endosomes or cell surface, that are recognized by autoimmune T cells. DM converts loosely bound complexes to tightly bound complexes; crystal structure advocated that DM bound form of MHC II generate novel threshold for peptide binding by creating a hydrogen bond inside the binding groove, that form loosely bound intermediate peptides complex. We used soluble I‐Ak and two peptides that differ in their length as a model system to detect such floating peptides. Fluorescein quenching study provided remarkable difference; fluorescein on the peptides gave about twice a strength signal with DM than without DM, suggests peptides floating half way from MHC II. Diffracted X‐ray tracking (DXT) that monitors real‐time motion of individual peptides/MHC in solution at micro‐second level, revealed that motions of peptides inside binding groove divided into two groups without DM. Addition of DM shifted the slow velocity peak to the fast one. Thus, we successfully detected the DM mediated loosely bound peptides intermediate by two distinct methods. We suggest that measuring the motion of peptide at micro‐second level presents new parameters for both peptide/MHC complex and DM function.
Management of amebiasis based on biochemical signaling and host pathogen interactions
Authors: Tashjian Joseph , Espinosa Avelina
Published: June 2021

The neglected tropical disease amebiasis, caused by Entamoeba histolytica, is considered a threat to public health. Combinatorial approaches could identify more efficient and less costly alternatives in managing the second leading parasitic disease worldwide. Aggregative signaling and metabolic pathways, two distinct biochemical strategies, are being explored. 1) In vitro identification of chemicals secreted for cellular communication could be manipulated to prevent pathogenesis. 2) An essential enzyme in the glycolytic pathway, Entameba histolytica alcohol dehydrogenase 2 (EhADH2), provides a novel target for anti‐amebic drugs. Several synthetic pyrazoline derivatives and a commercial iron chelator were capable of restricting amebic growth via blocking EhADH2 activities. A holistic understanding of an infective agent could contribute to an improved management of infectious diseases based on ecological and evolutionary principles.
Glycosylation of Human MUC1 Transcript Variant 2 Expressed in HeLa Cells
Authors: Warren Janine , Piefer Andrew
Published: June 2021

The purpose of this project is to study the localization and glycosylation of MUC1 a potentially important antigen found on tumor cells. Naïve and carcinogenic cells produce this protein, but there are very distinct differences between MUC1 isoforms found on these cells. MUC1 has a 20 amino acid tandem repeat where the bulk of O‐linked glycosylation occurs. There are five spots in each tandem repeat (TR) for glycosylation, but the typical MUC1 isoform only fills about 2.5 glycosylations per TR. Recognition of an altered cancer specific MUC1 glycosylation may lead to an effective target for anti‐cancer therapies. A recombinant, epitope‐tagged Human MUC1 transcript variant 2 cDNA will be transiently expressed in HeLa cells to study protein glycosylation. MUC1 will be isolated via immunoprecipitation and subjected to glycosidase treatment to determine presence of sugars. SDS‐PAGE and western blotting will be used to detect MUC1. We hypothesize that the glycosylation of recombinant MUC1 produced in HeLa cells will be similar to the glycosylation patterns found on MUC1 present in epithelial tumors. This project was funded by the Hartwick College Department of Chemistry.
Tamm‐Horsfall Protein Binds Properdin
Authors: Vargas Carlos , Rhodes Diana
Published: June 2021

The purpose of this study was to determine if Tamm‐Horsfall protein (THP), an abundant urinary protein that interacts with various molecules of the immune system, binds to properdin (Factor P), a positive regulator of complement activation which stabilizes the alternative pathway C3 convertase. Enzyme‐linked immunosorbent assays (ELISA) were used to evaluate the binding of soluble human properdin to immobilized human THP. In low ionic strength buffers containing magnesium and calcium, THP bound properdin strongly (KD ~ 10‐8 M). Removing divalent cations from the buffer markedly reduced the ability of THP to bind properdin. Ionic strength also was important in this interaction. Properdin binding to THP was stronger in low ionic strength (20mM NaCl) and intermediate ionic strength buffers (90mM NaCl), than in physiologic strength (154mM NaCl) buffers. By ELISA, soluble THP effectively competed with immobilized THP for binding to properdin, with 0.1µM soluble THP inhibiting 50% of 0.05µM properdin's binding to immobilized THP. Western blot analysis demonstrated that soluble THP could bind to properdin after the properdin had been separated by SDS‐PAGE and transferred to nitrocellulose paper. In conclusion, this study shows that THP binds to properdin in the presence of divalent cations. This interaction is influenced by NaCl concentration, suggesting that electrostatic interactions between the two molecules are important. Properdin appears to recognize THP in solution, as well as immobilized THP, and THP binds to soluble properdin, as well as properdin that has been partially denatured by SDS during electrophoresis and Western blotting. The functional significance of this interaction remains to be evaluated. Funded by the PNWU Research Seed Program.
Exploration of the Catalytic and Kinetic Mechanism of ATP Hydrolysis by Bacteriophage T4 Rad50
Authors: Herdendorf Timothy , Nelson Scott
Published: June 2021

DNA double‐strand breaks are among the most harmful forms of DNA damage and unless correctly repaired they may lead to the development of cancer. A well‐conserved protein complex, Mre11/Rad50, plays a primary role in the initial steps of DNA double‐strand break repair. Rad50 is a member of the ABC protein super‐family, which is defined by six conserved motifs that make up the two ATP active sites that are shared across subunits of the protein dimer. The identity of the catalytic base has been controversial and the kinetic mechanism for only a small number of super‐family members has been determined. We have used a combination of steady state and pre‐steady kinetics, along with site‐directed mutagenesis, solvent isotope and viscosity effects, and pH rate profiles to probe the kinetic and catalytic mechanism of this important ABC protein super‐family member. The results suggest that the catalytic mechanism of Rad50 proceeds thru a dissociative‐like, asymmetric transition state and that enzyme chemistry is the rate‐limiting step of the catalytic cycle. Mutations of the Walker B glutamate and the H‐loop histidine suggest that although these two residues are likely involved in the positioning of the attacking water molecule, neither act to abstract a proton during the transition state of the reaction. In addition, mutation of these residues results in a substantial loss of ATP cooperativity, which when combined with previous results from our laboratory, suggests two routes of communication between ATP active sites. While there does not appear to be a single unifying theme for the rate‐limiting step of the reaction, our conclusions for Rad50 regarding the catalytic mechanism and cooperativity are expected to apply more broadly to the ABC protein super‐family.
Investigation of the Radical SAM Methylthiotransferase MiaB Reaction Mechanism
Authors: Arcinas Arthur , Booker Squire
Published: June 2021

MiaB catalyzes the transfer of a methylthio (‐SCH3) group to C2 of N6‐(isopentenyl)adenosine (i6A) in the anticodon stem loop of many prokaryotic tRNAs. The product of the reaction, C2‐methylthio‐N6‐(isopentenyl)adenosine (ms2i6A) tRNA, is involved in reading frame maintenance and translational fidelity. MiaB is a member of the methylthiotransferase (MTTase) subclass of the radical S‐adenosyl‐L‐methionine (SAM) super‐family of enzymes, and as such, contains a canonical [4Fe‐4S] cluster involved in the reductive cleavage of SAM to generate a 5′‐deoxyadenosyl‐5′‐radical (5′dA) required for catalysis. In addition, the enzyme harbors an auxiliary N‐terminal [4Fe‐4S] that is thought to be the source of the sulfur atom inserted at C2, with the methyl group originating from a second molecule of SAM. Although it was anticipated that the MiaB mechanism involved the initial insertion of a sulfur atom prior to the methylation step by SAM to form ms2i6A, our work indicates that the sequence of mechanistic steps occurs in reversed order. Evidence for the initial methyl‐transfer from SAM to an acid‐labile sulfur atom acceptor present in MiaB was obtained under conditions that preclude the formation of the 5′dA radical species and sulfur insertion. The kinetic competence of the methylated intermediate was assessed by LC/MS of ms2i6A formation in assays using SAM during the initial methylation of MiaB, and S‐adenosyl‐L‐[methyl‐d3]methionine (d3‐SAM) under turnover conditions. Utilizing a combination of radioisotope‐labeling studies, mass spectrometry and spectroscopic techniques, insights into the MiaB mechanism presented here illustrates a new strategy in the diverse repertoire employed by radical SAM enzymes.
Enzyme ‘Social’ Networks: Tracking Sequence / Function Relationships to Elucidate Determinants of Enzyme Specificity and Mechanism
Authors: Akiva Eyal , Brown Shoshana , Holliday Gemma , Babbitt Patricia
Published: June 2021

Integrated analysis of sequence, structure and functional properties of enzymes is an established approach for determining their mechanisms and specificities. As sequence data continue to mount, analyzing the natural variation of homologs offers a powerful context for guiding experiments, while turning studies of single enzymes or large superfamilies into bioinformatic 'big data' problems. Many current tools designed to tease out potential functional residues are not sensitive enough to accommodate the specific intricacies of enzyme superfamilies, yielding biased predictions and misannotation. Here, we present the Structure‐Function Linkage Database (http://sfld.rbvi.ucsf.edu), a hierarchical classification resource and bioinformatic infrastructure that enables research of structure‐function relationships in functionally diverse enzyme superfamilies. It offers manually curated and automatic classification of enzymes, informed by the mechanistic blueprint of their corresponding superfamilies. Importantly, annotation and data exploration in the database takes advantage of sequence similarity networks to visualize functional trends mapped to the context of sequence similarity. We will illustrate how networks are used for enzyme classification in the glutathione transferase superfamily and show how mapping active site residue variation across a large number of proteins aids in elucidating specificity determinants in the Sirtuin superfamily of deacetylases. In summary, our approach allows a unique way to effectively utilize massive multidimensional data for generating hypotheses about enzyme mechanism and specificity.
Exploration of the Protein Cleaving Ability and Specificity of 1,4,7‐triazacyclononanecopper(II) Chloride
Authors: Sweet Patrick , Bodsgard Brett
Published: June 2021

In the study of protein structure, one route of analysis is peptide bond cleavage. Biological enzymes are the commonly used as the cleaving agent, but they have setbacks in that they tend to be rather expensive, unstable, nonspecific, and autolytic. Alternatively, synthetic proteases may have the potential to overcome these limitations. The protein cleaving ability of the small metal complex 1,4,7‐triazacyclononanecopper(II) chloride (1) was explored by reacting several proteins with under a variety of conditions including concentration of 1, time, and temperature. Progress was made toward the elucidation of the amino acid sequence specificity of 1.
Characterization and Complementation of a Potential Novel Phenotype for Saccharomyces cerevisiae PHO13
Authors: Gehret Austin , Mills Kimbria , Kellogg Courtney , O'Handley Suzanne
Published: June 2021

PHO13 in Saccharomyces cerevisiae is a member of the p‐nitrophenylphosphatase (p‐NPPase) family of the Haloalkanoic Acid Dehalogenase (HAD) superfamily. While the physiological role of this enzyme remains debated, our lab has demonstrated PHO13 possesses specific activity towards 2‐phosphoglycolate, a by‐product of the Calvin Cycle. Phosphoglycolate phosphatases (PGPases) are required in photosynthetic organisms to recycle this metabolite, yet their presence in non‐photosynthetic organisms suggests 2‐phosphoglycolate is generated in other pathways. Regardless of its origin in yeast, 2‐phosphoglycolate must be catabolized due to its tendency to strongly inhibit triose phosphate isomerase. We have identified a potential growth phenotype for pho13 knockout (KO) cells, a delay in growth upon hyperosmotic (1M NaCl) shock compared to wild‐type cells. Studies are currently underway to attempt complementation of this phenotype in pho13‐KO cells by PHO13, gph (a previously characterized PGPase from E. coli), and PGP2 (a PGPase from the green alga C. reinhardtii). Though the pho13‐KO phenotype is not a direct measure of PGPase activity, it could serve as a useful tool in assessing whether an un‐characterized HAD phosphatase possesses physiological PGPase activity.
Structural Properties in the FMN Reductase of the Alkanesulfonate Monooxygenase System that Dictate Oligomeric States and Catalysis
Authors: Musila Jonathan , Ellis Holly
Published: June 2021

The alkanesulfonate monooxygenase system is expressed in many bacteria when sulfur is limiting to enable the organism to utilize a diverse range of alkanesulfonates. This system is comprised of a NADPH‐dependent FMN reductase (SsuE) that reduces flavin and transfers it to the monooxygenase enzyme (SsuD) for the desulfonation of alkanesulfonates 1. The SsuE enzyme exists in different oligomeric states, but it is unclear how these oligomeric states relate to catalysis.

A conserved π‐helix is located in the tetramer interface of SsuE which results from the insertion of Tyr118 in the α4‐helix. This Tyr118 residue forms a hydrogen bond with the carboxyl group of Ala78 across the tetramer interface to enhance the protein architecture 2. Variants of Y118 SsuE were generated to evaluate the role of the π‐helix in SsuE. Interestingly, the Y118A and Y118S SsuE variants were FMN‐bound as purified, while the Y118F and ΔY118 SsuE variants were flavin‐free. The Y118 SsuE variants were able to effectively bind FMN giving similar Kd values as wild‐type SsuE. This flavin affinity correlated with the ability of Y118 SsuE to reduce flavin with kcat/Km values similar to that of wild‐type SsuE in flavin reductase assays. While desulfonation activity was observed with the Y118F SsuE variant, there was no activity in the coupled assays monitoring SsuD desulfonation with the Y118A, Y118S and the ΔY118 variants of SsuE. This would indicate that the Tyr118 is vital for the transfer of FMNH2 to SsuD. The results from these studies suggest that multiple non‐covalent interactions are important for the structural stability of SsuE. These findings provide insights on the role of Tyr118 in SsuE.
1. Eichhorn, E. et al. (1999) J. Biol. Chem. 274, 26639‐46.
2. Driggers, C. M. et al (2014) Biochem. 53, 3509‐19.
Unraveling the Biosynthesis of Dimethyl Indolic Acid, A Key Moiety of the Antibacterial Nosiheptide
Authors: Badding Edward , Grove Tyler , Booker Squire
Published: June 2021

Nosiheptide (NOS) belongs to a unique class of thiazole‐containing peptide antibiotics that display formidable activity against a number of pathogenic bacteria. Such bacteria include methicillin resistant Staphylococcus aureus, penicillin‐resistant Streptococcus pneumoniae, and vancomycin‐resistant enterococci. One key structural feature of NOS is an indolic acid ring system. The indolic acid moiety is derived from tryptophan via the action of at least four enzymes. In the first step of the proposed pathway, which is catalyzed by NosL, tryptophan is converted into 3‐methyl indolic acid (MIA). The remaining steps and enzymes involved in formation and installation of MIA into the NOS core have not been definitively determined, but are believed to be catalyzed by the sequential action of NosI, NosK, and NosN. The research described herein is focused on elucidating the mechanism by which the indolic acid moiety is formed and installed with the immediate goal of characterizing NosI, NosK, and NosN and the reactions they catalyze. Additionally through bioinformatic analyses, a protein in the nos operon, NosJ, has a sequence similarity to acyl carrier proteins. We believe that this protein carries the product of NosI to either NosK or NosN.
Proton Tunneling Accelerates ATP Hydrolysis in Kinesin‐5 Proteins
Authors: Kim Sunyoung , Richard Jessica , Parke Courtney , Worthylake David , Wojcik Edward
Published: June 2021

It is well established that, for ATP hydrolysis, a proton from the water nucleophile is abstracted to create a hydroxide capable of attacking the substrate. Evidence for the physical basis for the catalytic power of this key reaction in biological systems has been limited until recently. X‐ray crystal structures capturing this initial step in biological ATP hydrolysis have revealed a two‐water cluster, with uncommon properties, in the active site of kinesin proteins. Herein, we tested the nature of the active‐site water cluster using solvent kinetic isotope experiments. The magnitude of the isotope effect, Arrhenius pre‐exponential factor ratios, and activation energy differences with human Kinesin‐5 proteins were determined. Our data show that the proton transfer between solvent molecules is not classical in description, but rather has a quantum mechanical tunneling component. Experiments with Drosophila Kinesin‐5 showed similar findings; moreover, X‐ray structures of the Drosophila Kinesin‐5 also captured two waters in the active site. Taken together, this is the first detection of enzymatic proton tunneling in an ATPase. More broadly, this study reveals that use of quantum biochemistry is not solely a characteristic of metalloenzymes and is more widespread than currently established. This work was supported by NIH GM097350 (SK).
Biochemical and Functional Characterization of the Mutagenic Cytidine Deaminase, APOBEC3B
Authors: Sasaki Tomoaki , Anderson Karen
Published: June 2021

Apolipoprotein B messenger RNA‐editing, catalytic polypeptide 3 (APOBEC3) family proteins are cellular cytidine deaminases that function as DNA/RNA mutators and play diverse roles in key physiological processes. Recent studies have shown that APOBEC3B, a unique member of this family that predominantly localizes to the nucleus, is implicated in a variety of cancers including squamous cell lung carcinoma, a cancer of which little options are available for targeted therapy. While speculations have been made for a possible association of APOBEC3B to carcinogenesis, little is known regarding the molecular mechanisms that link APOBEC3B to cancer. Biochemical experiments have been conducted to characterize this protein enzymatically, investigating its enzymatic capacity and function, its ability to differentially bind DNA and RNA, and the diverse ways in which APOBEC3B is regulated at the protein level. Accompanying these biochemical experiments, we simultaneously conducted a series of cellular based assays to validate the role APOBEC3B plays in carcinogenesis. These studies seek to understand the molecular mechanisms of APOBEC3B‐induced carcinogenesis, while also providing insight into the development of possible targeted treatment strategies for non‐small cell lung cancer (NSCLC).
Exploring the Role of Proline Metabolism in Helicobacter Pathogenicity
Authors: Christgen Shelbi , Ramer‐Tait Amanda , Becker Don
Published: June 2021

Helicobacters have been associated with many diseases; including inflammatory bowel disease (Shomer, N et. al. 1997 Infec & Immun 65: 4858‐64). It is thought that the chronic inflammatory response experienced by the host in response to Helicobacter infection is the cause of subsequent host disease (Maggio‐Price L et. al. 2006 Cancer Research 66: 828‐38). One of the preferred respiratory substrates of Helicobacters is the amino acid proline. In some bacteria, proline is metabolized by proline utilization A (PutA). PutAs have been shown to utilize substrate channeling between two active site domains: a proline dehydrogenase, and a pyrroline‐5‐carboxylate dehydrogenase (Tanner, JJ. 2008 Amino Acids 35: 719‐30). The physiological importance of substrate channeling in any system is unknown. Though bioinformatical analysis indicates that Helicobacter PutAs have both domains, it is unknown if they exhibit substrate channeling, and what advantage channeling would confer. Previously, it has been shown that PutAs are required for H. pylori infection and that disrupting the putA gene in H. hepaticus leads to decreased host inflammation (Nakajima, K et. al. 2008 Biomedical Research 29: 9‐18; Krishnan, N et. al. 2008 Infec & Immun 76: 3037). Collagenase is also required for H. pylori infection (Kavermann, H et. al. (2003) J Exp Med 197:813‐22). Due to the high levels of proline in collagen, we hypothesize that collagenase is required for infection because it provides the pathogen with a steady source of proline, and the collagen breakdown leads to host inflammation. We therefore seek to explore the relationship between collagenase and proline metabolism, as well as the importance of substrate channeling, in H. bilis.
Electron Microscopic Analysis of Lens Gap Junction Connexin50 Hemichannels and Channels
Authors: Sosinsky Gina , Ren Cynthia , Ambrosi Cinzia
Published: June 2021

The mammalian lens is an avascular organelle that depends on gap junction (GJ) mediated intercellular communication for homeostasis, growth and development. Connexin50 (Cx50) is one of three lens GJ proteins and one of the largest members of the connexin protein family. Cx50 is present in all cells of the lens, first synthesized in the epithelium and persisting at high levels in differentiating fibers. Cases of human hereditary cataracts caused by >20 Cx50 distinct mutations demonstrate its crucial role in lens function and Cx50 knockout mice develop cataracts. Little is known about Cx50 structure except through homology with Cx26, a small connexin solved to 3.5 Å resolution by X‐ray crystallography and NMR of Cx50 C‐terminal peptides. Cx26 and Cx50 have strong sequence conservation and folding in their transmembrane and extracellular domains, but Cx26 lacks the extensive, non‐conserved C‐terminus present in Cx50. We have examined the full‐length Cx50 hemichannels and channels by single particle EM methods, the only structural biology technique that directly addresses how the large Cx50 C‐terminus is organized within the full‐length channel. Using baculovirus expression in Sf9 cells, we have purified both full‐length mouse Cx50 (440 amino acids) and a truncated form (Cx50del250‐440, 249 amino acids long) missing most of the C‐terminus such that it is slightly longer than Cx26 (226 amino acids). Electron micrographs of purified Cx50 hemichannels show a range of morphologies due to the way the oligomers are oriented on the grid. In contrast, Cx26 and Cx50del250‐440 channels are more homogeneous, displaying the canonical “doughnut‐like” appearance in projection because their structure.

Funding: NIH grants GM065937 (GS) & GM103412 (NCMIR)
PIP2 Stabilizes Dopamine Transporter Surface Expression
Authors: Fagan Rita , Hofmaier Tina , Sitte Harald , Melikian Haley
Published: June 2021

Dopamine (DA) signaling is central to movement, cognition, and reward pathways in the CNS and presynaptic DA reuptake mediated by the DA transporter (DAT) terminates DA signaling. DAT is potently inhibited by addictive psychostimulants cocaine and amphetamine (AMPH), and psychostimulant reward requires this inhibition. Thus, DAT availability is a critical factor for normal neurotransmission and reward, and mechanisms limiting DAT are likely to significantly impact these processes. Membrane trafficking acutely regulates DAT surface expression, and AMPH exposure and PKC activation reduce DAT surface expression by releasing an endocytic brake. However, the mechanisms constituting the DAT endocytic brake are undefined. A recent report found that phosphatidylinositol‐4,5‐bisphosphate (PIP2) interacts with DAT and facilitates AMPH‐stimulated DA efflux. Moreover, DAT/PIP2 interactions appeared to influence DAT surface stability. In the current study we directly tested whether PIP2 impacts DAT internalization. Acute PIP2 depletion decreased DA uptake and PKC activation did not further downregulate DAT, suggesting that PKC and PIP2 converge on a similar mechanism. Decreased DAT activity was accompanied by significantly increased DAT internalization rates, consistent with a requisite role for PIP2 in the DAT endocytic brake. Furthermore, DAT mutants lacking PIP2 binding displayed increased basal internalization rates. Ongoing experiments will test whether PIP2 depletion results in DAT surface losses and whether molecular components of the DAT endocytic brake work in concert with PIP2 to restrict DAT internalization.
Exosomal AnnexinA2′s Role In Angiogenesis And Breast Cancer Metastasis
Authors: Maji Sayantan , Vishwanatha Jamboor
Published: June 2021

Early detection of cancer using circulating biomarkers is a realistic possibility with the discovery of exosomes which are 40‐100nm in size and comprise a major portion of secretory vesicles. Annexin A2 (AnxA2) is a 36 KD Ca+2 dependent phospholipid‐binding protein up‐regulated in many cancer types and implicated in promoting tumorigenesis and angiogenesis. Although AnxA2 is highly expressed in exosomes, its function has never been studied. We propose to explore the correlation and functionality of exosomal AnxA2 (exo‐AnxA2) in breast cancer

Methods

Exosomes were isolated from MCF10 progression model,MDA‐MB‐231 and its organ specific metastatic variants and characterized via Western blotting and particle size analyzer. Transmission Electron Microscopy (TEM) and Atomic Force Microscopy (AFM) were used to study the correlation between exo‐AnxA2 and breast cancer progression. In vitro and in vivo angiogenesis assays explored the role of exo‐AnxA2 in angiogenesis. Experimentally induced (intracardiac injection) and spontaneous (mammary fat pad) metastasis models were used to study the role of exo‐AnxA2 in metastasis. To study AnxA2 specific function, LCKLSL inhibitory peptide or shAnxA2 were used.

Results

AFM and Western analysis showed increased levels of exo‐AnxA2 along the progression model

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Angiogenesis studies with LCKLSL showed exo‐AnxA2 to be a potent inducer of angiogenesis

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Both the metastasis models showed exo‐AnxA2 to be a potent inducer of metastasis

Conclusion

We found increased expression of exo‐AnxA2 in cancer exosomes vs. normal. Further, we found that exo‐AnxA2 is a potent inducer of angiogenesis and breast cancer metastasis indicating a possible role of exo‐AnxA2 in tumor‐ microenvironment signaling and cancer progression

(Supported by NIMHHD grant under Award Number P20MD006882)
Elucidating the structural consequences of CQC‐mediated dimerization of MUC1
Authors: Stachowski Tim , Li Edwin
Published: June 2021

Mucin 1 (MUC1) is a highly glycosylated single pass membrane protein that serves as a selective barrier against pathogens and engages in signal transduction influencing cellular growth and differentiation. Overexpression of mucins have been linked to 80‐90% of all human solid tissue cancers. Constitutive homodimerization, as a result of overexpression, leads to the formation of complexes with growth factor receptors and targeting to the nucleus, where MUC1 interacts with effector proteins regulating gene expression.

Recent studies have shown that the MUC1 cytosolic membrane proximal CQC motif promotes dimerization under oxidizing conditions, suggesting that the motif may act as a redox switch in response to changes of oxidant levels. Preliminary studies have shown that the transmembrane domain (TMD) of MUC1 lacking the CQC motif form weak dimers, confirming their contribution to dimerization. Also, results show that TMDs containing the CQC in an oxidizing environment exhibit increased dimerization, further enforcing the role of the motif acts as a redox switch.

Using the ToxR dimerization assay, the effects of single amino acid substitutions in TMDs with the CQC motif and TMDs with the CQC‐>AQA motif are being measured. Our goal is to determine (1) whether certain amino acids in the TMD play a key role in forming MUC1 homodimers, and (2) whether the CQC motif induces a structural change when it functions as a redox switch. These conclusions will contribute to our understanding of MUC1 dimerization and provide insights for the development of novel therapeutic strategies.
TORC1 Inhibition Using a Drug Combination Approach in Saccharomyces cerevisiae
Authors: Bravo Priscilla , Manandhar Surya , Gharkhanian Editte
Published: June 2021

The yeast vacuole is functionally analogous to the mammalian lysosome, a dynamic organelle responsible for macromolecule degradation, receptor down regulation and stress survival. As with lysosomes, the vacuole also serves as the platform for Target of Rapamycin Complex 1 (TORC1) signaling. TORC1 is the master regulator of cell growth and proliferation and its hyperactivity is associated with cancer and metabolic diseases. Inhibition of TORC1 signaling by rapamycin is associated with severe toxicities. Therefore, combination of drugs that can regulate TORC1 at lower, less toxic concentrations can be advantageous in treatments of TORC1 hyperactivity. A yeast genomic deletion strain screen in our lab uncovered deletion strains with hypersensitivity to hygromycin B (hhy mutants), all of which exhibited defects in vacuole trafficking, morphology and/or function. Recent findings in our lab indicate that hhy mutants also have compromised TORC1 signaling, and that intact lysosomal trafficking is a prerequisite for this signaling. Thus, drugs that affect lysosomal trafficking may have potential for use in combination with lower levels of rapamycin in TORC1 inhibition. We are currently testing hygromycin B and other vesicular trafficking inhibitory drugs in combination with sub‐inhibitory levels of rapamycin and assessing cumulative effects on growth. To date, we have established such a cumulative effect for hygromycin B and rapamycin drug combination. Additionally, our prior microscopic studies show that vacuolar Tor1‐GFP is mislocalized in hhy mutants especially in presence of hygromycin B. Biochemical localization assays of native Tor1 following drug treatments in wild type and hhy mutants are in progress.
Investigation of the Mechanism of Action of Oxazolidinones
Authors: Koleva Bilyana , Aggen James , Beuning Penny
Published: June 2021

The need to develop broad spectrum antibacterial agents is urgent as multidrug resistant bacteria have emerged in hospitals worldwide. The number of efficacious antibiotics is diminishing because bacteria develop mechanisms to confer resistance. Oxazolidinones work by interfering with protein translation and are mainly active against gram‐positive bacteria. The Aggen research group at Northeastern University is focused on optimizing the properties of oxazolidinones to be able to penetrate the lipopolysaccharide layer of gram‐negative bacteria while maintaining their target potency. The rational lead optimization campaign based on the linezolid scaffold, involves keeping a low molecular weight (MW<400), and high polarity (ClogD <1) to allow passage through the porins and to be able to escape the bacterial efflux pumps. The goal is to determine the mechanism of action of oxazolidinones in order to provide insight into the development of the next‐generation oxazolidinones. We aim to determine the effect of oxazolidinones on the translation function of ribosomes, defining which step of translation is inhibited and determining the binding site of the novel oxazolidinones.
Pharmaceutical Control of the Microbiome
Authors: Pollet Rebecca , Wallace Bret , Robers Adam , Boelsterli Urs , Mani Sridhar , Redinbo Matthew
Published: June 2021

The intestinal microbiota encode millions of genes that represent unique pharmaceutical targets for human diseases from neurological disorders to drug toxicity. We are interested in the role intestinal microbiota play in reactivating compounds that have been inactivated through glucuronidation. In particular, the chemotherapy drug CPT‐11 is inactivated by UGTs in the liver and then reactivated by bacterial Β‐glucuronidases in the large intestine resulting in dose‐limiting diarrhea. We characterized the structure and function of three Β‐glucuronidase enzymes from different bacteria and showed that a key bacterial loop allows for selective inhibition of these enzymes versus the human enzyme orthologue. We have developed, using chemical biology, novel inhibitors that potently and non‐lethally inhibit bacterial Β‐glucuronidases and stop drug toxicity of CPT‐11 in mouse models. We also examined the toxicity caused by the nonsteroidal anti‐inflammatory drugs (NSAIDs) diclofenac, ketoprofen, and indomethacin. These drugs were also hypothesized to be reactivated by bacterial Β‐glucuronidases, resulting in the acute longitudinal small intestinal ulcers that causer 40% of NSAID‐associated deaths. We found our inhibitors are also effective against this type of drug toxicity and alleviated the intestinal ulcers seen in mouse models. Thus, we have established that the microbiome contains targets for non‐lethal pharmacology to address acute problems of human health.

Funding from NIH CA98468 and NSF Graduate Research Fellowship
Characterization of an Alternative Starch Utilization System from the Human Gut Bacterium Bacteroides thetaiotaomicron and its Implications in Human Nutrition
Authors: Chaudet Marcia , Rose David
Published: June 2021

Bacteroides thetaiotaomicron is a dominant member of the human gut microbiota and has been shown to play an important role in the physiological state of the human body with influences on nutrition, pathology and immunological development. This bacterium has been recorded to have an extensive array of genes involved in the acquisition and utilization of dietary and human mucosal glycans. The ability to utilization such dietary carbohydrates were first recognized with the Starch Utilization System (Sus) and demonstrated an important metabolic cascade that allows B.thetaiotaomicron to thrive in the gut. The objective of our study is to investigate an additional starch system in B.thetaiotaomicron and critically analyze its contribution to starch utilization in the human colon. This system is predicted to function similarly to Sus with outer membrane binding of starch glycans by a SusD‐homolog, transportation of glycans into the bacterial cell by a SusC‐homolog, periplasmic hydrolysis by a GH31 and GH97 glycoside hydrolases and gene up‐regulation by a Two‐Component System regulator. Using biochemical techniques, our research has demonstrated for the first time that a SusD homolog has the ability to hydrolyze starch glycans outside the cell and assist in the utilization of starch as an energy source. Furthermore, through biochemical and structural studies, we are able to show maltooligosaccharide hydrolysis from a GH31 enzyme, complemented with a GH 97 enzyme, revealing dual alpha‐glucosidic activity in the periplasm. These mechanisms demonstrate a divergence of starch utilization and further provide supporting evidence of the evolutionary advantage this bacterium has in the human colon.
Evaluation of Different Oxidative Stress Biomarkers in Endometriosis
Authors: Castruccio Castracani Carlo , Li Volti Giovanni , Cianci Antonino , Barbagallo Ignazio , Di Simone Giuliana , Vanella Luca , Iraci Marco
Published: June 2021

Endometriosis is an estrogen‐dependent chronic inflammatory disease in which oxidative stress is one of the key factors for the disease progression. Our aim was to assess oxidative stress parameters secondary to chronic inflammation in women with endometriosis by the determination of tissue thiol groups (GSH), lipid peroxidation (LOOH) and the expression of different genes related to oxidative stress (heme oxygenase‐1 (HO‐1), inducible nitric oxide synthase (iNOS) and inflammation (IL‐6 and IL‐1b). GSH and LOOH were determined by spectrophotometric analysis whereas HO‐1, iNOS, IL‐6 and IL‐1b gene expressions were determined by real time PCR. Our results showed that GSH was significantly reduced in endometriosis group compared to controls; whereas LOOH were surprisingly lower in the endometriosis group. No significant differences were observed in GSH and LOOH levels among the different stages of the disease. In addition, our results showed that HO‐1, iNOS, IL‐6 and IL‐1b genes were more expressed in the endometriosis group. In particular IL‐6 was significantly higher in the moderate groups compared to mild and severe group whereas IL‐1b was significantly higher in the mild group compared to moderate and severe. Furthermore, oxidative stress and inflammatory biomarkers were not dependent on the localization of the disease. Taken all together, our data suggest that oxidative stress is increased in endometriosis and it is not depending on the stadiation or localization of the disease whereas oxidative stress‐related gene expression is depending on the stage of the disease.
Functional evaluation of the effects of missense and non‐sense SNPs in the p25 subunit of human dynactin complex
Authors: Misra Smita , Mittal M , Singhal A , Chaudhuri G
Published: June 2021

The working hypothesis driving this research is that a molecular defect in the retrograde receptor recycling pathway involving the dynactin complex in the cancer cells elevates the level of growth factor receptors on their outer surface resulting in aggressive growth of these cells. We have identified that suppression of the p25 subunit (DCTN5) of the dynactin complex as the defect. Aggressive breast cancer cells lacking DCTN5 have significantly higher levels of growth factor receptors such as EGFR and PAR1 on their surface. Forced expression of wild‐type DCTN5 in these breast cancer cells complemented the defect and restored the level of the growth factor receptors tested. Bioinformatics analysis revealed 10 missense and one nonsense SNPs in the coding sequence of human DCTN5. We have generated these eleven mutant derivatives of DCTN5 and evaluated the ability of each of these variants to complement DCTN5‐defect in the aggressive breast cancer cells. Forced expression of the mutant carrying the nonsense mutated SNP generated a DCTN5 variant with a C‐terminal truncation of 24 amino acids and was unable to decrease EGFR/PAR1 levels on MDA‐MB‐231 cells. These data indicate that: (i) DCTN5 is perhaps essential for the receptor recycling function of the dynactin complex; (ii) the C‐terminal 24 amino acids of human DCTN5 protein are important for the folding and/or for its interaction with other subunits in the dynactin complex; and, (iii) individuals carrying this mutation in their DCTN5 gene may have cells with high abundance of surface growth factor receptors predisposing them to the incidence to cancer. This research is supported in part by DOD‐CDMRP IDEA Expansion Grant#BC103645 and NIH/NCI grant 1R21CA181920‐01 to GC.
Specific Macrophage Targeting for MRI Using Amyloid β Oligomer‐Labeled Nanoparticles
Authors: Fuchs Ann‐Kathrin , Kumar Senthil , Büchele Berthold , Syrovets Tatiana , Fändrich Marcus , Simmet Thomas
Published: June 2021

Amyloid β (Aβ) fibrils are strongly involved in the pathogenesis of Alzheimer's diseases. The Aβ oligomers represent a non‐fibrillar intermediate state of fibril formation.Here we explored the potential of Aβ peptide oligomers as a novel class of biological nanoparticles for macrophage targeting. This study shows that Aβ(1‐40) peptide oligomers have a near‐spheroid shape and due to a β‐sheet assembly possess a compact, quasi‐crystalline architecture. Additionally, FRET analysis revealed that Aβ(1‐40) are structurally dynamic. Hence, they represent highly structured and biocompatible nanoparticles, which can be readily degraded by natural enzymes. Macrophages play a major role in immunity and tissue repair. However, recent evidence implicates them in progression of atherosclerosis, fibrosis, tumor initiation and development. In this context, specific labeling and imaging of macrophages are of special interest for diagnostic and therapeutic applications. We found that oligomeric Aβ(1‐40) is preferentially taken up by macrophages compared to peripheral mononuclear cells. This prompted us to conjugate oligomeric Aβ(1‐40) to nanoparticles composed of a superparamagnetic iron oxide (SPIO) core and a polymeric shell used to increase contrast in magnetic resonance imaging (MRI). Such functionalization enabled the preferential uptake of SPIO‐Aβ contrast agents by macrophages. Thus, functionalization with Aβ oligomers might be used to diagnose disease‐associated macrophage accumulation by MRI.
Monitoring Cellular Adenine Nucleotide Levels and Their Relevance to Cellular Metabolic Functions Using Bioluminescence based Strategies
Authors: Goueli Said , Hsiao Kevin , Mondal Subhanjan
Published: June 2021

Adenine nucleotides are major determinants of the energy status of the cell and thus any modulation of their cellular concentration has significant consequences to cellular metabolism, cellular growth and cell death. ATP generating enzymes are usually involved in catabolic processes while ATP consuming enzymes are involved in anabolic processes. Changes in the ratio of AMP: ATP tends to increase in concert with, and to an even greater extent than the ADP/ATP ratio. Therefore, the ratio of AMP: ATP could be monitored instead of/or in addition to ADP: ATP, although AMP measurement may be problematic since it is two orders of magnitude lower than those of ADP and ATP. Consequently, improving existing technologies is important for better understanding of the role of these nucleotides in metabolic regulation. Although HPLC has been the most commonly used method for determining the concentrations of several metabolites, it is fraught with problems such as the inability to accurately determine low concentrations of metabolites, existence of overlapping peaks, high baseline, need for large number of cells and long time to perform, and inability to be used for HTS programs. We developed a homogenous and bioluminescent strategy to monitor these cellular metabolites using robust HTS formatted and easy to use protocol. We have demonstrated the application of this technology in monitoring the cellular adenine nucleotides levels under various physiological conditions. We will also show the effect of altered cellular metabolites on cell viability and other enzymatic activities in response to changes in cellular adenine nucleotides
The Role of Non‐coding RNAs in Breast Cancer Resistance to EGFR Therapy
Authors: Borrero‐García Luis , Dharmawardhane Suranganie
Published: June 2021

To achieve the objective of characterizing the miRNA‐mediated mechanisms of resistance to current epidermal growth factor receptor (EGFR) targeted therapies, therapy resistant breast cancer (BC) cell lines were created by exposing therapy sensitive SKBR3 and BT‐474 human BC cells to varying concentrations of gefitinib or lapatinib. The fold resistance, as determined from the IC50s, was 2.5 and 21.5 respectively, for the SKBR3 gefitinib and lapatinib resistant cell lines and 1.9 for the BT‐474 gefitinib resistant cells. The therapy sensitive cells demonstrated higher levels of apoptosis in the presence of the therapeutics compared to the therapy resistant variants. Diferentially expressed miRNAs in therapy resistant and sensitive variants were identified from qRT‐PCR and their targets detected by Western blotting. The oncomiRs miR‐221/222 that promotes the epithelial to mesenchymal (EMT) transition, were upregulated, while miR‐200a, a miRNA that maintains the epithelial phenotype, was downregulated in the therapy resistant cell lines. Accordingly, the miR‐221/222 targets and tumor suppressors PTEN and p27 were downregulated, and the miR‐200 target β‐catenin, a pro‐cancer protein, was upregulated in the therapy resistant variants. In conclusion, this data suggest that downregulation of miR‐200 family, and upregulation of miR‐221/222 is a general mechanism of EGFR therapy resistance in BC.
An RNA Aptamer Binding With Tumor Endothelial Marker I
Authors: Kumari Abha , Yadava Pramod
Published: June 2021

The commonest strategy of dealing with cancer depends on availability of tools to detect them at early stage and selectively eliminate the cancer cells. Certain molecules differentially expressed on cancer cell surface offer a basis for developing diagnostic as well as therapeutic tools. Tumor endothelial marker I (TEM I) is one such marker expressed on surface of solid tumors. Beginning with a random sequence library we have selected for RNA molecules specifically binding with a TEMI peptide following a SELEX protocol. Surface plasmon resonance assays showed Kd for some of the TEMI‐binding aptamers to be in nanomolar range. We have also put this molecule in context with cytotoxin‐coding sequence along with an internal ribosome entry site borrowed from ras I. We hope to use this chimeric construct for targeted delivery of cytotoxin to tumor cells. This would result in targeted killing of the tumor cells and can also be used as supplement to surgical removal to ensure any transformed cells escaping surgical removal. We are also developing chimeric molecules with fluorophores for detection of TEM I on cell surface.
Milk Osmolality and Body Hydration Status in Lactating Mothers from the Western Highlands of Guatemala
Authors: Soto Mendez Maria , Maldonado Alejandra , Burgunder Lauren , Scieszka Laura , Gil Angel , Solomons Noel
Published: June 2021

Background

Human plasma osmolality is tightly regulated in the 275 – 295 mOsm/kg range and breast milk osmolality (Mosm) oscillates through a slightly higher range. Studies in dairy cows demonstrate an influence of systemic hydration on bovine Mosm

Objective

To describe hydration status in lactating women and explore any relation with Mosm

Methods

31 low‐ and middle‐income women gave a full‐breast expression after 90 min of nursing abstinence along with a concurrent spot sample of urine, 15 of whom repeated ~7 days later. Milk volume (Mvol) was measured and aliquots of urine and milk stored from 1 to 12 wks. Osmolality was determined in both fluids using a Vogel, Löser 815 osmometer expressed as mOsm/kg. Data were analyzed using SPSS version 20

Results

The Mvol ranged from 2‐100 mL, and Mosm from 288‐448 mOsm/kg, whereas Uosm ranged from 93‐1678 mOsm/kg. Nevertheless, the Spearmann rank‐order correlation coefficient was r=0.214 (p=0.153) [n=46]; Mosm was not associated with maternal age r=0.243 (p=0.104), nor with lactation age r= ‐0.280 (p=0.060). Pearson correlation for repeat Mosm was r=0.204 (p=0.233) and for Uosm was r=0.642 (p=0.005) [n=15]

Conclusions

Lactating women have higher Uosm than other sectors in Western Guatemala. Mosm is not associated with systemic hydration as reflected by Uosm. There is a high association of systemic hydration status on repeated measurement

Supported by

The European Hydration Institute, Madrid, Spain; Fundación Iberoamericana de Nutricion, Granada, Spain; and Hildegard Grunow Foundation, Munich, Germany