bluuebunny/crossref_metadata_2025 huggingface dataset:
Loss of Sil1, an ER Co‐Chaperone, Causes an Age‐Dependent Collapse of Skeletal Muscle Proteostasis, Affecting Pathways Critical for Muscle Physiology
Authors: Ichhaporia Viraj P. , Vogel Peter , Frase Sharon , Horner Linda , Hendershot Linda M.
Published: November 2023

Objective

Decipher molecular mechanism(s) underlying the myopathy associated with Marinesco‐Sjögren syndrome.

Background

Marinesco‐Sjögren syndrome (MSS) is an autosomal recessive, multisystem disorder clinically characterized by cerebellar ataxia, bilateral cataracts, and progressive myopathy. MSS is attributed to loss‐of‐function mutations in Sil1, a nucleotide exchange factor for the ER resident Hsp70 molecular chaperone‐BiP.

Methods

Using a combination of an inverted screen test, histopathology, electron microscopy, LC‐MS/MS‐based proteomics, real‐time PCR, and western blotting, we investigated the effects of Sil1 disruption in skeletal muscles of the MSS mouse model (Sil1Gt).

Results

Sil1Gt mice exhibited progressive loss of skeletal muscle mass and strength beginning around six months of age. Histopathological and ultrastructural investigations confirmed a multifocal, myogenic myopathy which predominantly affected fast myofibers and phenocopied the MSS‐myopathy. Electron microscopy (EM) revealed a severe swelling of sarcoplasmic reticular triads, autophagic vesicles, electron‐dense material suggestive of protein aggregates, and disruption of the overall cytoarchitecture.

Quantitative proteomic profiling performed on quadriceps from six‐month‐old Sil1Gt and wild‐type mice identified that 490 proteins were significantly altered, including secretory pathway proteins, as well as proteins residing in all major cellular organelles. Importantly, multiple pathways critical to muscle physiology were strikingly affected.

To validate these changes in protein expression and to determine their onset and progression, we performed western blotting and mRNA expression analyses on quadriceps derived from mice ranging from 1–18 months of age. Prior to the onset of muscle weakness, we detected increased levels of BiP and the lectin chaperones, accompanied by activation of the unfolded protein response, which became profound after six months. We observed robust increases in markers of autophagy, lysosomal biogenesis, and cytoskeletal remodeling. Conversely, components of glucose metabolism and pentose phosphate pathways were dramatically decreased. Interestingly, while levels of Insulin and Igf1 receptors ‐which are synthesized in the ER‐ were decreased, there was a progressive increase in the insulin‐regulated glucose transporter, Glut4. These changes were accompanied by increased levels of both total and phospho‐Akt, similar to data observed in murine skeletal muscles with genetic ablation of both the Insulin and Igf1 receptors.

Conclusion

These findings provide evidence for extensive cellular perturbations upon loss of Sil1 in murine skeletal muscles due to a wide‐spread proteostasis collapse, offering critical insights into pathomechanisms underlying the MSS‐myopathy.
Chaperoning the Proteome
Authors: Houry Walid A
Published: November 2023

Molecular chaperones are critical to maintaining cellular protein homeostasis. There are several families of chaperones in the cell with some being specific for certain substrates, while others act on many protein targets. As a result, chaperones are expected to be involved in many cellular pathways with only a few being well elucidated. To obtain a comprehensive view of chaperone function in the cell, we developed an integrative approach based on systematic physical and genetic interaction mapping to decipher interactions involving all main chaperones (67) and cochaperones (15) of Saccharomyces cerevisiae. The analysis revealed novel chaperone substrates and novel chaperone activities. As part of this analysis, we unexpectedly found that many interactors of the Hsp90 chaperone system are proteins that form foci under stress conditions. Such foci forming tendency was then demonstrated for the highly conserved AAA+ ATPases Rvb1 and Rvb2 that are part of the R2TP complex that interacts with Hsp90. In response to nutrient deprivation, a fraction of Rvb1 and Rvb2 colocalize to perinuclear foci that dissolve upon nutrient addition. We propose that foci formation might be a feature of many chaperone substrates.

Support or Funding Information

Canadian Institutes of Health Research
Thiol Addition to Conjugated Nitrolinoleic Acid
Authors: Alvarez Beatriz , Turell Lucia , Vitturi Dario A , Coitiño Elena Laura , Lebrato Lourdes , Moller Matias N , Sagasti Camila , Salvatore Sonia R , Woodcock Steven R , Schopfer Francisco J
Published: November 2023

Nitroalkene fatty acids are formed endogenously after nitration of unsaturated fatty acids and exert protective effects via reversible Michael addition to thiol‐containing proteins in signaling pathways. Conjugated linoleic acid is nitrated preferentially and forms 9‐ and 12‐nitro‐octadecadienoic acid (NO2‐CLA). In this work, we characterized the reactivity of NO2‐CLA with low molecular weight thiols (glutathione, cysteine, homocysteine, cysteinylglycine and β‐mercaptoethanol) and with human serum albumin (HSA). Reactions with low molecular weight thiols followed reversible biphasic kinetics, consistent with the presence of two electrophilic centers in NO2‐CLA. LC‐UV‐Vis and LC‐MS analyses showed that the fast reaction corresponds to the formation of the β‐adduct (the kinetic product) while the slow reaction corresponds to the δ‐adduct (the thermodynamic product). The rates (kon and koff) and equilibrium constants (Kd) for both reactions were determined. The pH dependencies of the rate constants, the correlations between intrinsic reactivity and thiol pKa and the absence of deuterium solvent kinetic isotope effects suggest stepwise mechanisms with thiolate attack on NO2‐CLA to form the anionic intermediates as rate‐controlling steps. The characteristics of NO2‐CLA, of the transition states and of the anionic intermediates were further investigated through computational modeling. In human urine, cysteine‐δ‐adducts were detected, which is consistent with the higher stability of the δ‐adducts. HSA was found to bind NO2‐CLA non‐covalently and to slowly form covalent adducts at Cys34. Our results provide new insights into the mechanisms that mediate not only the signaling actions of NO2‐CLA, but also its transport and storage.

Support or Funding Information

This work was supported by grants and fellowships from CSIC (Universidad de la República, Uruguay) (to LT, MNM and BA), Agencia Nacional de Investigación e Innovación (ANII, Uruguay) (to LL), NIH grants K01‐HL133331 (DAV), R01‐AT006822 (FJS) and AHA #14GRNT20170024 (FJS).
Differential Protein Splicing of Salt Dependent Inteins from Haloquadratum Walsybi
Authors: Lynch Abigeal Kathleen , Amunya Shirley , Reitter Julie , Mills Kenneth
Published: November 2023

Protein splicing is a self‐catalyzed, post‐translational process in which an intervening polypeptide (intein) catalyzes its own removal from the flanking polypeptides (exteins), as well as the ligation of the exteins. We are interested in the splicing of two inteins interrupting the cdc21 protein from Haloquadratum walsbyi (Hwa). Hwa is a halophilic archaeon. Because of this, we hypothesized that protein splicing would be salt dependent. The first intein, Cdc21a, does not splice on overexpression in E. coli, but can be induced to splice in vitro on incubation with 2.5 M sodium chloride at 28°C. The second intein, Cdc21d, splices in vivo, and further splicing can be induced in vitro with higher concentrations of salt. Although the first intein requires 2.5 M salt to splice, Cdc21d requires only 500 mM salt to induce additional splicing. Given that both inteins interrupt the same protein, this differential activity could result in different protein products due to alternative splicing in the full precursor. We are studying this relative activity when the two inteins are expressed in the same precursor fusion protein.

Support or Funding Information

This work was supported by the National Science Foundation (grants MCB‐1244089 and MCB‐1517138 to KVM) and the Dreyfus Foundation (KVM).
A structural investigation and splicing study of inteins from Halobacteria
Authors: Zhang Wanlin , Lynch Abigeal K. , Gomez Alvin O. , Reitter Julie N. , Mills Kenneth V.
Published: November 2023

Protein splicing is a post‐translational modification by which a segment of protein, known as an intein, self‐catalyzes its excision from the flanking protein while simultaneously ligating the two flanking exteins. Inteins can be found in many types of archaea, such as halophiles, which specifically thrive in environments with high salt concentrations. The lab previously reported that the Halobacterium salinarum (Hsa) DNA polymerase II (Pol II) intein promotes conditional protein splicing at high concentrations of sodium chloride. To study the structure of the intein, we attempted to find conditions for the formation of protein crystals of the unspliced Hsa fusion protein under conditions of different precipitating solutions. Furthermore, we also attempted to learn if splicing of an intein interrupting the Replication Factor C (RFC) from Haloquadratum walsbyi (Hwa) is also salt dependent.

Support or Funding Information

This work was supported by the National Science Foundation (grants MCB‐1244089 and MCB‐1517138 to KVM) and the Dreyfus Foundation (KVM).
Role of Cys292 in Coupling ATP hydrolysis to RNA Unwinding Catalyzed by the Hepatitis C Virus Helicase
Authors: Yerukhimovich Mark M. , Frick David , Marohnic Christopher
Published: November 2023

The DECH sequence, in the second conserved motif, shared by all superfamily II helicases, is well known to assist the hydrolysis of ATP needed to fuel helicase movements. The aspartate, glutamate and histidine are clearly needed to activate the attacking water molecule and coordinate the divalent metal cation that bridges the protein to ATP. However, the role of the cysteine is not clear from available crystal structures. In order to examine the role of the cysteine, we used site directed mutagenesis to change Cys292 to either a serine or a glycine in the hepatitis C virus NS3 helicase, which is one of the best studied enzymes in this family. These C292S, and C292G, proteins were then compared to wildtype and proteins in which other cysteines were altered (C374S, C499S, C525S, C622S). We also analyzed the activity of three double mutants (C292S+C374S, C292S+C499S, C292S+C525S, and C292S + C622S) to explore the possibility that Cys292 is needed for a critical disulfide bond formation. All proteins had a similar affinity for ATP and RNA (or DNA). All also unwound DNA and hydrolyzed ATP in the presence of RNA at similar rates. However, in the absence of RNA, all the proteins lacking a cysteine at position 292 hydrolyzed ATP 5–10 times faster than the others, suggesting Cys292 is critical for reorienting the Asp, Glu, and His residues in the DECH sequence upon RNA binding to the helicase. We are presently analyzing how the Cys292 mutation influences the helicase step size and processivity.
Purification and Characterization of Flavin Reductase, DszD, from Rhodococcus erythropolis.
Authors: Mendez Gilberto
Published: November 2023

Flavin reductases are found in all organisms, and catalyze essential reactions in all domains of life. Flavins such as flavin mononucleotide, flavin adenine dinucleotide, and riboflavin are essential for many metabolic processes and are best known for their role in oxidation‐reduction reactions. We are working on crystallizing and assessing the catalytic capabilities of the NADH‐dependent flavin reductase, DszD, from the biodesulfurizing bacterium Rhodococcus erythropolis. In our work, DszD has been successfully overexpressed and purified essentially to homogeneity from E. coli. We have verified the substrate preferences of DszD, showing the enzyme has maximal activity with flavin mononucleotide. DszD was subjected to a sparse matrix crystallization experiment using the hanging drop vapor diffusion method. Promising crystallization conditions were identified, replicated, and are currently being optimized to improve crystal quality. Once solved, the structure of DszD will lead to the identification of the active site residues important for substrate binding and catalysis.

Support or Funding Information

This work was funded by the Research Corporation through CCSA 22672 and the NIH through Grant 5SC2AI109500.
Preliminary Characterization of Pyrimidine Biosynthesis Protein Aspartate Transcarbamoylase (ATC) in Pseudomonas aeruginosa
Authors: Patel Chandni , Hachem Fatme A , Vaishnav Asmita , Edwards Brian FP , Evans Hedeel Guy , Evans David R
Published: November 2023

Aspartate transcarbamoylase (ATC) in Pseudomonadaceae is a class A enzyme, a dodecamer consisting of six copies of a 36‐kDa catalytic chain and six copies of 45‐kDa polypeptide of unknown function. The 45‐kDa polypeptide is homologous to dihydroorotase but lacks dihydroorotate activity. Pseudomonas aeruginosa aspartate transcarbamoylase catalytic and pseudo‐dihydroorotate (pDHO) subunits containing his‐tag were overexpressed separately in Escherichia coli and purified using nickel affinity column chromatography. ATC catalytic subunit is inactive in the absence of pDHO suggesting that ATC catalytic subunit and p‐DHO need to form a complex in order to form a functional and active ATC. Size exclusion chromatography result suggests that a complex, likely a dodecamer, is formed between ATC and pDHO. The carbamoyl phosphate saturation curve of P. aeruginosa at fixed concentration of aspartate was hyperbolic with a Km for carbamoyl phosphate of 2.2 mM and a Vmax of 33.3 μmol/min/mg. When carbamoyl phosphate saturation assays were performed with PALA (N‐phoshonacetyl)‐L‐aspartate), a bisubstrate analog that resembles an intermediate along the pathway of catalysis, 0.5 μM PALA completely inhibited the enzyme, whereas 1 nM had no inhibitory effect. With increasing PALA concentrations, carbamoyl phosphate saturation curves became strongly sigmoidal suggesting strong cooperative interactions between subunits. The N‐ and C‐terminal regions of P. aeruginosa ATC are highly disordered. Our hypothesis is that the disordered regions of ATC might be responsible for its inactivity and that the deletion of these highly disordered terminal ends of ATC will lead to its activation without the need of pDHO.
Using Dynamics and Structure to Understand Allostery in Signaling Enzymes
Authors: Peti Wolfgang
Published: November 2023

Protein function originates from a cooperation of structural rigidity, dynamics at different timescales and allostery. However, how these three pillars of protein function are integrated is still only poorly understood. Here we show how these pillars are connected in phosphorylation enzymes, specifically in the protein tyrosine phosphatase PTP1B and in serine/threonine kinases. Only by combining a large array of biophysical tools with NMR having a leading role, it is possible to unravel how these enzymes are regulated and function. We will present how dynamics at different timescales drive the enzymatic cycle of these enzymes as well as show how allostery controls their enzymatic activity.

Support or Funding Information

This work was supported by the American Diabetes Association Pathway to Stop Diabetes Grant 1‐14‐ACN‐31, NIH grants R01GM098482, R01NS091336 and grants from the Lundbeck and Novo Nordisk Foundations.
A Structure‐Based Mechanism for Oxidative Decarboxylation Reactions Mediated by Amino Acids and Heme Propionates
Authors: Celis Arianna Ixcheel
Published: November 2023

Coproheme decarboxylase catalyzes two sequential oxidative decarboxylations with H2O2 as the oxidant, coproheme III as substrate and cofactor, and heme b as the product. Each reaction breaks a C‐C bond and results in net loss of hydride, via steps that are not clear. Solution and solid‐state structural characterization of the protein in complex with a substrate analog has revealed a highly unconventional H2O2‐activating distal environment with the reactive propionic acids (2 and 4) on the opposite side of the porphyrin plane. This suggests that, in contrast to direct C‐H bond cleavage catalyzed by a high‐valent iron intermediate, the coproheme oxidations must occur through mediating amino acid residues. A tyrosine that hydrogen bonds to propionate 2 in a position analogous to the substrate in ascorbate peroxidase is essential for both decarboxylations, while a lysine that salt bridges to propionate 4 is required solely for the second. A mechanism is proposed in which propionate 2 relays an oxidizing equivalent from a coproheme compound I intermediate to the reactive deprotonated tyrosine, forming Tyr■. This residue then abstracts a net hydrogen atom (H■) from propionate 2, followed by migration of the unpaired propionyl electron to the coproheme iron to yield the ferric harderoheme and CO2 products. A similar pathway is proposed for decarboxylation of propionate 4, but with a lysine residue as an essential proton shuttle. The proposed reaction suggests an unprecedented relay of heme‐mediated e−/H+ transfers and a novel route for the conversion of carboxylic acids to alkenes.
An Essential Oil Blend Specifically Enhances Immune Responses in Human Cancerous Co‐Cultures
Authors: Han Xuesheng , Parker Tory
Published: November 2023

Essential oils, composed of aromatic compounds naturally found in plants, have been used in folk medicines to prevent and treat a variety of diseases in humans all over the world for many hundreds of years. Despite growing scientific evidence that essential oils possess important therapeutic benefits, research on their biological activities in complex human disease models is scarce. To contribute to this understanding, we analyzed the biological activities of an essential oil blend (EOB) in validated human cell co‐cultures, with or without tumor cells. These disease models allow for measurement of changes in protein biomarkers induced by EOB treatment. The EOB showed significant effects on important biomarkers related to inflammation, immune system function, wound healing, and tumor biology. In tumor co‐cultures, the EOB elevated inflammatory and immune‐related biomarkers, including sIL‐17A, sIL‐2, sIL‐6, VCAM‐1, CD‐40, CD‐69, sGranB, and sIFNγ, but many of these same biomarkers were decreased by the EOB in non‐tumor cell co‐cultures, suggesting that the EOB shows tumor‐specific immune enhancement. The EOB also broadly impacted genome‐wide gene expression in a dermal fibroblast cell model, as measured via total mRNA microarray assays. Finally, the EOB also pleiotropically regulated multiple signaling pathways in human cells, such as the hepatic fibrosis/hepatic stellate cell activation pathway, the antigen presentation pathway, the mitotic roles of polo‐like kinase pathway, and the cyclins and cell cycle regulation pathway. In summary, the study provided evidence suggesting that the EOB can beneficially impact human cells through anti‐inflammatory activities, immune enhancing functions, modulation of wound healing, and anticancer properties.

Support or Funding Information

This study was funded by dōTERRA International LLC, Pleasant Grove, UT, USA.
Mechanism and in vivo Activity of a Covalent Inhibitor of ERK Docking
Authors: Dalby Kevin N , Anslyn Eric V , Zamora‐Olivares Diana , Kaoud Tamer
Published: November 2023

ERK is a member of the mitogen‐activated protein kinase (MAPK) family, which consists of three main classes of enzyme; the extracellular signal regulated kinases (ERKs), the Jun N terminal kinases (JNKs), and the p38 MAPKs. Melanoma is often driven by ERK pathway activation, with high ERK activity, a hallmark of melanomas harboring activating BRAF and NRAS mutations. Vemurafenib and dabrafenib are BRAF inhibitors, and trametinib is a MEK inhibitor used for the treatment of BRAF mutant melanoma. These drugs are clinically effective in the short term and result in ERK pathway inhibition. Importantly, a high degree of ERK inhibition correlates with clinical response (tumor shrinkage). ERK pathway reactivation is a hallmark of many melanomas that have acquired resistance to BRAF and MEK inhibitors and consequently progress. Therefore, it is of interest to develop novel methods to inhibit ERK activity directly. ERK signaling is mediated by a critical protein docking site that lies outside the catalytic domain. We describe a small molecule inhibitor of ERK that binds this docking site covalently, to suppress ERK signaling in drug resistant melanoma cells and in a xenograft model of melanoma. We provide detailed characterization of its mechanism of inhibition and also describe new approaches for the quantification of ERK activity in tumor samples using fluorescent sensor arrays.

Support or Funding Information

RP 140648, RP 140649, RP160657, Cancer Prevention Research Institute of Texas (CPRIT)
Characterization of a cardiac drug‐inactivating enzyme from the prominent human gut microbe, Eggerthella lenta
Authors: Koppel Nitzan , Bisanz Jordan , Turnbaugh Peter , Balskus Emily P
Published: November 2023

Metabolism of pharmaceuticals by the human gut microbiota has been appreciated for several decades. Currently, over 50 examples are known in which the pharmacological properties, bioactivities, and toxicities of a drug are altered by the gut microbiota. However, only a few of these examples have been linked to specific bacteria or enzymes, and the precise mechanisms of these transformations remain largely unknown. The gut microbiota harbors approximately 150× more genes than the human genome providing additional metabolic capacity to modify drugs beyond host metabolism. In particular, gut microbial chemistry tends to be reductive and hydrolytic, in contrast to oxidative and conjugative host metabolism of xenobiotics. Additionally, the composition of the microbiota can vary substantially among individuals, leading to differences in drug metabolism. A deeper knowledge of microbial drug metabolism at the molecular level is thus crucial for understanding variability in patient response to drugs, and improving efficacy in the clinic.

In our work, we biochemically confirmed that Cgr2, a putative cardiac glycoside reductase from the prominent gut microbe Eggerthella lenta, is responsible for reduction and inactivation of the widely used cardiac drug, digoxin. Cgr2 is a divergent homolog of FAD‐dependent fumarate reductases and harbors Fe‐S clusters with non‐canonical binding motifs that could not be predicted computationally. Bioinformatics and network analyses also revealed that Cgr2 is a highly unique enzyme that likely employs a novel mechanism of substrate binding and reduction. Through microbial isolation and sequencing of human‐associated isolates, we determined that the cgr2 gene is found exclusively in a subset of E. lenta strains, and is remarkably conserved at the amino acid sequence level (>95% identity). Digoxin metabolism does not enhance E. lenta growth, and more work is required to understand the physiological implications of this highly conserved gene. We are also actively working to determine the mechanism of substrate reduction through biochemical and structural methods.

Support or Funding Information

Funded by Harvard University, National Science Foundation, Packard Fellowship for Science and Engineering, George W. Merck Fund.
Affinity Ligand for the Separation of Glycosylated Proteins from Non‐Glycosylated Proteins
Authors: Gunter Amy , Mammoser Claire , Dhar Shilpa , Rowe Laura
Published: November 2023

When studying glycosylated proteins, a method of separating glycosylated proteins from other proteins is crucial for purifying the sample. Affinity chromatography is currently performed most commonly for glycoprotein purification, through the use of boronic acid based, or lectin derived, ligands. However, more specific and economical ligands would be preferred. One such ligand described here is an affinity ligand that binds to glycosylated proteins and the monosaccharide α‐D‐mannopyranoside, synthesized through an Ugi‐multicomponent reaction. The ligand is composed of tryptamine, 1‐benzyl‐1H‐indole‐3‐carboxylic acid, isopropyl isocyanide, and an aldehyde‐functionalized Sepharose bead. The ligand's structure was selected via performance in a previously produced combinatorial library. For this work, after aldehyde functionalizing Sepharose beads and completing the Ugi reaction, the binding ability of the ligand is tested with gravity packed chromatography columns using glucose oxidase as the protein of interest. Then a Bradford assay is conducted in order to determine the amount of glycosylated protein (glucose oxidase) that bound to the affinity ligand during a positive and negative screening process. The ligand was initially synthesized and found to bind the glycoprotein glucose oxidase well in an inert buffer, with glycoprotein binding being reduced when a competitive ligand was added to the equilibrium buffer. Reproducibility of ligand synthesis and binding behavior is being assessed with multiple, separate ligand synthesis reactions and screens in order to determine optimal synthetic steps and assess the degree of sugar specificity in the ligands ability to bind the glycoprotein glucose oxidase.
New strategies for the expression of human P‐glycoprotein (MDR1) in the yeast Pichia pastoris
Authors: de Oliveira Maisa C. , Lavigne Collette A. , Thornton Henry A. , Tran Brandon M. , Wise John G. , Vogel Pia D.
Published: November 2023

P‐glycoprotein (P‐gp) overexpression is often correlated with multidrug‐resistance (MDR) of cancer. The search for P‐gp inhibitors as co‐therapeutics to combat MDR has had little success after decades of studies. We previously used high‐throughput in silico ligand docking studies and identified drug‐like compounds that reversed MDR in human cancer cells in culture. Effects on P‐gp ATPase hydrolysis activity was used as an initial biochemical screen for inhibitor effectiveness followed by assays of MDR‐reversal of a prostate cancer cell line ((Brewer et al. in Mol Pharmacol. 2014; Follit et al in Pharmacol Res Perspect. 2015). More recently, computationally identified compounds were shown to reverse MDR in cancer cells, but did not appear to inhibit ATP hydrolysis by isolated P‐gp. One reason may be that mouse P‐gp rather than human P‐gp was previously used in our biochemical tests. Expression of human P‐gp in Pichia pastoris in a biologically active form was previously reported, but unfortunately, we experienced very modest expression levels of the human P‐gp expression construct for P. pastoris available in our lab. Codon optimization of mouse P‐gp was previously shown by others to result in increased protein yield, therefore a gene for human P‐gp was designed with optimized codon usage for translation in P. pastoris. A strong Kozak sequence was incorporated at the 5′ end of the coding frame. Unique internal restriction sites were incorporated that allow subcloning of individual sections of the gene, if necessary for troubleshooting and future mutagenesis. The DNA was synthesized using standard Gibson assembly methods and cloned into a pUC57 cloning vector. When transformed into E. coli, DNA rearrangement was observed. To identify whether a specific section of the insert was the cause of counter‐selection in E. coli, the insert DNA was digested into 5 component pieces and each one was subcloned. One of the sections was found to be more susceptible to rearrangements. We report here our attempts at constructing a stable, codon‐optimized, human P‐gp gene for high yield protein production in Pichia pastoris.

Support or Funding Information

This work is supported by NIH NIGMS [R15GM094771‐02] to JGW/PDV, SMU University Research Council, SMU Engaged Learning program, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.
Determining the LC50 of a Novel MetAP Inhibitor in Lung Carcinoma Cells
Authors: Addison Angela Patricia , Hayden Nicholas Francis , Perli Elias , Erwin Emily , Chow Gordon , Vu Lan , Bhalla Sean , Condic Natalia , Olaleye Omonike , Rosell Rosemarie
Published: November 2023

The rise of multi‐drug resistant bacterial infections grows at an alarming rate, thus necessitating the discovery of new antibiotics and antimicrobials. Dr. Omonike Olaleye, a colleague from Texas State University, discovered 75 compound inhibitors against Methionine aminopeptidase (MetAP) after a high‐throughput screen. MetAP is a universally conserved, essential protein that removes the initial methionine from a newly synthesized polypeptide, which allows for further modification to form a completed protein; studies have indicated that MetAP deletion is lethal. Interestingly, MetAP is conserved across species, and prokaryotic and eukaryotic MetAP are dissimilar enough to make MetAP an attractive potential therapeutic target. This project is currently focused on a compound (UST‐001) efficacious in inhibiting Mycobacterium tuberculosis; however, the effect on mammalian cells is unknown. Relative toxicities must be determined before further tests can be conducted. We determined the in‐vitro LC50 of UST‐001 on a human lung carcinoma (H1299) line to be 3.2μM by using a combination of an MTT assay and flow cytometry. However, because the H1299s are a cancerous and thus drug resistant line, the experimental calculated value may not be representative of the MetAP inhibitor's toxicity in a non‐cancerous cell line. We plan to overcome this limitation in the future by using a primary lung cell line, and to compare the relative toxicities between both cell lines. The data gathered from this project will facilitate further research in determining whether or not this UST‐001 will be a viable clinical candidate.
Optimization of a Medium‐Throughput Cathepsin K Inhibition Assay for NAAA Inhibitors
Authors: Golden Emily Rose , Miyabe Christina , Wood JodiAnne , Malamas Michael , Alapafuja Shakiru , Makriyannis Alexandros
Published: November 2023

Cathepsin K and N‐acylethanolamine‐hydrolyzing acid amidase (NAAA) are members of the cysteine protease family. While NAAA is a metabolic enzyme in the endocannabinoid system, Cathepsin K is involved in bone reabsorption and the release of calcium into the bloodstream. Our interest in the endocannabinoid system has resulted in significant NAAA inhibitor development for the treatment of inflammation and the resulting chronic pain and has prompted our interest in Cathepsin K for off‐target selectivity screening. A medium‐throughput fluorescent assay to determine Cathepsin K inhibition was optimized to screen NAAA‐lead compounds using Omnicathepsin (aka Z‐Phe‐Arg‐AMC) as the fluorogenic substrate. The concentrations of enzyme (2 nM Cathepsin K) and substrate (25 μM Omnicathepsin) were optimized and the Km of Cathepsin K and Omnicathepsin was found to be 9.8 μM. Two published compounds, Odanacatib and BML‐244 with IC50 values of 0.54 ± 0.4 nM and 317 ± 41 nM respectively, were developed to be reference compounds in the inhibition 3‐point screen. The optimized assay was then used to screen NAAA‐inhibitors for competitive inhibition of Cathepsin K in order to create more potent, specific drugs for NAAA.

Support or Funding Information

This project was funded by NIH: 2‐R44‐DK104636, NAAA Inhibitors as Anti‐Inflammatory Agents, Shakiru Alapafuja, PI; Northeastern PI, JodiAnne T. Wood.
Viable Strategy for Inhibition of Death Receptor 5 Signaling by Disrupting Receptor‐Receptor Interactions
Authors: Vunnam Nagamani , Lo Chih Hung , Grant Benjamin D. , Thomas David D. , Sachs Jonathan N.
Published: November 2023

Therapeutic targeting of TNFR signaling in inflammatory diseases is a billion‐dollar industry. Current treatments for TNF‐related diseases function either by sequestering ligand or blocking ligand‐receptor interactions, which can cause adverse effects by inhibiting the receptors that are not involved in the disease condition. In the current study, we prove the viability of an alternative approach, namely the targeting of receptor‐receptor interactions. We use the soluble extracellular domain of Death Receptor 5 to block endogenous receptor assembly, mimicking the biological effect of decoy receptors that lack the intracellular signaling machinery to trigger apoptosis. Such a strategy should be generally applicable to other members of the TNFR superfamily, as well as to oligomeric receptors in general.

Support or Funding Information

This work is supported by National Institutes of Health Grant R01 GM107175
Improving the Sensitivity of P‐Glycoprotein to Drug‐Like Inhibitors in ATPase Assays and ESR Studies
Authors: Chen Gang , Ballou‐Crawford Jett , Wise John G. , Vogel Pia D.
Published: November 2023

Multidrug resistance (MDR) in cancers is frequently associated with transmembrane efflux proteins including P‐glycoprotein (P‐gp), multidrug resistance associated protein (MRP1), and breast cancer resistance protein (BCRP). Over‐expression of these transporters in cancer cells increases efflux of therapeutic drugs rendering them ineffective. In order to re‐sensitize multidrug resistant cancers to chemotherapy, we have found inhibitors of P‐gp by in silico screening methods (Brewer et al, Mol. Pharmacol. 2014). To investigate the mechanism underlying the inhibition, we have employed biochemical assays and electron spin resonance spectroscopy (ESR), and have established nanodisc technologies that incorporate and stabilize P‐gp in native‐like phospholipid bilayer environments for our drug finding and mechanistic studies. In ATP hydrolysis assays using P‐gp in micelles, we found that the concentration of detergents used to solubilize P‐gp during purification had a significant impact on the sensitivity of P‐gp to the putative inhibitors. We hypothesized that partitioning of the compounds into empty micelles may decrease the effective concentration of the potential inhibitors. Carefully decreasing the amount of detergents used during purification resulted in significantly increased sensitivity of P‐gp to the inhibitors, while overall protein activity and stability were not affected. Similarly, excess lipids used during reconstitution of P‐gp into nanodisc may cause partitioning of inhibitors into empty nanodiscs or empty lipid vesicles, resulting in lowered sensitivity of P‐gp to the inhibitors. By optimizing the amount of lipids used during nanodisc assembly, we restored the sensitivity of P‐gp to the inhibitory compounds. Biophysical studies using ESR and a spin‐labeled analog of ATP showed that the computationally identified compounds inhibited P‐gp by binding to the ATP binding domain of P‐gp, which was consistent with our predictions by in silico screening method.

Support or Funding Information

This work is supported by NIH NIGMS [R15GM09477102] to JGW/PDV, SMU University Research Council, SMU Engaged Learning program, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.
Enzyme‐catalyzed Expressed Protein Ligation
Authors: Henager Samuel , Chu Nam , Chen Zan , Bolduc David , Dempsey Daniel , Hwang Yousang , Wells James , Cole Philip
Published: November 2023

Expressed protein ligation (EPL) is an established method that exploits a chemoselective ligation between a recombinant protein thioester generated with an intein, and an N‐Cys‐containing synthetic peptide. However, the requirement of a cysteine at the ligation site limits the utility of EPL. Here we show the ligation of cys‐free peptides to protein thioesters by subtiligase, a previously described, engineered form of the bacterial protease subtilisin that has been converted to a peptide ligase. This method allows us to more accurately determine how C‐terminal phosphorylation affects the tumor suppressor protein PTEN.
Analysis of the Stability of Natural and Unnatural Amino Acids in Extraterrestrial Conditions
Authors: Mammoser Claire , Brown Bayland , Dhar Shilpa , Rowe Laura
Published: November 2023

In addition to the 20 natural amino acids used in the genetic codes of most biological organisms, there are hundreds more non‐proteinogenic, or “unnatural” amino acids which could be used in the genetic codes of organisms evolving under conditions different than those on Earth. However, in order for a protein based extraterrestrial life‐form to have evolved in the first place, the amino acids within the organism must be relatively stable in the extraterrestrial environmental conditions In this work, the stability of ten unnatural amino acids and five representative natural amino acids has been tested under a variety of extraterrestrial conditions, such as heat, cold, pH variation, and UV radiation. The environmental conditions selected were intended to potentially mimic the conditions on the three most likely locations of life development within our Solar System: the ice caps of Mars, and the subsurface oceans of the moons Europa and Enceladus. After the amino acids were subjected to these conditions while in solution, their stability was quantified using Ultra‐Performance Liquid Chromatography with UV‐Vis and MS detectors. The area of the parent amino acid peak (determined through MS) on the UV‐Vis chromatogram could then be described as a percentage of the area of all non‐solvent peaks, with any non‐solvent, nonparent peaks representing degradation of the original amino acid. Ultimately, trends in stability could indicate favorable traits for amino acids to possess in order to survive on other planets and moons.

Support or Funding Information

This work has been supported by an Indiana Space Grant consortium grant.
An interdisciplinary investigation of antimicrobial peptides
Authors: Wadhwa Raoul Rajesh , Stevens‐Truss Regina
Published: November 2023

Antimicrobial peptides (AMPs) have become a subject of research interest as a novel class of antimicrobial agents to complement conventional antibiotics. Because of their versatility and the low rate at which resistance against them develops, new research interest in them has been spurred. This study used an interdisciplinary approach to investigate the effect that the primary sequence of a short, cationic, alpha‐helical peptide has on antimicrobial activity. Computational and statistical methods were used to design peptide sequences that were complemented by experimental methods designed to assess antimicrobial activity. An analysis of data contained in two AMP databases (Antimicrobial Peptide Database (APD) and Collection of Anti‐Microbial Peptides (CAMP)) revealed that AMPs possess a number of factors in common that can explain their actions. Based on these factors, two test peptides were designed based on hydrophobic moment optimization and overall hydrophobicity, and tested for their ability to prevent bacterial cell growth, lyse red blood cells (RBCs), and liposomes. Software created for peptide structural analysis allowed for prediction of short AMP secondary structure through periodicity in the hydrophobicity as a function of the angle subtended. The experimentally tested peptides were both found to possess antimicrobial properties, but were also found to lyse RBCs and liposomes making them unfit for clinical use in their current state. However, the results of this study demonstrate that one can design short peptide sequences that mimic the actions of natural AMPs. With additional manipulation of specific properties, the primary sequence of the AMPs could be potentially tweaked to affect only microbes. This could open up a world of potential therapeutic agents useful in combating a host of microbial organisms, in particular multidrug‐resistant bacterial strains.

Support or Funding Information

F.W. & Elsie Heyl Foundation Summer Grant.
Lasertherapy after induction of myocardial infarction by coronary artery occlusion in rats: a genetic signature
Authors: Feliciano Regiane Santos , Manchini Martha T , Santana Eduardo T , Maretti Joao V , Caproni Larissa I , Antonio Ednei , Serra Andrey J , Tucci Paulo J , Silva Jose A
Published: November 2023

Myocardial infarction (MI) promoted by occlusion of the anterior descending coronary artery is the most widely used model in experimental research that seeks to elucidate the functional, structural and molecular changes associated with ischemic heart disease. Previously, we observed that low‐level laser therapy (LLLT) was able to decrease MI area and ameliorate cardiac function pot‐MI. The aim of this study was to assess a molecular signature regarding to mRNA expression of several signal transduction pathways after lasertherapy. Thus, we analyzed 48 rats that underwent to left ventricle coronary artery occlusion followed by laser irradiation in the occlusion site. Three days after surgery, we measured the infarct area and function by echocardiography followed by euthanasia. Three groups were studied as followed: Sham (n=5), MI (n=23) and MI+Laser (n=20). Remote area from MI site (septum) was removed from each rat and changes in mRNA expression were analyzed by microarray TaqMan Real‐time PCR. In‐silico analyses were used to identify genes involved in metabolic pathways related to MI in rats resulting in 47 genes of 9 metabolic pathways (angiogenesis and cell survival, hypertrophy, oxidative stress, apoptosis, extracellular matrix, Ca2+ kinetics, cell metabolism and inflammation). MI and the MI+Laser groups presented 22 (44.60%) and 12 (25.53%) differentially expressed genes, respectively, compared to Sham group. 15 genes (71.42%) of the IM group were upregulated and 6 (28.57%) were downregulated. In the IM+Laser group, 6 genes (50%) were upregulated and 6 (50%) were downregulated. IM + Laser group, when compared to the IM Group, presented 14 differentially expressed genes, all of them downregulated. Our results suggest that lasertherapy was able to modulate several post‐MI processes, including apoptosis, inflammation, cardiac hypertrophy, calcium kinetics and especially those related to cardiac remodeling. To the best of our knowledge, this was the first study that comprised a global analysis of several mRNA expression post‐MI and lasertherapy. Thus, the screening of initial mRNA expression profiles after laser irradiation in acute MI contributed to better understanding the beneficial effect of lasertherapy in the myocardium.

Support or Funding Information

This work was supported by grants number 2009/54225‐8, 2012/15808‐0, and 2015/11028‐9 from São Paulo Research Foundation (FAPESP) and National Council for Scientific and Technological (CNPq grant numbers 477458/2009‐2 and 479395/2012‐8). The authors would like FAPESP, CNPq, UNINOVE and UNIFESP for all support.
Using Transcriptomics for Biomarker Discovery Enabling High‐Throughput Drug Screening of iPSC‐Cardiomyocytes Modeling a Cardiomyopathy
Authors: Rowland Teisha J , Hashem Sherin I. , Jones Kenneth , Bonham Andrew J. , Jett Susan , Adler Eric , LaBarbera Daniel , Mestroni Luisa , Taylor Matthew
Published: November 2023

Danon disease is an untreatable and severe X‐linked form of cardiomyopathy that leads to profound cardiac hypertrophy, causing death or a need for cardiac transplantation in males by the third decade. Danon disease is known to be caused by loss of the lysosome‐associated membrane protein‐2 (LAMP‐2) protein, which is a membrane glycoprotein likely involved in lysosome‐autophagosome fusion and vesicle trafficking during autophagy. However, it is unknown how LAMP‐2 protein deficiency leads to the development of Danon disease. To better characterize the molecular and cellular pathogenic mechanisms involved and enable biomarker discovery for high‐throughput screening (HTS) of compound libraries, human induced pluripotent stem cells (iPSCs) were generated from patients with Danon disease and differentiated into cardiomyocytes. RNA transcription levels from the Danon iPSC‐derived cardiomyocytes (iPSC‐CMs) were analyzed using RNA‐Sequencing (RNA‐Seq) and expression of key genes confirmed via quantitative real‐time PCR (qRT‐PCR). The data show significant upregulation of genes involved in cellular respiration and sarcomere expression in Danon iPSC‐CMs compared to unaffected control iPSC‐CMs. The upregulation of cellular respiration transcripts in our data align with published data suggesting mitochondrial function is affected in these cells, including increased levels of mitochondrial oxidative stress. While biomarker discovery has previously been a hurdle for successfully undertaking HTS drug discovery using these cells, our RNA‐Seq findings of potentially impacted cellular functions in the Danon iPSC‐CM model can be used to enable HTS drug discovery by identifying novel biomarkers related to mitochondrial function and mitophagy. Following development and validation of a primary HTS assay, a pilot HTS using a library of 355 kinase inhibitors and then a HTS drug discovery using a custom designed diversity library of 20,000 compounds will be performed and hits confirmed using secondary and tertiary assays. After additional in vivo validation, these confirmed hits may offer novel potential therapeutic treatments for individuals with this currently devastating and untreatable cardiomyopathy.

Support or Funding Information

This work was supported by European Union grant FP7‐PEOPLE‐2011‐IRSES 291834 SarcoSI and U.S. National Institutes of Health grants UL1 RR025780, UL1 TR001082, R01 HL69071, and R01 116906 (to Dr. Mestroni); and grants CCTSI K23 JL067915 and R01HL109209 (to Dr. Taylor). This work was supported in part by a Trans‐Atlantic Network of Excellence grant from the Leducq Foundation (14‐CVD 03).
Validation of single nucleotide polymorphisms in Physcomitrella patens
Authors: Adler Keith , Hicks Karen A
Published: November 2023

In this experiment we use the moss, Physcomitrella patens, to investigate the effect of seasonal cues on sexual maturation. In particular we are interested in uncovering the ancestral origins of this mechanism. Sexual maturation in angiosperms (flowering plants) is regulated by both photoperiod and ambient temperature. These cues influence the regulation of genes involved in the sexual development pathway. Variation in the influence of seasonal cues on the sexual maturation between P. patens populations suggests that genetic variation may influence the different responses to seasonal cues. We sequenced and aligned some of the known European P. patens accessions due to these differences. This sequencing identified a dataset of single nucleotide polymorphisms (SNPs) between the individual samples. In this study, we are confirming the quality of the SNP data set previously generated. We identified candidate genes, based on high SNP prevalence or potential influences in the pathway, for validation of proposed SNPs by Sanger Sequencing. This knowledge will be useful in identifying specific genes of interest that participate in this mechanism.
Analysis of the rates of transcriptional coupling and translational fusion between hydroxylase and rubredoxin subunits of alkane monooxygenase in bacterial genomes
Authors: Masuda Hisako , Rikard Matthew , Tupa Peter R
Published: November 2023

Two functionally related proteins typically can be translationally fused to form a large protein or encoded as separate polypeptides, yet share transcriptional regulation by forming an operonic structure. In operon, multiple proteins are encoded from a single transcript, allowing these proteins to be expressed at the relatively fixed stoichiometric ratio. The goal of this study was to assess the relative rate of formation and disassembly of operons (transcriptional coupling) and gene fusions (translational fusion) of the hydroxylase and rubredoxin subunits of alkane monooxygenase in published bacterial genomes. Alkane monooxygenase is a non‐heme diiron oxygenase that catalyzes the initial, rate limiting step of linear alkane metabolism in aerobic bacteria. It is widely found both in gram negative and gram positive bacteria. The relative abundance of various genetic structure of alkane monooxygenases has not been studied previously. Our study indicates that the transcriptional coupling proceeds translational fusion in all detected cases. The rate of disassembly was faster than the formation in both processes. Moreover, the rate of disassembly of operon and fusion occurred at an equivalent rate. The possible biological basis for the faster rate of disassembly and its implications in horizontal gene transfer will be discussed.