bluuebunny/crossref_metadata_2025 huggingface dataset:
Fine‐tuning the Selectivity of Pore‐forming Peptides from Loss‐of‐function High Throughput Screening
Authors: Krauson Aram Julian , Hall Morgan , Fuselier Taylor , Starr Charlie G. , Kauffman W. Berkeley , Wimley William C
Published: November 2023

While screening a combinatorial peptide library of pore‐forming peptides for gain‐of‐function, we also screened for loss‐of‐function in parallel. We used melittin, a 26 amino acid long lytic peptide from honey bee venom, as a template. For the loss‐of‐function screen we assayed library members for failure to cause leakage of entrapped contents from lipid vesicles at a peptide to lipid ratio (P:L) of 1:20, ten‐fold higher than the concentration at which melittin efficiently permeabilizes. Surprisingly, about one third of the library members are inactive under these conditions. In the selected loss‐of‐function peptides, two changes of hydrophobic residues to glycine were especially abundant. We show that a single residue change from a leucine to a glycine at position 16 completely prevents lytic activity. Unlike the potently lytic melittin, the loss‐of‐function peptides, including the single site variant, are essentially inactive against phosphatidylcholine vesicles and multiple types of eukaryotic cells. Circular dichroism spectra reveal the loss‐of‐function peptides to shift in the binding‐folding equilibrium away from the bound, a‐helical state towards the unbound, random coil state. Accordingly, the addition of anionic lipids to synthetic lipid vesicles restored binding, a‐helical secondary structure and potent activity of the “negative” peptides. The single site variant had potent, melittin‐like antimicrobial activity against Gram‐positive Staph aureus and Gram‐negative E. Coli, with minimum sterilization concentration (MIC) of 10 μM and 7.5 μM, respectively. These results show that conformational fine‐tuning of pore‐forming peptides is a powerful way to modulate potent cytotoxic peptides toward selective antimicrobial activity.
N‐linked glycosylation of Hemagglutinin Globular Head Modulates the Antibody Response to H3N2 Influenza A Virus
Authors: Alymova Irina
Published: November 2023

Molecular evolution of humanH3N2 influenza A viruses (IAV) has included the progressive addition of N‐linked glycans onto the hemagglutinin(HA) globular head. The pandemic H3N2 virus A/Hong Kong/1/1968 (HK68) possessed only two glycosylation sites on the HA globular head at residues 81 and 165. In 1975 and 1999, periods of significant antigenic change, H3N2 IAV acquiredglycans at residues 63 and 126 (HK68+2) and residues 63, 126, 133, and 246(HK68+4), respectively. Differences in receptor binding, pathogenicity, and induction of T‐cell‐mediated immunopathology in mice between the HK68 and its glycosylation variants have previously been demonstrated. Here, we evaluated the consequences of this incremental glycosylation on antibody responses directed to HA. Total serum antibody titers against HA were measured 15 days after infection of mice with 105 TCID50 of each virus by hemagglutination inhibition assay (HI) with various red blood cells (RBC) at room temperature (RT). The avidity was measured at 37°C. Sera from mice infected with any virus showed reactivity against all tested HAs. However, sera from mice infected with HK68 and HK68+2 (lower glycosylation) demonstrated relatively low reactivity against higher glycosylated HK68+4. Sera from HK68+4‐infected mice recognized all tested HAs at similar levels. Viral pathogenicity did not correlate with antibody titers or avidity, since moderate titers and avidity were observed with viruses of highest and lowest pathogenicity (HK68 and HK69+4 respectively), while higher titers and avidity were seen in sera of mice infected with HK68+2, whose pathogenicity is intermediate between HK69 and HK68+4. The data suggest that HA antibody responses may be influenced by HA glycan composition in ways that are unconnected to pathogenicity. Modification of N‐linked glycosylation of the HA globular head may present a new approach for improvement of IAV vaccine efficacy.
The Role of N‐linked Glycosylation in Drosophila Development
Authors: Thomas Morgan D. , Selva Erica M.
Published: November 2023

Asparagine‐linked or N‐linked glycosylation is an important post‐translational modification pathway that adds 14‐sugar oligosaccharide chains to the Asparagine of specific Asn‐Xaa‐Ser/Thr sequences in target proteins on the luminal side of the endoplasmic reticulum. These glycan “tags” are necessary for multiple cell functions including cell‐cell recognition, extracellular signal regulation, and proper protein folding. The focus of this study is to examine the effect of loss of function mutations in the N‐linked pathway during development. A group of human diseases, congenital disorders of glycosylation (CDG), arise from mutations in the genes involved in various steps of this pathway. CDGs display severe and pleotropic phenotypes, which reflect the universal importance of this protein modification, but most cases display phenotypes that arise from impaired neuronal function. The two specific genes under study in this project are alg9 and alg10. Each gene encodes a glycosyltranferase that adds a sugar residue to the growing oligosaccharide chain before it is transferred en masse to a target protein. The Drosophila eye is used as a model organ to study the effects of loss‐of‐function mutations in these genes as it serves as a surrogate for neuronal development. In adult flies, these mutations yield a small rough eye phenotype, which is more severe in alg9, as it acts five steps before alg10 in the pathway. In order to determine the molecular basis of this phenotype, larval eye discs that are homozygous mutate for alg9 and alg10 were dissected, stained for different glycoprotein and neuronal markers, and then imaged using confocal microscopy. Using ELAV, a neuron specific marker, we observed normally differentiated neurons in the eye disc. However, these mutations interrupt proper glycoprotein trafficking, as Chaoptin, a photoreceptor specific cell surface adhesion molecule, accumulates in the cell bodies of alg9 and alg10 mutant photoreceptors. Presence of cleaved Caspase‐3 later in eye development suggests that the accumulation of glycoprotein in the cell body eventually leads to photoreceptor apoptosis. Photoreceptor death likely continues through pupal development resulting in the reduced number of photoreceptors seen in alg10 adult eyes and an almost complete absence in alg9 adult eyes. These results indicate that CDG patients may have normal neuronal specification and differentiation, but experience neuronal deficits due to intracellular accumulation of glycoproteins leading to cell death. These data also suggest induction of the unfolded protein response (UPR) resulting in apoptosis may play a role in this process. Markers of endoplasmic reticulum stress and the unfolded protein response such as XBP1 and BiP will be studied in the future.

Support or Funding Information

Howard Hughes Medical Institute Summer Fellowship, 4 University of Delaware Supply and Expense Grants
Downregulating FUK to Get Unstuck: Altered Fucosylation in Melanoma Promotes Tumor Development and Metastasis
Authors: Lau Eric Kirk , Feng Yongmei , Claps Giuseppina , Fukuda Michiko N , Perlina Ally , Donn Dylan , Jilaveanu Lucia , Kluger Harriet , Freeze Hudson H , Ronai Ze'ev A
Published: November 2023

Melanoma, one of the most lethal skin cancers worldwide, is characterized by a propensity to metastasize. Thus, defining mechanisms that govern metastatic propensity represents an urgent clinical need. Here, we report that Protein Kinase C‐epsilon (PKCe)‐Activating Transcription Factor‐2 (ATF2) signaling controls the motility and invasiveness of melanoma. PKCɛ‐phosphorylated ATF2 transcriptionally represses fucokinase (FUK), a key upstream regulatory kinase of the fucose salvage pathway, which directs the conversion of L‐fucose to GDP‐L‐fucose that is used to fucosylate proteins in cells. Primary melanocytes and early stage melanoma cell lines express low levels of PKCe‐phosphorylated ATF2, which permit the expression of FUK, enabling cellular fucosylation, which promotes cellular adhesion and reduces motility. In contrast, increased levels of PKCɛ and transcriptionally active ATF2, as observed in advanced melanomas, repress FUK transcription. Consequently, reduced levels of cellular fucosylation attenuate adhesion and increase motility. Significantly, increasing tumor fucosylation either by dietary L‐fucose supplementation or by genetic manipulation of murine Fuk expression blunts primary tumor growth, increases intratumoral NK cell populations, and decreases metastatic spread to the lung in murine isograft models. Tumor microarray analysis of > 300 human melanoma specimens confirmed reduced fucosylation in metastatic vs. primary tumors and revealed that high levels of fucosylation in primary melanomas connote increased overall survival rates. Collectively, our findings identify the regulation of fucosylation by ATF2 and its importance in melanoma development and metastasis.

Support or Funding Information

We would like to gratefully acknowledge support from NCI PO1 (CA128814), RO1 (CA179170), Hervey Family Non‐endowment Fund at the San Diego Foundation and from a Melanoma Research Foundation grant to Z.A.R. and K99 (CA172705) grant and T32 (CA121949) fellowship to E.L., and from NIH (R01DK9955) to H.H.F. This work was also supported by the Assistant Secretary of Defense for Health Affairs through the Peer‐Reviewed Cancer Program under Award No. W81XWH‐14‐1‐0127 to Z.A.R.
Transcriptomic and MicroRNA Analyses Identify Gene Networks Regulated by Eicosapentaenoic Acid in Brown Adipose Tissue from Diet‐Induced Obese Mice
Authors: Pahlavani Mandana , Wijayatunga Nadeeja N , Kottapalli Rao , Ramalingam Latha , Gunaratne Preethi H , Coarfa Cristian , Rajapakshe Kimal , Kalupahana Nishan S , Moustaid‐Moussa Naima
Published: November 2023

Brown adipose tissue (BAT) dissipates chemical energy as heat and protects against obesity by increasing energy expenditure. In recent years, interest in BAT as an anti‐obesity target has increased. However mechanisms mediating regulation of BAT by dietary factors remain largely unexplored at the mechanistic level. Hence, we used RNA sequencing (RNA‐Seq) and microRNA (miRNA) profiling, as powerful methods to identify novel regulators of BAT transcriptome. These techniques were applied on BAT from C57BL/6J mice fed either a high fat diet (HF, 45% kcal fat) or HF diet supplemented with EPA (HF‐EPA containing 36g EPA/kg diet) for 11 weeks, to characterize differential gene and miRNA expression between HF and HF‐EPA fed mice. RNA sequencing was performed using Illumina Hi‐Seq and pathway analyses were performed using Ingenuity Pathway Analysis software (IPA®). We identified 831 genes that were differentially expressed (95% confidence and p<0.05) in BAT from HF compared to HF‐EPA fed mice. HF‐EPA had significantly higher expression of genes in fatty acid oxidation and thermogenesis such as peroxisome proliferator‐activated receptor‐α (Ppar‐α), retinoid X receptor (Rxr), phosphatase and tensin homolog (Pten) and reduced expression of inflammatory genes such as nuclear factor kappa‐light‐chain‐enhancer of activated B cells (Nf‐κb), high‐mobility group protein B1(Hmgb1) and Tumor necrosis factor receptor 1 (Tnfr1). Moreover, miRNA profiling identified nine miRNAs that were upregulated and six miRNAs that were downregulated by EPA. Few miRNAs including miR‐455‐3 and miR‐150‐5p are key regulators of BAT thermogenic function. These data are currently being further validated using quantitative PCR. In summary, combining RNA‐Seq and miRNA profiling is a powerful approach to identify specific pathways regulated by omega 3 fatty acids, which may help identify novel biomarkers mediating nutritional regulation of thermogenesis.

Support or Funding Information

startup funds from Texas Tech University
Association of serum sex hormone‐binding globulin with features of metabolic syndrome in children
Authors: Al‐Daghri Nasser , Khan Nasiruddin , Al‐Attas Omar , Sabico Shaun , Alokail Majed , Kumar Sudhesh
Published: November 2023

Background

Sex hormone binding globulin (SHBG) has been proposed as a biomarker of MetS in children and adults. We aim to determine the association of SHBG with features of MetS in children in the Middle‐East where prevalence of MetS has been documented to be very high.

Materials and methods

In this cross‐sectional observational study, a total of 509 randomly selected school children aged 12–16 years were recruited. Fasting blood glucose and lipid profile were determined using routine laboratory procedures. Serum SHBG is measured with Cobas e411 using an electrochemiluminescence immuno assay. The modified definition of ATP‐III (NHANES III) was used for the diagnosis of MetS.

Results

Among 509 children, 23.4% had metabolic syndrome. Boys had a significantly higher waist circumference and systolic blood pressure (p<0.032, 0.024, respectively) than girls, while levels of glucose (p<0.029), and SHBG (p<0.003) were significantly higher in girls than in boys. In overall population, a significant negative correlation of SHBG level with age, BMI, systolic blood pressure and triglycerides and a significant direct correlation between SHBG level and HDL‐c was exhibited. Children with the lowest tertile of serum SHBG had significantly higher prevalence of MetS (p<0.05.). ROC curve shows SHBG level as more sensitive marker of MetS in boys (AUC=0.70, p<0.001).

Conclusions

SHBG levels not only suggest its promising use as a possible biomarker, but also as a predictor of CVD risk, with more accuracy in boys. Gender dimorphism seems to play a role with higher levels of SHBG in girls than boys.

Support or Funding Information

The authors thank the Deanship of Scientific Research, Prolific Research Group Program (PRG‐1436‐15), Vice Rectorate for Graduate Studies and Scientific Research in King Saud University (KSU), Riyadh, Saudi Arabia for funding the study.
Variation in Inositol Utilization in Closely Related Drosophilids
Authors: Smith Matthew Barrett Toshio , Eldon Elizabeth , Klig Lisa
Published: November 2023

Myo‐inositol is a six‐carbon sugar alcohol essential in cellular metabolism, osmoregulation, signal transduction pathways, and is a precursor of the membrane phospholipid phosphatidylinositol. Inositol can serve as a carbon and energy source via the glucuronate‐xylulose‐pentose phosphate pathway. The glucuronate‐xylulose pathway has been proposed to be the only pathway for myo‐inositol catabolism in eukaryotes. Myo‐inositol oxygenase (MIOX) catalyzes the initial step of myo‐inositol catabolism and is involved in regulating myo‐inositol levels in vivo. To establish a structure‐function relationship for MIOX and to examine its regulation, the viability of six drosphilids when provided either sucrose or inositol as the sole nutritional energy source was compared. D. yakuba and D. sechellia do not suvive with inositol as the sole energy source and appear similar to an RNAi knockdown of MIOX in D. melanogaster. D. ananassae and D. simulans, similar to D. melanogaster, survive slightly better on sucrose than inositol. In contrast, D. willistoni, which has two copies of the MIOX gene, appears to thrive on inositol‐containing semi‐defined food. This study annotates the MIOX gene and compares the primary MIOX transcripts, including the untranslated regions (UTRs), in these species using RT‐PCR, 5′ RACE, 3′ RACE, and sequencing. As expected, the coding regions are similar among the species and the protein sequences were between 90–99% identical. The RACE experiments, however, revealed variations in the lengths and sequences of the 5′ and 3′ UTRs of the MIOX transcripts among all species studied. The role of these UTRs in MIOX regulation has also been explored. These studies elucidate the relationship between MIOX gene structure and inositol catabolism.
Development and Characterization of Dual‐Site PPARγ Antagonists
Authors: Brust Richard , Asteian Alice , Kamenecka Theodore , Kojetin Douglas
Published: November 2023

Peroxisome proliferator‐activated receptor gamma (PPARγ) has been established as a drug target for type 2 diabetes. Insulin‐sensitizing drugs such as glitazones, which improve plasma glucose maintenance in patients with diabetes, have been shown to target PPARγ. Two commercially available antagonists, T0070907 and GW9662, are used as chemical tools to block ligand binding to PPARγ to show functional specificity of ligands that bind PPARγ. These compounds covalently modify residues Cys285 in the ligand‐binding domain (LBD) of PPARγ. GW9662 and T0070907 share a similar warhead but differ slightly, namely via the presence of a phenyl (GW9662) vs. a pyridyl (T0070907) group. However, in a recent study, we identified a second, alternative ligand‐binding site in PPARγ—one that is not blocked by GW9662 or T0070907. While alternate site ligand binding was not abolished, the phenyl to pyridyl group modification did reduce ligand‐binding affinity to the alternate site, indicating that making further modifications of this scaffold are worth exploring. With this information, we have generated new analogs that target and covalently modify PPARγ. These analogs decrease the binding affinity of PPARγ agonists to the alternate site while also reducing alternate site binding‐induced transactivation in cell based assays. These dual‐site PPARγ antagonists should prove to be a useful chemical tool for the many laboratories that use PPARγ antagonists to demonstrate functional specificity.

Support or Funding Information

Funding provided by NIH/NIGMS grant R01‐DK101871.
HMG box‐containing protein 1 (HBP1) modulates matrix metalloproteinase‐13 (MMP‐13)‐mediated cellular migration and invasion in oral cancer
Authors: Chan Chien Yi , Wu Wen‐Chieh , Lin Tzu‐Yuan , Lee Ming‐Fen , Huang Chun‐Yin
Published: November 2023

Transcription repressor HMG box‐containing protein 1 (HBP1) functions as a potential tumor suppressor in various types of cancer. Previously, we demonstrated that HBP1 modulates cell growth in epidermal growth factor receptor (EGFR)‐overexpressing oral cancer cells; however, whether HBP1 also regulates metastatic potential remains unclear. Analysis of a subset of oral tumor specimens revealed that mean HBP1 expression level in tumors was significantly lower than that of control tissues; moreover, low HBP1/high EGFR status was significantly associated with metastatic potential. When ectopically over‐expressing HBP1 in both HSC‐3 and FaDu oral cancer cells with elevated EGFR levels, we found that HBP1 potently inhibited cell invasion. In contrast, HBP1 knockdown enhanced cell migration and invasion with a concomitant increase in the expression and activity of matrix metalloproteinase (MMP) family members, MMP‐2 and ‐9, as well as a strong induction of MMP‐13. Both chemical inhibitors and MMP‐13 siRNA significantly suppressed MMP‐13 activity and cell invasion in HSC‐3 cells, suggesting a crucial role of MMP‐13 in metastasis. Further, HBP1 was not only a potent repressor of MMP‐13 transcription as shown by the MMP‐13 reporter gene assay, but also a modulator of Akt‐mediated MMP‐13 expression and invasion. Taken together, our study demonstrated that HBP1 is a negative regulator of cell migration and invasion as well as anchorage‐independent growth, supporting HBP1 as a potential predictor of the aggressiveness in the development of oral cancer.

Support or Funding Information

This work was supported by the grant MOST 103‐2320‐B‐039‐031‐MY3 to C‐Y Huang.
Pentoxifylline modulates the AKT/mTOR/AMPK pathway: potential role as an adjunct in cancer chemotherapy
Authors: Le Gloria T , Canatsey Ryan , Tran Kelvin , Monks Terrence J , Lau Serrine
Published: November 2023

Targeting specific pathways involved in the growth and death of cells, such as AKT (signal transduction pathway promoting growth), mTOR (intracellular signaling pathway regulating cell cycle), and AMPK (coordination of growth, autophagy, and metabolism), is an established, effective strategy to inhibit cancer cell growth. Previously, we have shown that pentoxifylline (PTX), a clinically‐utilized non‐specific phosphodiesterase inhibitor, can inhibit growth and induce G1 arrest in prostate cancer cells. In the present studies, we examined the mechanism of PTX induced growth inhibition via modulation of growth/metabolic pathways, and the potential use of PTX as an adjunct in cancer chemotherapy. In prostate adenocarcinoma cells (LNCaP), we show PTX modulates the AKT pathway, as evidenced by the decreased phosphorylation of AKT. Downstream modulation of the mTOR pathway is indicated by decreased phosphorylation of mTOR, ribosomal S6K, and 4EBP1, and total cyclin D1. P‐mTOR levels return to baseline by 24h, while p‐S6K, p‐4EBP1, and cyclin D1 levels are suppressed for at least 48h, indicating a sustained decrease in the activity of cell growth pathway proteins. PTX also modulates the AMPK pathway, as revealed by modulation of downstream p‐RAPTOR, p‐ACC and p‐ULK. Both the AKT and AMPK pathways regulate the activation of cellular autophagy, an intracellular degradation system and determinant of cell survival. Treatment of LNCaP cells with PTX alone, and in combination with bafilomycin, a lysosomal degradation inhibitor, increased LC3‐II expression, indicating an induction of autophagic flux. Overall, PTX modulates both AKT and AMPK pathways, decreasing cell growth and increasing autophagy. Moreover, LNCaP cells treated with PTX were more susceptible to docetaxel (DOC; first‐line chemotherapy) induced cytotoxicity than DOC alone. Interestingly, the PTX/DOC combination decreased the expression of LC3 relative to PTX alone, suggesting activation of an alternate cell death pathway. Indeed, assessment of caspase cleavage, pBcl‐2, and annexin staining revealed a synergistic effect of PTX/DOC treatment on the induction of apoptosis. In summary, the findings provide a mechanistic basis for the potential use of PTX as an adjunct in the chemotherapy of prostate cancer. The ability of PTX to potentiate the effects of DOC on tumor growth inhibition in a mouse xenograft model is currently being assessed.

Support or Funding Information

ASPET SURF, P30ES006694, T32ES006694
B‐Raf is required for normal cardiomyocyte growth
Authors: Rees Katherina , Chattergoon Natasha
Published: November 2023

Objectives

The postnatal heart does not retain the proliferative capacity it had during fetal life. Unlike large mammals, murine cardiomyocytes (CM) continue to divide into the first week of life before terminal differentiation and binucleation. This postnatal proliferation and maturation window offers the ability to study the mechanisms of CM terminal differentiation. We hypothesize that B‐Raf regulates ERK (extracellular‐regulated kinase) activation in newborn CM and that loss of B‐Raf suppresses cyclin levels and reduces CM proliferation. To test this, we determined whether the loss of B‐Raf disrupts the ERK cascade and impairs CM growth.

Methods

CM specific knockout (KO) of B‐Raf was generated using CRE/lox (floxed B‐Raf x αMHC CRE) resulting in a truncated, unstable B‐Raf and a null phenotype. KO mice (α‐MHC‐CRE/B‐Raf lox/lox) were compared to CRE negative/B‐Raf lox/lox mice (wild type; WT). Hearts from 1d, 3d, 8d and 14d old pups were harvested for molecular analysis of B‐Raf signaling and cell cycle markers (p21, p27, p53, cyclins A1, B1, D1 and E). Mid‐wall sections of the heart were stained with Masson's trichrome to determine changes in tissue composition.

Results

Heart weight to body weight (HW/BW) ratio was less in 3d KO versus 3d WT (n=50, p<0.05). HW/BW ratio became greater in 8d KO; there was no difference in 3d and 8d HW/BW in WT animals. B‐Raf and phosphorylated ERK levels were reduced in KO hearts (*p<0.05). Cell cycle inhibitors p21 and p53 were increased in 3d KO hearts with decreased levels of all cyclins (Cyclin A1 and B1, p<0.05). In 8d KO hearts, increased p21, p27, and p53 expression was accompanied with decreased cyclin levels (p<0.05). In 14d KO hearts, gene expression of p21, p27, and p53 are increased (p<0.05), along with cyclins D1 and E. Tissue composition of cardiomyocytes decreased and collagen increased in 3d and 8d hearts (p<0.05).

Conclusions

ERK activation was suppressed in KO hearts resulting in smaller newborn hearts but which exceeded normal HW/BW by 8d. This is may represent premature hypertrophy as the proliferative period of CM development had ended. Cell cycle analysis supports reduced CM mitosis among 8d CM. Trichrome analysis supports proliferation has ended prematurely by the reduction of the CM fraction. Such early disturbances in normal CM growth may increase susceptibility for reduced cardiac function in the face of increased postnatal load stress.
Proteomic Analysis of the Mammalian Katanin Family of Microtubule‐severing Enzymes Defines KATNBL1 as a Regulator of Mammalian Katanin Microtubule‐severing
Authors: Cheung Keith C. , Torres Jorge Z.
Published: November 2023

The Katanin family of microtubule‐severing enzymes are critical for remodeling microtubule‐based structures that influence cell division, motility, morphogenesis and signaling. In lower eukaryotes, Katanin is composed of a catalytic p60 subunit (A subunit, KATNA1) and a regulatory p80 subunit (B subunit, KATNB1). However, the mammalian genome encodes two additional A‐like subunits (KATNAL1 and KATNAL2) and one additional B‐like subunit (KATNBL1) that have remained poorly characterized. To better understand the factors and mechanisms controlling mammalian microtubule‐severing, we have taken a mass proteomic approach to define the protein interaction module for each mammalian Katanin subunit and to generate the mammalian Katanin family interaction network (Katan‐ome). Further, we have analyzed the function of the KATNBL1 subunit and determined that it associates with KATNA1 and KATNAL1, it localizes to the spindle poles only during mitosis and it regulates Katanin A subunit microtubule‐severing activity in vitro. Interestingly, during interphase, KATNBL1 is actively sequestered in the nucleus through an N‐terminal nuclear localization signal. Finally KATNB1 was able to compete the interaction of KATNBL1 with KATNA1 and KATNAL1. These data indicate that KATNBL1 functions as a regulator of Katanin A subunit microtubule‐severing activity during mitosis and that it likely coordinates with KATNB1 to perform this function.

Support or Funding Information

National Science Foundation and UCLA Whitcome fellowship
Electrostatics and N‐Glycan‐mediated Membrane Tethering of SCUBE1 Is Critical for Promoting Bone Morphogenetic Protein Signaling
Authors: Liao Wei‐Ju
Published: November 2023

SCUBE1, a secreted and membrane‐bound glycoprotein, has a modular protein structure composed of an NH2‐terminal signal peptide sequence followed by 9 epidermal growth factor (EGF)‐like repeats, a spacer region and 3 cysteine‐rich (CR) motifs with multiple potential N‐linked glycosylation sites, and one CUB domain at the COOH terminus. Soluble SCUBE1 is a biomarker of platelet activation but an active participant of thrombosis via its adhesive EGF‐like repeats, whereas its membrane form acts as a bone morphogenetic protein (BMP) co‐receptor in promoting BMP signal activity. However, the mechanism responsible for the membrane tethering and biological importance of N‐glycosylation of SCUBE1 remains largely unknown. In this study, molecular mapping analysis identified a polycationic segment (amino acids 501–550) in the spacer region required for its membrane tethering via electrostatic interactions possibly with the anionic heparan sulfate proteoglycans. Furthermore, deglycosylation by peptide‐N‐glycosidase F treatment revealed that N‐glycans within the CR motif are essential for membrane recruitment through lectin‐mediated surface retention. Injection of mRNA encoding zebrafish wild‐type but not N‐glycan–deficient scube1 restores the expression of hematopoietic and erythroid markers (scl and gata1) in scube1‐knockdown embryos. We describe novel mechanisms in targeting SCUBE1 to the plasma membrane and demonstrate that N‐glycans are required for SCUBE1 functions during primitive hematopoiesis in zebrafish.
AKAP7 and Calcineurin Control CaM Kinases and Cell Growth
Authors: Papenfuse Lael , McFarland Hannah M , Dodge‐Kafka Kimberly , Schmitt John M
Published: November 2023

Estrogen (E2) and the agonist ionomycin elevate intracellular calcium and activate the calcium/calmodulin‐dependent protein kinases (CaM Kinases) in breast cancer cells. CaM Kinases regulate ERK leading to transcription and cell growth. Inhibition of CaM KK blocks breast cancer cell growth and protein phosphatases such as Calcineurin (CN) may negatively regulate CaM KK pathways. CN regulates CaM Kinases and associates with A‐kinase‐anchoring‐proteins (AKAPs). AKAPs are used to link together and regulate enzymes within in a signaling pathway. One specific AKAP, AKAP7, is involved in calcium signaling and may regulate CaM Kinase control of ERK activation in cancer cells. Our goal was to determine if CN and AKAP7 associated in breast cancer cells, and to examine the role of CN in regulating E2 and calcium activation of CaM KK/KI, ERK and cell growth in MCF‐7 and MDA‐MB‐231 cells. Our results suggest that E2 treatment of MCF‐7 cells activates CaM KI and ERK, an effect that is blocked by the selective CaM KK‐inhibitor STO‐609. Immunoprecipitation with purified STag‐AKAP7 but not STag‐AKAP5 revealed CaM KK and CaM KI association from E2‐treated MCF‐7 extracts. Interestingly, purified AKAP7 directly binds purified CaM KK and endogenous AKAP7 co‐localizes with CaM KK in MCF‐7 and MDA‐MB‐231 cells. Ionomycin stimulation of MCF‐7 cells also increased CaM KI and ERK activation an effect that was enhanced by treating cells with the CN inhibitor cyclosporin A (CsA). Similarly, ionomycin treatment enhanced the binding of endogenous CaM KK‐AKAP7‐CN as determined by both immunoprecipitation and confocal microscopy. Interestingly, siRNA knockdown of AKAP7 disrupted CaM KK association with CN and increased basal ERK phosphorylation in MCF‐7 cells. Finally, E2‐stimulated cell growth was inhibited by pre‐treatment with STO‐609 but not the CN inhibitor CsA. Taken together, these results suggest that AKAP7 directly associates with CaM KK/KI and forms a complex with CN that may function to negatively regulate CaM KK and CaM KI activation of ERK and cell growth.

Support or Funding Information

The Paul K. and Evalyn E. C. Richter Memorial Fund.

This work was supported through a Life Science grant from the M.J. Murdock Charitable Trust to J.M.S. grant #2011267.
PEPTIDE LIGAND C391 AS A POTENTIAL PAR‐2 β‐ARRESTIN BIASED ANTAGONIST
Authors: Ming Monica , Yee Michael C , Lee Kyu J , Siegler Michael D. , Arrellano Marisol , Aghayan Sara , DeFea Kathryn
Published: November 2023

Protease Activated Receptor‐2 (PAR‐2) promotes two independent and opposing pathways: G‐alpha‐q (Gαq), and β‐arrestin. Studies characterizing the role of PAR‐2 in airway inflammation have shown that the Gαq pathway has protective effects by the release of prostaglandin E2 (PGE2), leading to bronchodilation. And that the β‐arrestin pathway has pro‐inflammatory effects through leukocyte infiltration into the lungs, epithelial thickening, and mucus production. These conflicting views led us to investigate whether a biased antagonist of PAR‐2, that preferentially silences β‐arrestin signaling, may also inhibit the inflammatory effects of allergenic proteases in the airway. Recent studies have identified a number of new, high affinity agonists and antagonists of PAR2. One of the peptide ligands found was C391, which was shown to be a weak agonist and antagonist of PAR‐2, however it was only tested for calcium (Ca2+) mobilization. Preliminary results from our lab suggest that C391 affects β‐arrestin‐2 recruitment to PAR‐2 at a lower concentration than it affects Ca2+ mobilization, suggesting it is a good candidate for a biased PAR‐2 antagonist. Using bioluminescent resonance energy transfer methods and protein analysis, this study demonstrates that C391 can affect both cofilin (an actin severing protein) dephosphorylation and β‐arrestin recruitment –elicited by trypsin and allergenic proteases from Alternaria Alternata and cockroach extracts in a dose dependent response– at concentrations where it does not affect Ca2+ mobilization or phosphatidylinositide 3‐kinase (PI3K) activation.
Janus kinase 3 (JAK3) increases phosphorylation of signal transducers and activators of transcription 3 (STAT3) in reproductive cells
Authors: Ndiaye Kalidou , Castonguay Amélie , Benoit Gabriel , Silversides David W , Lussier Jacques G
Published: November 2023

JAK3 is a member of the membrane‐associated non‐receptor tyrosine kinase protein family and is considered predominantly expressed in hematopoietic cells. We previously identified JAK3 as a differentially expressed gene in granulosa cells (GC) of bovine preovulatory follicles. This study aimed to further investigate JAK3 mRNA and protein regulation and to identify its binding partners in GC of bovine dominant follicles. GC were obtained from small follicles (SF), dominant follicles at day 5 of the estrous cycle (DF), and ovulatory follicles, 24 hours following hCG injection (OF). RT‐PCR analyses showed greatest expression of JAK3 in GC of DF, while the weakest expression was in OF (P < 0.0001). There was a 5‐ and 20‐fold reduction of JAK3 steady‐state mRNA levels in follicular walls, respectively at 12 and 24 hours post‐hCG as compared to 0 hour (P < 0.05). These results were confirmed in western blot analysis showing weakest JAK3 protein levels in OF as compared to DF. Yeast two‐hybrid screening of a DF‐cDNA library resulted in the identification of 23 JAK3 partners in GC. Moreover, the amount of phosphorylated STAT3 in GC was greatest in SF and DF as compared to OF while the opposite was observed for non‐phosphorylated STAT3. In functional studies using bovine endometrial cells, we discovered that phosphorylation of STAT3 (pSTAT3) in a bovine endometrial cell line was increased by JAK3 while inhibition of JAK3 by JANEX‐1 decreased the amount of pSTAT3. In the same study, we showed that JAK3 increased endometrial cell proliferation as well. We found a similar effect of JAK3 on STAT3 phosphorylation using an immortalized murine granulosa cell line. These results support a physiologically relevant role of JAK3 in follicular development and provide insights into the mode of action and function of JAK3 in reproductive tissues.

Support or Funding Information

This work was supported in part by internal funds from Université de Montréal to KN and by a Discovery Grant from Natural Sciences and Engineering Research Council of Canada (NSERC) to JGL.
Activation of membrane estrogen receptor: MAPK signaling by cadmium chloride and sodium arsenite in human lung adenocarcinoma cells
Authors: Hyzer Jeffrey M. , Krauss Katazyna A. , Black Austin T. , Todd Sarah L. , Elpers Julia T. , Hoerter Jacob E. , Klinge Carolyn M. , Huff Mary O.
Published: November 2023

Estrogens have been implicated in non‐small cell lung cancer (NSCLC) risk but the contribution and possible mechanisms of the endocrine disruptors, cadmium chloride (CdCl2) and sodium arsenate (NaAsO2), have not been delineated. In this study, cell proliferation of NCI‐H1793 human lung adenocarcinoma cells increased following treatment with nanomolar concentrations of CdCl2 and NaAsO2, and like 17β‐estadiol (E2), 10 min treatment stimulated phosphorylation of ERK1/2. In the presence of ICI 182,780, an estrogen receptor (ER) antagonist, CdCl2 and NaAsO2‐stimulated phosphorylation of ERK1/2 was decreased suggesting that activation is mediated through ERs. To identify the role of the ERs in CdCl2 and NaAsO2‐induced ERK1/2 phosphorylation, cells were treated with specific inhibitors for ERα, ERβ, and the G‐protein linked estrogen receptor (GPER), and ERK1/2 phosphorylation was measured by immunoblot analysis. The results suggest that CdCl2 and E2 activation of MAPK phosphorylation likely involves ERβ whereas ERα may play a minor role in NaAsO2‐stimulated MAPK activation. This study supports the involvement of membrane ER and GPER signaling in mediating cellular responses to environmentally relevant nanomolar concentrations of CdCl2 and NaAsO2 in lung adenocarcinoma cells.
The Role of Molecular Chaperonin CCT and its Co‐chaperone PhLP1 in the Assembly of mTOR Complexes
Authors: Dhavale Madhura , Plimpton Rebecca , Aoba Takuma , Tensmeyer Nicole
Published: November 2023

The mechanistic target of rapamycin (mTOR) kinase signaling pathway controls cell proliferation, metabolism and survival. Mutations in components of the mTOR pathway lead to tumerogenesis, angiogenesis and metastasis and therefore are implicated in many types of cancers. mTOR signaling is mediated through two signaling complexes, namely mTORC1 (consisting of mTOR, mLST8, Raptor, Deptor and PRAS40) and mTORC2 (consisting of mTOR, mLST8, Rictor, mSin1, Deptor and Protor1). In these complexes, mTOR is the central kinase whereas the other components are accessory proteins that assist in regulation and recognition of mTOR substrates. The mechanism by which the mTOR components are assembled into functional complexes from nascent polypeptides is a fundamental question yet to be answered. We have evidence indicating that the chaperonin CCT (also called TRiC) and its co‐chaperone PhLP1 assist in mTORC1 assembly by folding two of the mTOR components, mLST8 and Raptor. CCT and PhLP1 form a ternary complex with mLST8, while Raptor binds CCT in the absence of PhLP1. Furthermore, cellular depletion of CCT or PhLP1 disrupts mTORC1 assembly, and CCT depletion inhibits mTORC2‐mediated phosphorylation of Ser473 of AKT. In contrast, the mTORC2 subunit Rictor, which is structurally homologous to Raptor, does not bind CCT. These results suggest that CCT and PhLP1 contribute to mLST8 folding and assembly into mTORC1 and mTORC2, while CCT alone assists in Raptor folding and assembly into mTORC1. Thus, the CCT chaperonin and its co‐chaperones may represent new physiological or therapeutic targets to regulate mTOR signaling.

Support or Funding Information

NIH Grants: EY 012287, GM 078550, Brigham Young University Cancer Research Center
The potential link of COMP‐ADAMTS and Aggrecanase‐ADAMTS Genes with Osteoarthrit
Authors: Demircan Kadir , Gürlü Gülsüm , Güven Kübra Nur , Fıstık Tuba , Çakırbay Haşim , Yanık Burcu
Published: November 2023

This project aimed to investigate the role of COMP‐ADAMTS and aggrecanase‐ADAMTS which are responsible for the destruction of cartilage in the ADAMTS (a disintegrin and metalloproteinase with trombospondin motifs) genes that are suspected to play role in the genetic etiology of the osteoarthritis (OA) which is the most common joint disease.

88 patients aged between 40–75 years, with the diagnosis of primary knee OA (according to American College of Rheumatology criteria for knee OA) and 88 healthy volunteers aged between 40–75 years, without OA enrolled in the study. Exclusion criteria include, previous knee surgery (total knee prosthesis etc.), inflammatory rheumatic diseases, secondary OA and malignancies. Radiologic staging of the participants is done, pain and functional status are assessed by visual analogue scale, WOMAC, Lequesne index. Blood samples obtained from the participants analyzed for ADAMTS gene family by ELISA and RT‐PCR.

Blood samples obtained from the participants analyzed for ADAMTS ‐1,‐4,‐5,‐7,‐8,‐9,‐12,‐15,‐16 ve ‐18 genes by ELISA. Compared OD levels determined by ELISA reader of groups, OD level of ADAMTS‐4,‐8,‐9 and ‐18 was determined significantly high in the patient group compared to the control group. When examined the patients splitting (dividing) into two groups as early–advanced OA, results of ADAMTS‐4,‐5,‐8,‐9,‐15 ve ‐16 according to control group in early OA group, results of ADAMTS‐15 and ‐16 according to advanced OA group in early OA group and results of ADAMTS‐8, ‐9 and ‐18 according to control group in advanced OA group was seen significantly high.

Statistical evaluation of RT‐PCR results showed that ADAMTS‐4,‐9 and ‐18 expressions are significantly decreased in the patient group compared to the control group, ADAMTS‐8 expression is not low enough to be meaningful statistically and there isn't any enough difference between two groups in ADAMTS‐5 expression level.

Consequently; we observed that in the early stages ADAMTS‐4, ‐5, ‐15 and ‐16, in advanced stages ADAMTS may be the main aggrecanase responsible for the destruction of cartilage in human knee OA. ADAMTS‐8 and ‐9 were determined to play a role in both early and advanced stages of cartilage destruction in OA. We have seen that ADAMTS‐1, ‐7 and ‐12 were not playing an important role in the pathogenesis of OA.

Support or Funding Information

Tubitak and Turgut Ozal University Hospital Project Management Ofice
Effect of Pathway‐Interconnectors in SEB‐Induced Apoptosis Related Events in Human PBMCs
Authors: Geddes Erin , Hendricks Michelle , Doyle Connor , Borgos Dylan , Burton Danielle , Mendis Chanaka
Published: November 2023

Staphylococcus enterotoxin B (SEB) is produced by the bacterium Staphylococcus aureus. SEB is a common cause of apoptosis within human peripheral blood mononuclear cells (PBMCs). The purpose of this study is to further investigate human PBMCs in order to better understand the apoptosis related events induced by SEB. C‐Jun N‐Terminal Kinase (JNK) has been previously identified to be induced by SEB, and has demonstrated that it inter‐connects multiple signaling cascades. As a crucial pathway inter‐connector in SEB‐induced human PBMCs, we believe that inhibiting JNK may possess inhibitory effects on SEB‐induced apoptosis. In this study, JNK specific inhibitor (SP600125) was used to inhibit JNK. We will further evaluate the gene expression profile using RT and PCR. Protein expression patterns of some genes will be examined through the use of enzyme‐linked immunosorbent assays (ELISAs). We believe that our work will allow us to better understand the complex interactions of multiple signal transduction pathways induced by SEB.

Support or Funding Information

We want to thank James Waldschmidt, Tricia Mason, Aaron Hopkins, Emily Freehauf, Amanda Smet, and Nicole Krausert who worked on different aspects of the project. We also wanted to thank our Collaborator Dr. Marti Jett, MEDCOM, Frederick, MD.

The project was funded by Centerpoint and PURF, SAIF grants (University of Wisconsin ‐Platteville).
Myocardin Regulated Genetic Pathway Modulates Mitochondrial Permeability Transition Pore Closure to Prevent Cell Death during Cardiac Differentiation
Authors: Mughal Wajihah , da Silva Rosa Simone , Chapman Donald , Hai Yan , Diehl‐Jones William , Gordon Joseph
Published: November 2023

Myocardin is a transcriptional co‐activator required for cardiovascular development and cardiomyocyte differentiation. Recent studies have shown that genetic inhibition of myocardin results in congenital heart defects associated with increased programmed cell death (PCD). In addition to its established role in regulating PCD, the mitochondrial permeability transition pore (PTP) has been implicated in cardiac calcium homeostasis and myocyte maturation during development. Utilizing fluorescent staining and biochemical assays, we demonstrate that myocardin, a modulator of cardiac gene function, regulates mitochondrial PTP closure during myocyte differentiation to oppose pathways of PCD induced by protein kinase‐A (PKA) activating agents. We show that a loss of myocardin function reduces microRNA‐133a (miR‐133a) mRNA levels and induces mitochondrial dysfunction by PTP opening and reducing mitochondrial membrane potential. Furthermore, myocardin knockdown increased mitochondrial oxidative stress and mitochondrial calcium uptake in differentiated myocytes. Conversely, restoring myocardin expression rescued mitochondrial function, concurrent with a decreased expression of a mitochondrial death protein, Nix. Furthermore, miR‐133a protects against PKA‐activating agents while inhibitory molecules opposing miR‐133a increase Nix expression. With the use of genetically encoded and organelle targeted calcium sensors, our data demonstrates that miR‐133a reverses endoplasmic reticulum calcium release and prevents mitochondrial calcium uptake, in a manner dependent on Nix repression. Finally, we provide mechanistic evidence that myocardin‐dependent miR‐133a expression is regulated by histone deactylase‐5 (HDAC5) and the PKA‐regulated salt inducible kinase‐1 (SIK1). These findings support the notion that myocardin opposes PCD through mitochondrial permeability transition by regulating calcium homeostasis; however, this genetic pathway may be attenuated by pathological PKA signaling.

Support or Funding Information

Supported by Children's Hospital Foundation of Manitoba, Research Manitoba and NSERC Canada.
Isolated Extracellular Domain of DR5 is a Potential inhibitor of Apoptosis
Authors: Vunnam Nagamani , Sachs Jonathan N
Published: November 2023

In recent years, there has been a great interest in pre‐ligand assembly of tumor necrosis factor receptors (TNFRs). TNF‐receptor superfamily plays a major role in regulation of apoptosis. Recent studies suggest that disrupting the pre‐ligand assembly of TNFRs maybe a useful approach for the treatment of TNF‐disorders like rheumatoid arthritis. Thus, strategies for the manipulation of pre‐ligand assembly of TNFRs maybe valuable in prevention and mediation of TNF‐receptor related ailments. In the current study, we targeted the pre‐ligand assembly of death receptor 5 (DR5) to inhibit the apoptosis induced by its cognate ligand. DR5 is a pro‐apoptotic TNF‐receptor protein with a N‐terminal extracellular domain (ECD), a transmembrane domain, and a cytoplasmic death domain, which is crucial for initiation of death signaling upon binding of TNF‐related apoptosis inducing ligand (TRAIL). We hypothesized that ECD, which does not have the death domain, of DR5 could prevent the TRAIL‐induced apoptosis by disrupting the pre‐ligand assembly of endogenous‐DR5 (eDR5) and forming the death‐inhibitory complex with it. To test this hypothesis we first determined the interactions between isolated ECD, eDR5 and TRAIL by using electrophoresis, spectroscopy, FRET and Coimmunoprecipitation (CoIP) studies. These studies show that isolated ECD interacts with both TRAIL and eDR5 and does not significantly influence the eDR5‐TRAIL interactions. We further investigated the effect of ECD on TRAIL‐induced apoptosis by using cell culture studies. Here, we show that ECD inhibits the TRAIL‐induced apoptosis by disrupting the pre‐ligand assembly of eDR5, which is required for induction of death signaling, and forming the anti‐apoptotic complex with eDR5. In summary, our biophysical and cell culture data suggest that the ECD is a potential inhibitor of apoptosis and may have a prospective therapeutic value for the treatment of TNF‐receptor related disorders.

Support or Funding Information

This work was supported by National Institutes of Health Grant GM107175‐02