bluuebunny/crossref_metadata_2025 huggingface dataset:
Sphingosine Kinase 1 Mediates Transmembrane Calcium Entry via Store‐Operated Calcium Channel
Authors: El‐Shewy Hesham , Wilson Parker , Luttrell Louis
Published: June 2021

Angiotensin II plays a critical role in the regulation of vascular tone and blood pressure mainly via regulation of Ca2+ mobilization. Several reports have implicated sphingosine kinase 1 (SK1)/sphingosine 1‐phosphate (S1P) in the mobilization of intracellular Ca2+ through a yet undefined mechanism. Here we demonstrate that angiotensin II induces biphasic calcium entry in vascular smooth muscle cells, consisting of an immediate peak due to inositol tris‐phosphate‐dependent release of intracellular calcium, followed by a sustained transmembrane Ca2+ influx through store operated calcium channels (SOC). Inhibition of SK1 attenuates the second phase of transmembrane Ca2+ influx, suggesting a role for SK1 in angiotensin II‐dependent activation of SOC. Intracellular S1P triggers SOC‐dependent Ca2+ influx independent of S1P receptors, while external application of S1P stimulated S1P receptor‐dependent Ca2+ influx that is insensitive to inhibitors of SOC, suggesting that the SK1/S1P axis regulates SOC entry via intracellular rather than extracellular actions. Collectively, these data implicate SK1 as the missing link that connects the angiotensin AT1A receptor to transmembrane Ca2+ influx and identify SOC as a potential intracellular target for SK1/S1P axis.
Specificity and competitive cation association to Phosphatidylinositol‐4,5‐bisphosphate model membranes
Authors: Kooijman Edgar , Wang Wenjie , Singh Gautam , Kuzmenko Ivan , Vaknin David , Graber Zachary
Published: June 2021

Phosphatidylinositol‐4,5‐bisphosphate (PIP2) is an active signaling lipid implicated, among other functions, in the regulation of cell growth by activating the tumor suppressor PTEN. Using synchrotron surface‐sensitive x‐ray diffraction and fluorescence techniques we determined the preferential cation binding to PIP2 monolayers. The natural, highly unsaturated PIP2 was spread as a Langmuir monolayer on a physiological buffer containing 100 mM KCl at pH 7.2 to which divalent cations (Ca2+ and Mg2+) were added. X‐ray fluorescence of the PIP2 monolayer on the buffer shows an eight fold surface enhancement of monovalent K+ compared to its bulk concentration. When physiological levels of calcium are added (1‐100 µM), the Ca2+ gradually replaces bound K+ ions, leading to a significant change in the organization of the PIP2 model membrane. At higher concentrations (100‐1000 µM), which might be achieved during calcium signaling, we observe a 1000 fold surface enhancement of Ca2+. Similar experiments with Mg2+ ions also show strong ion binding to PIP2 at physiological levels (1 mM) with a lesser structural effect on the monolayer compared to that induced by Ca2+. For mixed solutions of Mg2+ and Ca2+ we find that Ca2+ occupies the majority of binding sites, and at mM concentrations completely removes the Mg2+ ions from the interface. Surprisingly, with both 1 mM Mg2+ and 1 mM Ca2+ in the subphase there is still a fourfold surface enrichment of K+ ions at the headgroup region.
Role of Macroautophagy in Anti‐Cancer Effects of the Novel Sphingolipid, Enigmol
Authors: Mays Suzanne , Bushnev Anatoliy , Baillie Mark , Yi Hong , Liotta Dennis , Petros John
Published: June 2021

Enigmol ([2S,3S,5S]‐2‐amino‐3,5‐dihydroxyoctadecane) is a novel sphingolipid analog that inhibits tumor growth in preclinical models of human prostate cancer; however, little is known about mechanisms contributing to this efficacy. Since sphingolipids can affect macroautophagy (hereafter “autophagy”), the purpose of this research was to investigate whether autophagy is involved in Enigmol's anti prostate cancer activity. Although the role of autophagy in cancer is complex, several studies have shown that blocking the pH‐dependent fusion of autophagosomes with lysosomes is detrimental to cancer cells. We demonstrate here a robust, dose‐dependent accumulation of autophagosomes in PC‐3 prostate cancer cells treated with Enigmol, evidenced by Western blot and immunofluorescence monitoring of an autophagosome marker (LC3‐II) and by electron microscopy. At doses near the EC50 for cancer cell killing, Enigmol neutralized lysosomal pH and inhibited autophagosome turnover. Enigmol also caused inhibition of Akt kinase and mammalian target of rapamycin (mTOR), which contributed to autophagosome accumulation. Enigmol analogs deficient in Akt/ mTOR inhibition caused a much lesser degree of LC3‐II accumulation and were markedly less cytotoxic to prostate cancer cells, linking these molecular actions to anti‐cancer effects. We propose a model in which Enigmol's blockade of lysosome‐mediated autophagosome degradation enhances the anti‐cancer effects of Akt/ mTOR inhibition.
Aging Yeast Cells Are Fat!
Authors: Kanagavijayan Dhanabalan , Srinivasan Malathi , Rajasekharan Ram
Published: June 2021

Saccharomyces cerevisiae is an excellent model organism to understand the molecular mechanism of aging. Here we have used yeast haploid RAD deletion strains to explore the relationship between aging and lipid storage. RAD genes are mainly involved in DNA repair mechanism and hence, their deletions result in shorter life span. Viable rad mutants were screened for neutral lipid content and some of the mutants showed significantly high amount of triacylglycerol and steryl ester, besides short chronological life span. Of these, RAD50, MRE11 and XRS2 form a complex denoted as MRX which is involved in homologous recombination and hence the mutant cells are hypersensitive to DNA damage. Deletion of any one of the three genes in MRX complex abolished the function of the complex and exhibited the same phenotype. Microarray data of single MRX deletions revealed that besides DNA damage signature genes, lipid metabolism genes were also up‐regulated. We observed that rad50Δ, mre11Δ, xrs2Δ and mrxΔ have more number of lipid droplets (LDs) at stationary phase compared to wild type yeast cells. These mutants also showed a peculiar phenotype as observed under a scanning electron microscope and had short chronological life span compared to wild‐type. Since this aging phenotype correlated directly with the increase in number and size of lipid droplets, lipid storage could be a possible indicator of aging cells. Further, this phenomenon was also observed in normal wild type aging cells grown for over a month, with huge lipid droplets of ~2.0 µm in diameter. While lipid accumulation in stressed conditions has been reported, our findings demonstrate that aging cells accumulate neutral lipids.
Differential Effects of Nanoparticle Surface‐Functionalization on the Polarization Profiles of M1 and M2 Macrophages
Authors: Syrovets Tatiana , Haas Karina , Loos Cornelia , Musyanovych Anna , Mailänder Volker , Landfester Katharina , Simmet Thomas
Published: June 2021

Exposure of macrophages to various cytokines initiates their polarization into either the classical proinflammatory M1 or the alternative anti‐inflammatory M2 phenotype, which promotes wound healing and supports cancer growth. Here, we explored the effects of particle surface functionalization on human M1 and M2 macrophages by using carboxyl‐ (PS‐COOH) and amino‐functionalized (PS‐NH2) polystyrene nanoparticles as model platform. M1 macrophages exhibit high expression of CD86 and release proinflammatory TNF‐α and IL‐1β. By contrast, M2 express CD200R, CD206, and phagocyte efficiently E. coli, and secrete anti‐inflammatory IL‐10. PS‐COOH led to an increase in protein contents and ATP levels without induction of proliferation, and did not compromise the cell viability of both macrophage subsets or the phagocytosis of E. coli by M2 macrophages.By contrast, PS‐NH2 significantly decreased release of IL‐10 by M2, reduced the ATP contents, impaired E. coli phagocytosis, and viability of both macrophage subsets. When nanoparticles were added to macrophages together with polarization stimuli, PS‐COOH slightly enhanced release of IL‐1β by M1, and significantly inhibited release of IL‐10 by both macrophage subsets. Thus, PS‐COOH might impair the M2 macrophage polarization without affecting phagocytosis. Given the importance of macrophage subsets and their products in health and disease, functionalized nanoparticles may provide a useful tool to reprogram their activation state possibly even for therapeutic purposes. Supported by the DFG, SPP1313.
Targeting Citrullination in the Injured Retina
Authors: Wizeman John , Nicholas Anthony , Mohan Royce
Published: June 2021

Objective

To define the contribution of citrullination on GFAP and vimentin in a traumatic eye injury model.

Methods: An ocular alkali injury model of retinal gliosis was employed in mice. Retinas from injured mice were examined at different times by western blot or immunochemistry for expression of GFAP, vimentin and citrullinated proteins. Posterior eye‐cups from 7‐day injured mice were placed into explant culture to investigate effects of peptidyl arginine deiminase (PAD) inhibitor Cl‐amidine.

Results: GFAP expression increases initially in the soluble extracts, starting as early as 1 hour after injury and continues over the 7‐day post‐injury period, followed by increased abundance of polymeric filaments in insoluble extracts. GFAP filament growth begins at the Muller glial end‐feet and advances towards the outer retina, showing punctate co‐staining for citrullinated precursors that become incorporated into vimentin and GFAP filaments from soluble pools. At later time points, these citrullinated protein species also become abundant in insoluble extracts. In posterior eye explant culture model from 7‐day injured eyes showing reactive gliosis, global increases in high molecular weight citrullinated proteins were detected. Cl‐amidine treatment strongly downregulates these hypercitrullinated proteins and high‐molecular weight citrullinated GFAP species.

Conclusions

GFAP appears to be a major target of hypercitrullination in this traumatic injury model of irreversible blindness because of its abundant overexpression. Our findings suggest that PADs are important targets for reversing pathological reactive gliosis.

Supported by EY016782 and Solomon Endowed Chair Fund.
O‐GlcNAcylation Modifies the Metastatic Properties of Prostate Cancer Cells
Authors: Liu Xin , mooney Steven , Hart Gerald
Published: June 2021

O‐GlcNAc is a ubiquitous and dynamic post‐translational modification that occurs on serine/threonine residues of nuclear and cytoplasmic proteins. It is regulated by O‐GlcNAc transferase, which attaches O‐GlcNAc to proteins and O‐GlcNAcase, which removes O‐GlcNAc.It serves as a nutrient sensor to regulate virtually all cellular processes, as well as playing roles in various diseases, including Alzheimer's disease, diabetes and cancer. The roles of O‐GlcNAc in the regulation of prostate cancer remain unclear. Herein, we investigate how the regulation of O‐GlcNAc may drive prostate cancer progression using normal prostate epithelial cells and prostate cancer cell lines as models, and human tissue arrays. Using western blotting, we demonstrate that O‐GlcNAc, OGT and OGA are highly elevated in LNCaP and PC‐3 when compared to PrEC. LNCaP exhibited the highest levels of O‐GlcNAc, OGT and OGA. OGT and OGA are differentially localized in lines with different metastatic properties. Additionally, 2D gel electrophoresis reveals significant differences in O‐GlcNAcylated protein profiles among the three cell lines. In order to address how O‐GlcNAc affects the biological properties of these cancer cells, including shapes, proliferation, invasion and metastasis, several assays were used. The result of soft agar assays indicates that cycling of O‐GlcNAc in cells influence the anchorage independent growth of prostate cancer. Interestingly, analyses of human tissues indicate that there is a field effect in O‐GlcNAc expression with higher levels in the cytoplasm of cancer and the cytoplasm of benign tissue in close proximity to the cancer.
N‐chlorination, a Reversible Post‐translational Modification That Activates Chaperone Function in RidA
Authors: Müller Alexandra , Langklotz Sina , Lupilova Nataliya , Kuhlmann Katja , Bandow Julia , Leichert Lars
Published: June 2021

RidA from Escherichia coli is a member of the highly conserved YjgF/YER057p/UK114 protein family. Members of this family have a multitude of assigned functions, which include translational inhibition (human), Hsp90‐like chaperone activity (fruit fly), and enamine/imine deaminase activity in the branched‐chain amino acid synthesis (Salmonella enterica).

In the present study, we describe a novel function of RidA from E. coli. When treated with HOCl (household bleach), RidA is converted into a highly effective chaperone holdase preventing aggregation of the model substrate citrate synthase. Activation of RidA is reversed by treatment with DTT, ascorbic acid, the thioredoxin system, and glutathione.

However, in contrast to Hsp33, another well‐described HOCl‐activated chaperone in E. coli, activation of RidA does not depend on cysteine modification. Instead, upon treatment with HOCl or chloramines, the amino group content of RidA decreases as a result of reversible N‐chlorination of positively charged residues. N‐chlorination increases surface hydrophobicity, which enables RidA to bind its unfolding client proteins. Deletion of ridA results in an HOCl‐sensitive phenotype of E. coli demonstrating its crucial role during HOCl stress. A proteomic analysis of RidA's client proteins revealed that N‐chlorinated RidA binds highly abundant cytosolic proteins to protect them from HOCl‐mediated precipitation. N‐chlorination of positively charged residues thus is a novel post‐translational modification that reversibly turns RidA into a chaperone holdase.
Protein Polyphosphorylation of Lysine Residues by Inorganic Polyphosphate
Authors: Azevedo Cristina , Saiardi Adolfo
Published: June 2021

Whole genome sequencing revealed that the complexity of higher organisms is not only linked to the number of genes but also to the evolution of a wide range of additional regulatory features such as post‐translational modification of proteins (PTMs). Almost all proteins can be modulated in this way, adding a level of complexity much greater than each gene product. PTMs can be very complex in nature and occur enzymatically and non‐enzymatically.
We identified and characterized a new PTM mediated by inorganic polyphosphate (polyP). This linear polymer of orthophosphate linked by ‘high‐energy’ phosphoanhydride bonds, analogous to those found in ATP, is ubiquitous in nature. We named this modification “polyphosphorylation” as it represents the covalent attachment of polyP to target proteins. We found that Nuclear signal recognition 1 (Nsr1) and its interacting partner, topoisomerase 1 (Top1) are polyphosphorylated at lysine residues within a conserved N‐terminal PolyAcidic serine (S) and lysine (K) rich (PASK) cluster. Polyphosphorylation negatively regulates Nsr1/Top1 interaction and impairs Top1 enzymatic activity of relaxing super coiled DNA. We demonstrate that modulation of cellular levels of polyP regulates Top1 activity by modifying its polyphosphorylation status. We propose that polyphosphorylation represents a new regulatory mechanism of acidic proteins known to be enriched in the nuclei of both yeast and mammalian cells. Polyphosphorylation of these proteins may add an additional layer of regulation to nuclear signaling, in particular to one of the most energy‐consuming and tightly regulated process in eukaryotic cells, ribosomal RNA biogenesis, where Nsr1 and Top1 play roles.
Arkadia Mediates Sumoylation‐dependent Stabilization of Nrf2
Authors: McIntosh Deneshia , Walters Treniqka , Arinze Ifeanyi
Published: June 2021

We have previously demonstrated that ubiquitylation of polysumoylated Nrf2 by RING finger protein 4 (RNF4), a SUMO‐targeted ubiquitin ligase (STUbL), targets Nrf2 for degradation in promyelocytic leukemia‐nuclear bodies (PML‐NBs) in HepG2 cells. Here, we show that Arkadia (also called RNF111), another SUMO‐targeted ubiquitin ligase, also ubiquitylated polysumoylated Nrf2 in these cells. The action of Arkadia stabilized Nrf2,measured in whole‐cell lysates or in PML‐NB‐enriched fractions. Using linkage (Lys 48‐ or Lys 63‐) specific ubiquitin antibodies, we show in co‐immunoprecipitation assays that Lys‐63 but not Lys‐48 is involved in Arkadia‐mediated ubiquitylation of polysumoylated Nrf2. Thus, Arkadia‐induced stabilization of Nrf2 is consistent with the notion that polyubiquitin chains linked through Lys‐63 residues do not target proteins for proteolytic destruction. Given that Arkadia has also been reported by others to ubiquitylate polysumoylated PML, thereby targeting it for proteolytic degradation, our study suggests that Arkadia functions as a dual specificity E3 ubiquitin ligase. Arkadia was able to enhance Nrf2‐mediated gene transcription from the heme‐oxygenase (HO‐1) luciferase gene construct whereas RNF4 decreased Nrf2‐mediated transcription from this reporter. RNF4 and Arkadia did not act synergistically suggesting that these two STUbLs affect Nrf2‐mediated gene transcription by two different mehanisms. Arkadia‐mediated ubiquitylation of polysumoylated Nrf2, resulting in stabilization of Nrf2, may explain the Arkadia‐induced increase in Nrf2‐dependent gene transcription.

Supported by NIH grants # SC1CA143985, 5T32HL007737, 2 SD1MD000104, and ULITR000445
cAMP Signaling: An Important Event Facilitating Survival and Infectivity of Leishmania.
Authors: Biswas Arunima , Bhattacharya Arijit , Vij Amit , Das pijush
Published: June 2021

Introduction

Leishmania donovani encounters striking shift in temperature and pH and these act as the key environmental trigger for differentiation. Moreover, the parasite also faces a huge oxidative stress inside macrophages. We showed that differentiation condition‐exposed parasites become resistant to oxidative damage due to increased intracellular cAMP‐mediated responses.

Objective

For comprehensive understanding of cAMP signaling, we studied all the enzymes associated with cAMP metabolism, viz. the synthesizing enzyme adenylate cyclase, the degrading enzyme, phosphodiesterase (PDE), the regulatory enzyme pyrophosphatase and the functional enzyme, cAMP‐dependent protein kinase (PKA).

Results

A stage‐specific isoform of receptor adenylate cyclase (LdRACA) showed to regulate differentiation‐coupled induction of cAMP. The membrane bound acidocalcisomal pyrophosphatase, LdV‐H+PPase, was the major isoform regulating cAMP level in association with LdRACA by modulating the total pyrophosphate pool. A differentially expressed soluble cytosolic cAMP phosphodiesterase (LdPDEA) might be related to infection establishment by shifting trypanothione pool utilization bias toward antioxidant defence. Another cytosolic phosphodiesterase, LdPDED though not differentially expressed, was found to down‐regulate cAMP‐mediated responses by inhibiting catalytic activity of PKA and by increasing its own PDEase activity through phosphorylation. Also, a functional cAMP‐binding effector molecule (a regulatory subunit of PKA, LdPKAR) seemed to modulate metacyclogenesis through induction of autophagy.

Conclusion

This study reveals the significance of cAMP signaling in parasite survival and infectivity.
M1 Protein from GAS Protects against Histone Killing
Authors: Döhrmann Simon , LaRock Chris , Anderson Ericka , Ryali Brinda , Ghosh Partho , Nizet Victor
Published: June 2021

Histones are an essential element of chromatin structure and necessary for eukaryotic survival. Highly positively charged, histones resemble small, cationicantimicrobial peptides (AMPs) of the mammalian innate immune system. The broad‐spectrum bactericidal action of histones against a variety of bacteria was first described in 1958. More recently, a framework for histone function in host defense in vivo was revealed with the discovery of neutrophil extracellular traps (NETs), DNA and histone‐based structures extruded by neutrophils to entrap and kill bacteria. Here, we explore histone resistance of several Gram‐positive bacterial pathogens and observe that one important human pathogen, group A Streptococcus (GAS), is highly resistant to histone killing. A screen of isogenic GAS mutants lacking various virulence factor genes revealed that the surface‐anchored M1 protein is associated with high‐level histone resistance. Studies utilizing purified protein and heterologous gene expression showed M1 to be both necessary and sufficient to confer histone resistance. The capacity of M1 to sequester histones and promote accumulatation of host fibrinogen as a further steric hindrance factor, blocked histone access to the bacterial cell wall. M1‐mediated histone resistance was correlated with enhanced GAS survival in NETs. This is the first description of a Gram‐positive bacterial resistance mechanism to histone killing, which in turn contributes to NET resistance. Histone resistance is likely a contributing factor to the well‐established virulence role of M1 in invasive GAS infection.

This research was supported by NIH grant (AI096837) to PG and VN.
From the ECM to Thrombosis: a New Role for the Matrix Enzyme Lysyl Oxidase
Authors: Matsuura Shinobu , Mi Rongjuan , Eliades Alexia , Toselli Paul , Thon Jonathan , Italiano Joseph , Trackman Philip , Papadantonakis Nikolaos , Ravid Katya
Published: June 2021

Introduction

Lysyl oxidase (LOX) catalyzes the oxidation of lysines to aldehydes, providing stability in connective tissues through spontaneous cross‐linking of collagen and elastin. LOX expression is elevated in sera of patients with myeloproliferative neoplasms (MPN), a group of disorders that have in common the proliferation of megakaryocytes and myelofibrosis in bone marrow. Thrombosis is a leading cause of morbidity and mortality in MPN, yet there is still controversy over contributors to the thrombotic events in MPN.

Objective

to test whether Lysyl Oxidase (LOX) is involved in the pathogenesis of thrombosis.

Methods

A transgenic mouse model expressing LOX in platelets (Pf4‐Loxtg/tg) was used to analyze platelet activation and thrombus formation.

Results

Vessel occlusion after endothelial injury was significantly shorter in Pf4‐Loxtg/tg mice, indicating a higher propensity for thrombus formation in vivo. While exploring mechanisms involved, we found that Pf4‐Loxtg/tg platelets adhere better to collagen. The higher affinity of Pf4‐Loxtg/tg platelets to monomeric compared to fibrillar collagen, as well as affinity to the GFOGER peptide implies integrin α2β1 as the collagen receptor targeted by LOX. Taken together, our study identified LOX as a new regulator of collagen receptor and thus platelet activation and thrombosis.
Cargo Receptor Traffics Many Matrix Proteins Out of the Endoplasmic Reticulum (ER) by Binding to N‐Terminal IleProVal (IPV)‐Like Motif
Authors: Yin Ying , Garcia Mekka , Novak Alex , Fisher Larry
Published: June 2021

Introduction

Matrix proteins assemble large complexes in the extracellular environment and may be at risk of forming undesired aggregates during intracellular trafficking. Patients with nonsyndromic Dentinogenesis Imperfecta have mutations in the dentin sialophosphoprotein (DSPP) gene. In half of the cases, the N‐terminal IPV tripeptide is changed (e.g., IPD, ITV). We have shown that IPV‐mutant DSPP inefficiently exits the ER and forms aggregates. Many secreted proteins of dentin (e.g., DSPP, DMP1), enamel (AMELX, AMBN, ENAM), bone (SPARC, OCN, OPN) and cartilage (BGN, DCN, ASPN), as well as enzymes (trypsin) and hormones (GH, LEP, PRL, FGF23) also start with conserved IPV‐like tripeptides. We propose that the same cargo receptor binds to these proteins through their IPV‐like motifs thereby trafficking them out of the ER before self‐ or inter‐molecular aggregates form.

Methods

The ER‐trafficking efficiency of DSPP (and other secreted proteins) expressing a series of single amino acid changes in their IPV motifs was studied in HEK293 and HeLa cells. To identify the cargo receptor, we expressed wildtype and mutant constructs in Saccharomyces cell lines each lacking a single ER‐associated protein.

Results

We have defined the position/property of amino acids in IPV‐like motif that promote ER exit in yeast and higher eukaryotes. Furthermore, gene deletion of a conserved ER‐associated yeast protein specifically delays ER exit of wildtype yeast and transfected matrix proteins.

Conclusion

We have defined the amino acids required to bind the proposed cargo receptor and have identified the yeast's receptor as a highly conserved, ER‐associated protein.

Supported by Division of Intramural Research of the NIDCR, NIH.
Integrin Dysregulation as a Driver of Matrix Remodeling in a Model of Congenital Muscular Dystrophy
Authors: Accorsi Anthony , Mehuron Thomas , Kumar Ajay , Rhee Younghwa , Girgenrath Mahasweta
Published: June 2021

Laminin‐deficient congenital muscular dystrophy (MDC1A) is the second most prevalent form of muscular dystrophy and is caused by a defect in the alpha chain of the muscle specific extracellular matrix protein Laminin‐211. Loss of this protein leads to vast signaling defects leading to failed regeneration, inflammation, fibrosis, and apoptosis. While the genetic cause is known, distinct pathomechanisms have yet to be elucidated. Integrins are heterodimeric membrane proteins that play key roles in many critical cell processes including cell adhesion and signal transduction. However, when dysregulated, they have been shown to be key pathological drivers in many disease scenarios. Using the DyW mouse model of MDC1A, qRT‐PCR, ELISA, and immunohistochemistry, we show for the first time that integrins, and most importantly Integrin‐αV, which is known to activate Transforming Growth Factor‐beta (TGF‐β) in fibrotic scenarios, are dysregulated in dystrophic muscle. Further, we found that protein levels of both latent and active TGF‐β are upregulated in the serum of DyW mice but only active TGF‐β is attenuated in response to treatment with Losartan (Angiotensin II type 1 receptor blocker known to be a potent antifibrotic agent). Interestingly, along with reduced levels of active TGF‐β, we also see a rescue of Integrin‐αV overexpression. Therefore, it is possible that the attenuation of TGF‐β signaling by Losartan could be mediated by downregulation of Integrin‐αV. These results suggest Integrin‐αV could be an important player in the induction and mediation of fibrotic pathology in MDC1A and could serve as a novel therapeutic target in this and other fibrotic, degenerative diseases.
Characterizing Fibrosis in Mouse Kidney Using Fluorescence Lifetime and Second Harmonic Generation Imaging Microscopy in Unilateral Ureteral Obstruction Model
Authors: Dobrinskikh Evgenia , Ranjit Suman , Montford John , Dvornikov Alexander , Lehman Allison , Nemenoff Raphael , Gratton Enrico , Levi Moshe , Furgeson Seth
Published: June 2021

Renal tubulointerstitial fibrosis is considered to be the final common pathway for most forms of chronic kidney disease (CKD). Fibrosis has been traditionally characterized by histological studies such as Picro‐Sirius Red (PSR) staining, Masson Trichrome staining or collagen immunohistochemistry. Such methods are limited due to variability in staining and pathologist scoring. The goal of this study is to compare histologic measures of renal fibrosis to Fluorescence Lifetime Imaging (FLIM) and Second Harmonic Generation (SHG) techniques in our deep tissue imaging microscope called DIVER. Male C57Bl6 mice were subjected to unilateral ureteral obstruction (UUO), a well‐characterized model of fibrosis. At 21 days, both kidneys were collected. Serial sections of both kidneys were analyzed by PSR staining or FLIM with SHG. Quantification of a whole kidney section from the PSR staining showed 34.32±0.99% area of fibrosis in the injured kidney compared to 5.55±1.07% in the control kidney. Using the Phasor approach to FLIM, comparisons between the two kidneys show that the auto fluorescence lifetime signature give rise to two well separate phasor clusters. Quantification of ten different fields of view for each kidney from SHG suggests the presence of more collagen I in the diseased kidneys (17.44±4.21% compared to 2.59±1.98% control kidney). In conclusion, the combined FLIM and SHG images let us establish a criterion for quantitative determination of fibrosis directly from the microscope images.
SUN2 accumulation contributes to vascular smooth muscle cell alterations induced by matrix rigidity
Authors: Bélaadi Néjma , Chadeuf Gilliane , Nguyen Luan , Rio Marc , Louarn Guy , Loirand Gervaise , Guilluy Christophe
Published: June 2021

In the vascular system, arterial wall cell phenotype and fate are driven by the mechanical properties of their surrounding environment. Changes in the mechanical environment, such as age‐related extracellular matrix (ECM) stiffening, alter vascular cell phenotype and growth, leading to inflammation and vascular remodeling, which favor cardiovascular diseases. Despite recent advances, how mechanical cues regulate cell phenotype remains poorly understood. By combining proteomic and biophysical assays, we recently identified nuclear proteins whose expression or post translational modifications are regulated by matrix rigidity. Among the identified candidates is Sad1/UNC‐84 domain‐containing protein 2 (SUN2), which belongs to the linker of nucleoskeleton and cytoskeleton complex and enables mechanical force transmission from the cytoskeleton to the nucleus. We observed increased SUN2 expression in vascular smooth muscle cells (VSMCs) cultured on rigid matrix. Interestingly, we found that SUN2 depletion prevented the increase in cell proliferation in response to matrix rigidity. Using a mouse model of endothelial injury, we observed SUN2 accumulation in the neointima at sites of cell proliferation. To analyze VSMC contractile function we used atomic force microscopy (AFM). We found that SUN2 depletion decreased VSMC contractile response to AFM pulling forces. All together these results indicate that upregulation of SUN2 contributes to the increased proliferation and altered contractility of VSCM in response to matrix stiffening.
Extracellular Matrix Fibronectin Attenuates PDGF‐Induced Intracellular Calcium Release
Authors: Farrar Christopher , Hocking Denise
Published: June 2021

The extracellular matrix (ECM) has structural and signaling roles within tissues. Fibronectin is an ECM protein that is secreted by fibroblasts in a protomeric form and assembled into a fibrillar matrix by a cell‐mediated process termed fibronectin matrix assembly. ECM proteins affect growth factor signaling. Platelet derived growth factor (PDGF) is a major mitogen for fibroblasts that promotes proliferation and migration. The objective of this study was to determine the effect of ECM fibronectin on the response of fibroblasts to PDGF. The response of cells to PDGF was quantified by measuring intracellular calcium release in Fluo‐4 loaded cells. Calcium release was measured in fibronectin‐null mouse embryonic fibroblasts, allowing for control over the amount of fibronectin to which cells were exposed and the initiation of fibronectin matrix assembly. Results showed that in the absence of fibronectin, addition of 50 ng/mL PDGF to cells transiently increased intracellular calcium concentration. In contrast, incubation of cells with 25 nM fibronectin for 20 hr significantly attenuated the PDGF‐induced increase in calcium release. Fibronectin‐mediated attenuation of PDGF‐induced calcium release was blocked by an inhibitor of fibronectin matrix assembly, indicating that the ECM form of fibronectin is required for fibronectin‐mediated attenuation of PDGF signaling. A fibronectin fragment engineered to mimic the ECM form of fibronectin also attenuated PDGF‐induced calcium release. Attenuation of PDGF‐induced calcium release was not due to downregulation of PDGF receptor β expression. This study demonstrates that fibronectin matrix assembly attenuates PDGF‐induced intracellular calcium release by an as‐yet unidentified mechanism.

Supported by: NIH/NHLBI: T32 HL066988
MAP1S‐enhanced Autophagy Alleviates Tissue Fibrosis and Sustains Mouse Survival under Stress Imposed by LC3‐induced Overexpression of Fibronectin
Authors: Liu Leyuan
Published: June 2021

Autophagy is a cellular process that controls and executes the turnover of misfolded and abnormally aggregated proteins. Microtubule‐associated protein 1S (MAP1S) interacts with autophagy marker light chain 3 (LC3) and positively regulates autophagy flux. LC3 also binds with fibronectin mRNA and facilitates its sorting along microtubules onto rough endoplasmic reticulum where it is translated into fibronectin protein. Fibronectin is exported to surface of plasma membrane to initiate the assembly of fibronectin extracellular matrix and other extracellular matrix proteins. Fibronectin matrix is degraded in lysosomes after it is engulfed into cytosol via endocytosis. Here we show that overexpression of LC3 in wildtype mice promoted the synthesis and degradation of fibronectin and consequently resulted in a balanced levels of fibronectin; therefore, the GFP‐LC3 transgenic mice had a normal length of lifespan as the wild‐type mice did. In GFP‐LC3 transgenic mice with MAP1S deleted, LC3 overexpression enhanced synthesis of fibronectin while MAP1S‐deficiency‐caused autophagy defect impaired the degradation of fibronectin. The accumulation of fibronectin protein led to enhanced levels of oxidative stress, genomic instability and tissue fibrosis, and to a dramatic 35% reduction in mouse lifespan. Therefore, autophagy may play an important role in maintaining mouse lifespan via regulating the homeostasis of fibronectin extracellular matrix.

This work was supported by a NIH R01CA142862 to Leyuan Liu.
Evaluating p97 inhibitor analogues for their domain‐selectivity and potency against the p97‐p47 complex
Authors: Gui Lin , Zhang Xiaoyi , Fang Chenjie , Moen Derek R. , Wang Daniel , Chou Tsui‐Fen
Published: June 2021

The p97 AAA is an important therapeutic target for cancer and neurodegenerative diseases. Each monomer is composed of an N domain followed by D1 and D2 ATPase domains. It is clear that the D2 domain hydrolyzes ATP in vitro. D1 shows ATPase activity in the full‐length protein only when the D2 domain is capable of binding nucleotides. A major role of the N domain is to recruit numerous p97 cofactors. Binding of p47‐one of the major p97 cofactors‐ to the N‐domain of p97 significantly inhibits wild type p97 ATPase activity.

Based on observations of enzymatic activity with small‐molecule inhibitors, we have recently evaluated that four p97 inhibitors have differential responses to disease‐causing IBMPFD mutations and to the presence of the N domain binding cofactor protein p47. DBeQ and NMS‐873 inhibited both ATPase domains, whereas ML240 and ML241 specifically target the D2‐domain of wild type p97. In addition, p47 significantly decreases their potency by ~50‐fold. In contrast,DBeQshows only a 4‐ to 6‐fold decrease in potency against the p97‐p47 complex.

To elucidate the structure‐activity relationships of 200 potential p97 inhibitor analogues, we characterize their ability to inhibit the p97 ATPase activity of the D1, D2, or both domains as well as the effect of p47 on potency. The selectivity of 29 of these compounds was further examined by 8‐dose titration.

These studies identify several potential starting points for future SAR analyses and also highlight the possibility of developing domain‐selective and complex‐specific p97 inhibitors. Together, our results provide the framework for developing domain, complex, and pathway specific inhibitors, with the ultimate goal of validating p97 as a potential therapeutic target.
Synthesis and Characterization of Triazine‐based Chemical Probes
Authors: Cole Kyle , Banerjee Ranjan , Pace Nicholas , Weerapana Eranthie
Published: June 2021

Rapid proliferation of cancer cells requires upregulated protein synthesis. Protein folding in the cell is assisted by chaperones and foldases– that accelerate protein folding and inhibit aggregation. One such family of chaperones is the protein disulfide isomerase (PDI) family. The first PDI discovered, PDIA1, is a 57‐kDa protein which is localized in the ER and has been shown to be critical for tumor growth in a mouse xenograft model of ovarian cancer. Several small molecule inhibitors of PDIA1 have been developed in the last few years, including one from our lab, RB‐11‐ca; which is equipotent, yet more site‐selective than the best current commercially available inhibitor, 16F16. Current efforts are to synthesize novel analogs of RB‐11‐ca to increase potency and selectivity against PDIA1. One compound, KSC‐7, has shown approximately a 20% increase in affinity for PDIA1 compared to RB‐11‐ca.

The triazine‐based scaffold has also become useful for targeting reactive lysines. A dichlorotriazine electrophile, RB7, has been discovered to label lysines, however, it is far too reactive for covalent‐inhibitor design. Efforts to replace the second chlorine with an amine substitution, to tune down the reactivity of the electrophile, led to a complete loss of protein labeling, Therefore, if we substitute a less electron donating substituent in the second position, then we would be able to tune down the reactivity of the electrophile enough to maintain the reactivity, but be able to selectively target reactive lysine residues.
Synthesis, Cytotoxicity, and DNA‐binding studies of the natural product eudistomin U
Authors: Giulietti Jennifer , Tate Patrick , Roggero Chad , Mulcahy Seann , Cho Bongsup
Published: June 2021

Natural products have been the leading source of potential drug leads and comprise approximately a third of the drugs on the market. β‐carbolines are a class of naturally occurring, tricyclic aromatic indole alkaloids that have been used to probe the biochemistry of a broad range of ailments. A subclass of β‐carbolines, known as the eudistomins, is reported to have diverse biological activity, as well as a high binding affinity to DNA. Through a novel five‐step synthesis, our lab successfully synthesized the natural product eudistomin U, a derivative of the eudistomin family. Furthermore, we have characterized eudistomin U's cytotoxic activity, as well as the compound's interaction with DNA via spectroscopic and biophysical techniques. We have determined that eudistomin U weakly binds to DNA in a nonspecific fashion, thus DNA binding is most likely not the cause of toxicity observed in the bacterial and cancer cell lines. We hope that future experiments will elucidate how eudistomin U disrupts biochemical pathways.
Curcumin Inhibits DLD‐1 Colorectal Cancer Cell Proliferation by Modulating MicroRNA and Tumor Suppressor Gene Expression
Authors: Adeyeni Temitope , Vattathara Kenn , Duggirala Rajat , Selvamani Vijayalingam , Ezekiel Uthayashanker
Published: June 2021

Phytochemicals are plant‐based, non‐toxic compounds that produce health benefits. Curcumin, a major phytochemical in turmeric, inhibits the proliferation of many types of cancer cells, both in vitro and in vivo. Phytochemicals subvert carcinogenesis by modifying methylation patterns normally maintained by cancer cells. This methylation modification mechanism indirectly influences the regulatory microRNA that maintains normal cell division. Earlier, we identified that curcumin exerts an antiproliferative effect on DLD‐1 colon cancer cells in a dose dependent manner. After 48‐hour treatment of DLD‐1 cells with curcumin, expression level of tumor suppressor gene, RASSF1A, and microRNA‐15a (miR‐15a) was evaluated. Normally, expression levels of both RASSF1A and miR‐15a were suppressed in DLD‐1 cells; however, upon curcumin treatment, expression levels of RASSF1A and miR‐15a were higher than those in vehicle‐treated controls. Also, the miR‐15a target, BMI1, was suppressed in curcumin‐treated cells. In summary, curcumin exerted an antiproliferative effect against colon cancer cells, possibly by an epigenetic mechanism.

KV and RD were supported by the DeNardo Foundation Fellowship.
Expanding the Utility of Sulfonated Fluorophores in cellulo
Authors: Choi Adam , Pauff Steven , Miller Stephen
Published: June 2021

Many of the best and brightest small molecule fluorophores are sulfonated, which greatly increases their water solubility, but precludes cellular permeability. Thus, these dyes are limited to in vitro and extracellular applications. To increase the utility of sulfonated fluorophores in cellulo, we have developed a modular delivery approach for sulfonated molecules. First, the negative charge on the free sulfonate is neutralized by forming a chemically stable sulfonyl ester, allowing membrane permeation. Then, upon cellular entry, a trigger that is labile to the intracellular environment causes the release of the free sulfonate. Using an esterase‐labile scaffold, we have delivered sulfonated fluorophores into mammalian cells. Here, we describe work to design reductively‐labile scaffolds that rely on the reducing intracellular environment to release the free sulfonate. These reductively‐labile scaffolds are complementary to the esterase‐labile approach, as they do not rely on enzyme activity or recognition. Both types of scaffolds can deliver otherwise impermeable sulfonated fluorophores into live mammalian cells. We envision that these strategies will be broadly applicable to the cellular delivery of a vast array of sulfonated molecules.
Identification and Synthesis of D. athabasca Cuticular Hydrocarbons
Authors: Bebernitz Christina , Kehlbeck Joanne , Harvey Taylor , Yukilevich Roman
Published: June 2021

We are investigating the role of chemical signals on courtship behavior of Drosophila. The chemical signals of Drosophila athabasca are cuticular hydrocarbons, derived from fatty acids, spanning 20‐40 carbons in length with variations in saturation and branching patterns. Drosophila athabasca has three distinct races that are geographically overlapping but reproductively isolated: WestNorthern, Eastern A, and Eastern B. We have identified several pheromones of Drosophila athabasca that may influence the sexual selection that necessarily precedes speciation. Using Gas Chromatography tandem Mass Spectrometry and dimethyl disulfide derivatization (DMDS), we have characterized the pheromone composition of several isofemale lines. Synthesis of the major pheromones was completed to confirm identity of the cuticular hydrocarbons. Synthesis of monounsaturated hydrocarbons utilized cis‐directed Wittig reactions, while diene hydrocarbon synthesis employed substitution chemistry with acetylenes followed by a cis‐selective reduction. Purification and isomeric separations of hydrocarbons was completed using silica followed by argentation chromatography. Behavioral studies using synthetic pheromones are underway and may provide insight into the relationship between cuticular hydrocarbons and mate discrimination.
Structural Mechanism of Laforin Function in Glycogen Dephosphorylation and Lafora Disease
Authors: Raththagala Madushi , Brewer M Kathryn , Sherwood Amanda , Parker Matthew , Vander Kooi Craig , Gentry Matthew
Published: June 2021

Glycogen is the major mammalian glucose storage cache and is critical for energy homeostasis. Glycogen synthesis in neurons must be tightly controlled, due to neuronal sensitivity to perturbations in glycogen metabolism. Lafora disease (LD) is a fatal, congenital, neurodegenerative epilepsy. Mutations in the gene encoding the glycogen phosphatase laforin result in hyperphosphorylated glycogen that forms water‐insoluble inclusions called Lafora bodies (LBs). LBs induce neuronal apoptosis and are the causative agent of LD. The mechanism of glycogen dephosphorylation by laforin and dysfunction in LD is unknown. We report the crystal structure of laforin bound to phosphoglucan product revealing its unique integrated tertiary and quaternary structure. Structure‐guided mutagenesis combined with biophysical and biochemical analyses reveal the basis for normal function of laforin in glycogen metabolism. Analyses of LD patient mutations define the mechanism by which subsets of mutations disrupt laforin function. These data provide fundamental insights connecting glycogen metabolism to neurodegenerative disease.
Identification of Env7 Phosphorylated Residues
Authors: Garfinkle Elizabeth , Gharakhanian Editte
Published: June 2021

The baker's yeast, Saccharomyces cerevisiae, vacuole is functionally analogous to the human lysosome. A novel genomic screen was performed in our laboratory to identify genes involved in the last stage of trafficking to the lysosome. ENV7 is one of four novel genes identified, and its product is involved in defects at the late endosome and vacuole interface. We have shown that Env7 is a phosphorylated palmitoylated protein kinase that is also conserved in humans. Based on bioinformatic studies in our lab on Env7, we hypothesize that Env7 phosphorylation is occurring at the specific C‐terminus serine residues 236, 287, 300, and 331. The objective is to isolate the phosphorylated C‐terminus fragment and identify its phosphorylated residues. A protease inhibitor assay was performed to maximize partially degraded C‐terminal fragments of Env7‐HA without excess protein degradation. The developed protocol was used to generate fragments of wild type Env7 and mutant Env7 Cys13‐15Ser, which does not autophosphorylate in vitro. The partially degraded protein samples were separated using SDS‐polyacrylamide gel electrophoresis and visualized using a Western blot. The phosphorylated C‐terminal fragments traveled a shorter distance relative to non‐phosphorylated Env7 due to their heavy weight. Wild type fragments showed a doublet band, whereas the corresponding fragment for Cys13‐15Ser showed a strong, lower single band. This shows that the strong, top band of the doublet present in wild type may be the phosphorylated residue of interest. It also proves that the Cys13‐15 track is necessary for kinase activity of Env7. More protocols will be developed to compare the phosphorylated fragments. Identification of the phosphorylated residues will be performed with mass spectroscopy of the isolated fragments.
The tyrosine kinase domains of the insulin and IGF1 receptors are functional dimers in the activated state
Authors: CABAIL MARIA , LI SHIQING , LEMMON ERIC , BOWEN MARK , HUBBARD STEVAN , MILLER TODD
Published: June 2021

Insulin Receptor (IR) and Insulin‐like growth factor 1 Receptor (IGF‐1R) are receptor tyrosine kinases (RTKs) ubiquitously distributed in human tissues. They are pre‐formed disulfide‐linked αβ homodimers. Binding of Insulin/IGF1 binding to the receptor ectodomain induces a conformational change in the quaternary structure of the receptors, which transforms the receptors from a basal state, in which trans‐phosphorylation on tyrosine residues is limited, to a trans‐phosphorylation‐competent state, and subsequently to a fully activated phosphorylated state, which stimulates catalytic activity and creates recruitment sites for downstream signaling proteins. The spatial relationships between the two kinase domains prior to and after transautophosphorylation are not known. In this study, we provide crystallographic, biophysical, and biochemical evidence demonstrating that, upon phosphorylation, the tyrosine kinase domains of IR and IGF1R form an activated kinase dimer.