Mammalian Isoforms Research Articles

Secreted Phospholipases A2: Drivers of Inflammation and Cancer.

Secreted phospholipase 2 (sPLA2) is the largest family of phospholipase A2 (PLA2) enzymes with 11 mammalian isoforms. Each sPLA2 exhibits different localizations and specific properties, being involved in a very wide spectrum of biological processes. The enzymatic activity of sPLA2 has been well described; however, recent findings have shown that they could regulate different signaling pathways by acting directly as ligands. Arachidonic acid (AA) and its derivatives are produced by sPLA2 in collaboration with other molecules in the extracellular space, making important impacts on the cellular environment, being especially relevant in the contexts of immunity and cancer. For these reasons, this review focuses on sPLA2 functions in processes such as the promotion of EMT, angiogenesis, and immunomodulation in the context of tumor initiation and progression. Finally, we will also describe how this knowledge has been applied in the search for new sPLA2 inhibitory compounds that can be used for cancer treatment.

Structural and Functional Biology of Mammalian ALOX Isoforms with Particular Emphasis on Enzyme Dimerization and Their Allosteric Properties.

The human genome involves six functional arachidonic acid (AA) lipoxygenase (ALOX) genes, and the corresponding enzymes (ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3, ALOX5) have been implicated in cell differentiations and in the pathogenesis of inflammatory, hyperproliferative, metabolic, and neurological disorders. Humans express two different AA 15-lipoxygenating ALOX isoforms, and these enzymes are called ALOX15 (15-LOX1) and ALOX15B (15-LOX2). Chromosomal localization, sequence alignments, and comparison of the enzyme properties suggest that pig and mouse ALOX15 orthologs (leukocyte-type 12-LOX) on the one hand and rabbit and human ALOX15 orthologs on the other (reticulocyte-type 15-LOX1) belong to the same enzyme family despite their different reaction specificities with AA as a substrate. In contrast, human ALOX12 (platelet-type 12-LOX), as well as pig and mouse ALOX15 (leukocyte-type 12-LOX), belong to different enzyme families, although they exhibit a similar reaction specificity with AA as a substrate. The complex multiplicity of mammalian ALOX isoforms and the controversial enzyme nomenclatures are highly confusing and prompted us to summarize the current knowledge on the biological functions, enzymatic properties, and allosteric regulation mechanisms of mammalian ALOX15, ALOX15B, and ALOX12 orthologs that belong to three different enzyme sub-families.

Application of the Linear Interaction Energy Method to Nitric Oxide Synthase Structure-Based Inhibitor Design.

The overproduction of nitric oxide by neuronal nitric oxide synthase (nNOS) is associated with several neuropathological conditions. As a result, inhibition of nNOS is a desirable therapeutic goal while avoiding the inhibition of endothelial NOS (eNOS) given its essential role in maintaining cardiovascular tone. Designing inhibitors with high specificity for nNOS over eNOS is challenging given the close similarity in the active site structure of all mammalian NOS isoforms. Computational methods like free energy perturbation (FEP) and thermodynamic integration (TI) offer attractive avenues for rational drug design, but application of these methods to NOS is hindered by several challenges, including proper handling of highly charged inhibitors with diverse structures as well as computational expense. To address these issues, we present a simplified approach combining continuum dielectric generalized born (GB) solvent models with linear interaction energy (LIE) calculations. Our method demonstrates excellent agreement with experimental data for charged inhibitors targeting mammalian NOS isoforms (mNOS). Our results highlight the utility of the GB-LIE method as a promising tool for screening NOS inhibitors and potentially other protein targets with charged active sites and diverse inhibitor structures.

The different roles of V-ATPase a subunits in phagocytosis/endocytosis and autophagy

ABSTRACT Microglia are specialized macrophages responsible for the clearance of dead neurons and pathogens by phagocytosis and degradation. The degradation requires phagosome maturation and acidification provided by the vesicular- or vacuolar-type H+-translocating adenosine triphosphatase (V-ATPase), which is composed of the cytoplasmic V1 domain and the membrane-embedded Vo domain. The V-ATPase a subunit, an integral part of the Vo domain, has four isoforms in mammals. The functions of different isoforms on phagosome maturation in different cells/species remain controversial. Here we show that mutations of both the V-ATPase Atp6v0a1 and Tcirg1b/Atp6v0a3 subunits lead to the accumulation of phagosomes in zebrafish microglia. However, their mechanisms are different. The V-ATPase Atp6v0a1 subunit is mainly distributed in early and late phagosomes. Defects of this subunit lead to a defective transition from early phagosomes to late phagosomes. In contrast, The V-ATPase Tcirg1b/Atp6v0a3 subunit is primarily located on lysosomes and regulates late phagosome-lysosomal fusion. Defective Tcirg1b/Atp6v0a3, but not Atp6v0a1 subunit leads to reduced acidification and impaired macroautophagy/autophagy in microglia. We further showed that ATP6V0A1/a1 and TCIRG1/a3 subunits in mouse macrophages preferentially located in endosomes and lysosomes, respectively. Blocking these subunits disrupted early-to-late endosome transition and endosome-to-lysosome fusion, respectively. Taken together, our results highlight the essential and conserved roles played by different V-ATPase subunits in multiple steps of phagocytosis and endocytosis across various species. Abbrevations: Apoe: apolipoprotein E; ANXA5/annexin V: annexin A5; ATP6V0A1/a1: ATPase H+-transporting V0 subunit a1; ATP6V0A2/a2: ATPase H+-transporting V0 subunit a2; ATP6V0A4/a4: ATPase H+-transporting V0 subunit a4; dpf: days post-fertilization; EEA1: early endosome antigen 1; HOPS: homotypic fusion and protein sorting; LAMP1: lysosomal associated membrane protein 1; Lcp1: lymphocyte cytosolic protein 1 (L-plastin); Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; NR: neutral red; PBS: phosphate-buffered saline; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,5)P2: phosphatidylinositol (3,5)-bisphosphate; RAB4: RAB4, member RAS oncogene family; RAB5: RAB5, member RAS oncogene family; RAB7: RAB7, member RAS oncogene family; TCIRG1/Atp6v0a3/a3: T cell immune regulator 1, ATPase H+-transporting V0 subunit a3; V-ATPase: vacuolar-type H+-translocating adenosine triphosphatase; Xla.Tubb2b/NBT: tubulin beta 2B class IIb.

Abstract 3356: Inhibiting phospholipase D1 reduces pancreatic carcinogenesis in mice

Abstract New therapeutic targets are needed for pancreatic ductal adenocarcinoma (PDAC), a leading cause of cancer-related death worldwide. Phospholipase D (PLD) is a lipid-signaling enzyme that appears to play a critical role in cell proliferation, survival/apoptosis, and downstream oncogenic transformation. Elevated PLD1 or PLD2 (the two classical mammalian isoforms) activity has been shown to promote tumor progression in various cancer types, however, the role and mechanisms of PLD in pancreatic cancer are not yet understood. Thus, the objective is to determine the role of PLD1 regulation in pancreatic carcinogenesis through genetic and pharmacological inhibition. To evaluate the role of PLD1 in tumor growth, we subcutaneously transplanted human MIA PaCa-2 pancreatic cancer cells expressing endogenous PLD1 levels (Ctr KD cells), or MIA PaCa-2 cells in which PLD1 was knocked down (Pld1 KD cells), into immunodeficient nude mice. After 18 days post implantation, tumors that arose from Pld1-KD cells presented a significantly smaller tumor size (p<0.05) and had reduced levels of Bcl-xL and Bcl-2, compared to controls (Ctr KD). Then, to assess the role of PLD1 in the tumor microenvironment, we implanted KPC pancreatic cancer cells into C57BL/6 wild-type (WT) or C57BL/6 PLD1 knockout (Pld1−/−) mice with and without treatment with gemcitabine. Fourteen days after tumor implantation, the growth of KPC tumors was attenuated in Pld1−/− mice by 49% (p<0.05). Treatment with gemcitabine reduced tumor growth by 51.6% (p<0.05) in WT mice and by 79.5% (p<0.03) in Pld1−/− mice. Moreover, we evaluated the effects of PLD1 genetic ablation by crossing LSL-KrasG12D/+; Ptf1Cre/+ (KC) mice into PLD1 knockout (Pld1−/−) mice to generate KC; Pld1−/− mice. At 8 months of age, KC; Pld1−/− mice had significantly smaller pancreases (p<0.05) compared to KC mice. Interestingly, PLD1 ablation increased phosphorylation of ERK and c-Jun in KC; Pld1−/− mice compared to KC mice. Finally, to assess whether pharmacological inhibition of PLD can inhibit pancreatic carcinogenesis, KC mice received daily intraperitoneal injections of a small molecule inhibitor of PLD1 and PLD2 (FIPI) at a dosage of 3 mg/kg of body weight for a duration of 5 weeks. Treatment with FIPI reduced acinar-to-ductal metaplasia (p<0.05) and reduced cell proliferation (p<0.01), when compared to vehicle-treated KC controls. Additionally, FIPI treatment was safe demonstrating no significant differences in body weight between the groups and no significant alteration in the serum levels of liver enzymes, including aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase. In conclusion, these findings reveal PLD1 plays a critical role in pancreatic carcinogenesis and might represent a novel therapeutic target. Citation Format: Hala Amer Addassi, Karen Matsukuma, Gerardo G. Mackenzie. Inhibiting phospholipase D1 reduces pancreatic carcinogenesis in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3356.

ESI-MS analysis of Cu(I) binding to apo and Zn7 human metallothionein 1A, 2, and 3 identifies the formation of a similar series of metallated species with no individual isoform optimization for Cu(I).

Metallothioneins (MTs) are cysteine-rich proteins involved in metal homeostasis, heavy metal detoxification, and protection against oxidative stress. Whether the four mammalian MT isoforms exhibit different metal binding properties is not clear. In this paper, the Cu(I) binding properties of the apo MT1A, apo MT2, and apo MT3 are compared and the relative Cu(I) binding affinities are reported. In all three isoforms, Cu4, Cu6, and Cu10 species form cooperatively, and MT1A and MT2 also form a Cu13 species. The Cu(I) binding properties of Zn7-MT1A, Zn7-MT2, and Zn7-MT3 are compared systematically using isotopically pure 63Cu(I) and 68Zn(II). The species formed in each MT isoform were detected through electrospray ionization-mass spectrometry and further characterized using room temperature phosphorescence spectroscopy. The mixed metal Cu, Zn species forming in MT1A, MT2, and MT3 have similar stoichiometries and their emission spectral properties indicate that analogous clusters form in the three isoforms. Three parallel metallation pathways have been proposed through analysis of the detailed Cu, Zn speciation in MT1A, MT2, and MT3. Pathway ① results in Cu5Zn5-MT and Cu9Zn3-MT. Pathway ② involves Cu6Zn4-MT and Cu10Zn2-MT. Pathway ③ includes Cu8Zn4-MT. Speciation analysis indicates that Pathway ② is the preferred pathway for MT2. This is also evident in the phosphorescence spectra with the 750nm emission from Cu6Zn4-MT being most prominent in MT2. We see no evidence for different MT isoforms being optimized or exhibiting preferences for certain metals. We discuss the probable stoichiometry for MTs in vivo based on the in vitro determined binding constants.

Tissue distribution of cysteine string protein/DNAJC5 in C. elegans analysed by CRISPR/Cas9-mediated tagging of endogenous DNJ-14.

Cysteine string protein (CSP) is a member of the DnaJ/Hsp40 family of molecular chaperones. CSP is enriched in neurons, where it mainly localises to synaptic vesicles. Mutations in CSP-encoding genes in flies, worms, mice and humans result in neuronal dysfunction, neurodegeneration and reduced lifespan. Most attention has therefore focused on CSP's neuronal functions, although CSP is also expressed in non-neuronal cells. Here, we used genome editing to fluorescently tag the Caenorhabditis elegans CSP orthologue, dnj-14, to identify which tissues preferentially express CSP and hence may contribute to the observed mutant phenotypes. Replacement of dnj-14 with wrmScarlet caused a strong chemotaxis defect, as seen with other dnj-14 null mutants. In contrast, inserting the reporter in-frame to create a DNJ-14-wrmScarlet fusion protein had no effect on chemotaxis, indicating that C-terminal tagging does not impair DNJ-14 function. WrmScarlet fluorescence appeared most obvious in the intestine, head/pharynx, spermathecae and vulva/uterus in the reporter strains, suggesting that DNJ-14 is preferentially expressed in these tissues. Crossing the DNJ-14-wrmScarlet strain with GFP marker strains confirmed the intestinal and pharyngeal expression, but only a partial overlap with neuronal GFP was observed. DNJ-14-wrmScarlet fluorescence in the intestine was increased in response to starvation, which may be relevant to mammalian CSPα's role in microautophagy. DNJ-14's enrichment in worm reproductive tissues (spermathecae and vulva/uterus) parallels the testis-specific expression of CSPβ and CSPγ isoforms in mammals. Furthermore, CSPα messenger RNA is highly expressed in the human proximal digestive tract, suggesting that CSP may have a conserved, but overlooked, function within the gastrointestinal system.

The CNK–HYP scaffolding complex promotes RAF activation by enhancing KSR–MEK interaction

The RAS–MAPK pathway regulates cell proliferation, differentiation and survival, and its dysregulation is associated with cancer development. The pathway minimally comprises the small GTPase RAS and the kinases RAF, MEK and ERK. Activation of RAF by RAS is notoriously intricate and remains only partially understood. There are three RAF isoforms in mammals (ARAF, BRAF and CRAF) and two related pseudokinases (KSR1 and KSR2). RAS-mediated activation of RAF depends on an allosteric mechanism driven by the dimerization of its kinase domain. Recent work on human RAFs showed that MEK binding to KSR1 promotes KSR1–BRAF heterodimerization, which leads to the phosphorylation of free MEK molecules by BRAF. Similar findings were made with the single Drosophila RAF homolog. Here we show that the fly scaffold proteins CNK and HYP stabilize the KSR–MEK interaction, which in turn enhances RAF–KSR heterodimerization and RAF activation. The cryogenic electron microscopy structure of the minimal KSR–MEK–CNK–HYP complex reveals a ring-like arrangement of the CNK–HYP complex allowing CNK to simultaneously engage KSR and MEK, thus stabilizing the binary interaction. Together, these results illuminate how CNK contributes to RAF activation by stimulating the allosteric function of KSR and highlight the diversity of mechanisms impacting RAF dimerization as well as the regulatory potential of the KSR–MEK interaction.

Charged Amino Acids in the Transmembrane Helix Strongly Affect the Enzyme Activity of Aromatase.

Estrogens play critical roles in embryonic development, gonadal sex differentiation, behavior, and reproduction in vertebrates and in several human cancers. Estrogens are synthesized from testosterone and androstenedione by the endoplasmic reticulum membrane-bound P450 aromatase/cytochrome P450 oxidoreductase complex (CYP19/CPR). Here, we report the characterization of novel mammalian CYP19 isoforms encoded by CYP19 gene copies. These CYP19 isoforms are all defined by a combination of mutations in the N-terminal transmembrane helix (E42K, D43N) and in helix C of the catalytic domain (P146T, F147Y). The mutant CYP19 isoforms show increased androgen conversion due to the KN transmembrane helix. In addition, the TY substitutions in helix C result in a substrate preference for androstenedione. Our structural models suggest that CYP19 mutants may interact differently with the membrane (affecting substrate uptake) and with CPR (affecting electron transfer), providing structural clues for the catalytic differences.

The wide world of non-mammalian phospholipase D enzymes

Phospholipase D (PLD) hydrolyses phosphatidylcholine (PtdCho) to produce free choline and the critically important lipid signaling molecule phosphatidic acid (PtdOH). Since the initial discovery of PLD activities in plants and bacteria, PLDs have been identified in a diverse range of organisms spanning the taxa. While widespread interest in these proteins grew following the discovery of mammalian isoforms, research into the PLDs of non-mammalian organisms has revealed a fascinating array of functions ranging from roles in microbial pathogenesis, to the stress responses of plants and the developmental patterning of flies. Furthermore, studies in non-mammalian model systems have aided our understanding of the entire PLD superfamily, with translational relevance to human biology and health. Increasingly, the promise for utilization of non-mammalian PLDs in biotechnology is also being recognized, with widespread potential applications ranging from roles in lipid synthesis, to their exploitation for agricultural and pharmaceutical applications.

In-silico and in-vitro functional validation of imidazole derivatives as potential sirtuin inhibitor.

Epigenetic enzymes can interact with a wide range of genes that actively participate in the progression or repression of a diseased condition, as they are involved in maintaining cellular homeostasis. Sirtuins are a family of Class III epigenetic modifying enzymes that regulate cellular processes by removing acetyl groups from proteins. They rely on NAD+ as a coenzyme in contrast to classical histone deacetylases (HDACs) (Class I, II, and IV) that depend on Zn+ for their activation, linking their function to cellular energy levels. There are seven mammalian sirtuin isoforms (Sirt1-7), each located in different subcellular compartments. Sirtuins have emerged as a promising target, given that inhibitors of natural and synthetic sources are highly warranted. Imidazole derivatives are often investigated as sirtuin regulators due to their ability to interact with the binding site and modulate their activity. Imidazole bestows many possible substitutions on its ring and neighboring atoms to design and synthesize derivatives with specific target selectivity and improved pharmacokinetic properties, optimizing drug development. Ligand preparation, protein preparation, molecular docking, molecular dynamics, density function theory (DFT) analysis, and absorption, distribution, metabolism, and excretion (ADME) analysis were performed to understand the interacting potential and effective stability of the ligand with the protein. RT-PCR and Western blot analyses were performed to understand the impact of ligands on the gene and protein expression of Class III HDAC enzymes. We evaluated the sirtuin inhibition activity of our in-house compound comprised of imidazole derivatives by docking the molecules with the protein data bank. ADME properties of all the compounds used in the study were evaluated, and it was found that all fall within the favorable range of being a potential drug. The molecule with the highest docking score was analyzed using DFT, and the specific compound was used to treat the non-small cell lung cancer (NSCLC) cell lines A549 and NCI-H460. The gene and protein expression data support the in-silico finding that the compound Ethyl 2-[5-(4-chlorophenyl)-2-methyl-1-H-Imidazole-4-yl) acetate has an inhibitory effect on nuclear sirtuins. In conclusion, targeting sirtuins is an emerging strategy to combat carcinogenesis. In this study, we establish that Ethyl 2-[5-(4-chlorophenyl)-2-methyl-1-H-Imidazole-4-yl) acetate possesses a strong inhibitory effect on nuclear sirtuins in NSCLC cell lines.

Purification of modified mammalian actin isoforms for in vitro reconstitution assays

In vitro reconstitution assays using purified actin have greatly improved our understanding of cytoskeletal dynamics and their regulation by actin-binding proteins. However, early purification methods consisted of harsh conditions to obtain pure actin and often did not include correct maturation and obligate modification of the isolated actin monomers. Novel insights into the folding requirements and N-terminal processing of actin as well as a better understanding of the interaction of actin with monomer sequestering proteins such as DNaseI, profilin and gelsolin, led to the development of more gentle approaches to obtain pure recombinant actin isoforms with known obligate modifications. This review summarizes the approaches that can be employed to isolate natively folded endogenous and recombinant actin from tissues and cells. We further emphasize the use and limitations of each method and describe how these methods can be implemented to study actin PTMs, disease-related actin mutations and novel actin-like proteins.

Structural analysis of human ATE1 isoforms and their interactions with Arg-tRNAArg

Posttranslational protein arginylation has been shown as a key regulator of cellular processes in eukaryotes by affecting protein stability, function, and interaction with macromolecules. Thus, the enzyme Arginyltransferase and its targets, are of immense interest to modulate cellular processes in the normal and diseased state. While the study on the effect of this posttranslational modification in mammalian systems gained momentum in the recent times, the detail structures of human ATE1 (hATE1) enzymes has not been investigated so far. Thus, the purpose of this study was to predict the overall structure and the structure function relationship of hATE1 enzyme and its four isoforms. The structure of four ATE1 isoforms were modelled and were docked with 3’end of the Arg-tRNAArg which acts as arginine donor in the arginylation reaction, followed by MD simulation. All the isoforms showed two distinct domains. A compact domain and a somewhat flexible domain as observed in the RMSF plot. A distinct similarity in the overall structure and interacting residues were observed between hATE1-1 and X4 compared to hATE1-2 and 5. While the putative active sites of all the hATE1 isoforms were located at the same pocket, differences were observed in the active site residues across hATE1 isoforms suggesting different substrate specificity. Mining of nsSNPs showed several nsSNPs including cancer associated SNPs with deleterious consequences on hATE1 structure and function. Thus, the current study for the first time shows the structural differences in the mammalian ATE1 isoforms and their possible implications in the function of these proteins. Communicated by Ramaswamy H. Sarma

The cytosolic N-terminal domain of V-ATPase a-subunits is a regulatory hub targeted by multiple signals.

Vacuolar H+-ATPases (V-ATPases) acidify several organelles in all eukaryotic cells and export protons across the plasma membrane in a subset of cell types. V-ATPases are multisubunit enzymes consisting of a peripheral subcomplex, V1, that is exposed to the cytosol and an integral membrane subcomplex, Vo, that contains the proton pore. The Vo a-subunit is the largest membrane subunit and consists of two domains. The N-terminal domain of the a-subunit (aNT) interacts with several V1 and Vo subunits and serves to bridge the V1 and Vo subcomplexes, while the C-terminal domain contains eight transmembrane helices, two of which are directly involved in proton transport. Although there can be multiple isoforms of several V-ATPase subunits, the a-subunit is encoded by the largest number of isoforms in most organisms. For example, the human genome encodes four a-subunit isoforms that exhibit a tissue- and organelle-specific distribution. In the yeast S. cerevisiae, the two a-subunit isoforms, Golgi-enriched Stv1 and vacuolar Vph1, are the only V-ATPase subunit isoforms. Current structural information indicates that a-subunit isoforms adopt a similar backbone structure but sequence variations allow for specific interactions during trafficking and in response to cellular signals. V-ATPases are subject to several types of environmental regulation that serve to tune their activity to their cellular location and environmental demands. The position of the aNT domain in the complex makes it an ideal target for modulating V1-Vo interactions and regulating enzyme activity. The yeast a-subunit isoforms have served as a paradigm for dissecting interactions of regulatory inputs with subunit isoforms. Importantly, structures of yeast V-ATPases containing each a-subunit isoform are available. Chimeric a-subunits combining elements of Stv1NT and Vph1NT have provided insights into how regulatory inputs can be integrated to allow V-ATPases to support cell growth under different stress conditions. Although the function and distribution of the four mammalian a-subunit isoforms present additional complexity, it is clear that the aNT domains of these isoforms are also subject to multiple regulatory interactions. Regulatory mechanisms that target mammalian a-subunit isoforms, and specifically the aNT domains, will be described. Altered V-ATPase function is associated with multiple diseases in humans. The possibility of regulating V-ATPase subpopulations via their isoform-specific regulatory interactions are discussed.

PKD phosphorylation and COP9/Signalosome modulate intracellular Spry2 protein stability

Spry2 is a molecular modulator of tyrosine kinase receptor signaling pathways that has cancer-type-specific effects. Mammalian Spry2 protein undergoes tyrosine and serine phosphorylation in response to growth factor stimulation. Spry2 expression is distinctly altered in various cancer types. Inhibition of the proteasome functionality results in reduced intracellular Spry2 degradation. Using in vitro and in vivo assays, we show that protein kinase D (PKD) phosphorylates Spry2 at serine 112 and interacts in vivo with the C-terminal half of this protein. Importantly, missense mutation of Ser112 decreases the rate of Spry2 intracellular protein degradation. Either knocking down the expression of all three mammalian PKD isoforms or blocking their kinase activity with a specific inhibitor contributes to the stabilization of Spry2 wild-type protein. Downregulation of CSN3, a component of the COP9/Signalosome that binds PKD, significantly increases the half-life of Spry2 wild-type protein but does not affect the stability of a Spry2 after mutating Ser112 to the non-phosphorylatable residue alanine. Our data demonstrate that both PKD and the COP9/Signalosome play a significant role in control of Spry2 intracellular stability and support the consideration of the PKD/COP9 complex as a potential therapeutic target in tumors where Spry2 expression is reduced.

TGF-β mediated drug resistance in solid cancer

Transforming growth factor β (TGF-β) is an important signaling molecule which is expressed in three different isoforms in mammals (i.e. TGF-β1, -β2, and -β3). The interaction between TGF-β and its receptor triggers several pathways, which are classified into SMAD-dependent (canonical) and SMAD-independent (non-canonical) signaling, whose activation/transduction is finely regulated by several mechanisms. TGF-β is involved in many physiological and pathological processes, assuming a dualistic role in cancer progression depending on tumor stage. Indeed, TGF-β inhibits cell proliferation in early-stage tumor cells, while it promotes cancer progression and invasion in advanced tumors, where high levels of TGF-β have been reported in both tumor and stromal cells. In particular, TGF-β signaling has been found to be strongly activated in cancers after treatment with chemotherapeutic agents and radiotherapy, resulting in the onset of drug resistance conditions. In this review we provide an up-to-date description of several mechanisms involved in TGF-β-mediated drug resistance, and we report different strategies that are currently under development in order to target TGF-β pathway and increase tumor sensitivity to therapy.

Modulation of immunity by the secreted phospholipase A2 family.

Among the phospholipase A2 (PLA2 ) superfamily, which typically catalyzes the sn-2 hydrolysis of phospholipids to yield fatty acids and lysophospholipids, the secreted PLA2 (sPLA2 ) family contains 11 isoforms in mammals. Individual sPLA2 s have unique enzymatic specificity toward fatty acids and polar heads of phospholipid substrates and display distinct tissue/cellular distributions, suggesting their distinct physiological functions. Recent studies using knockout and/or transgenic mice for a full set of sPLA2 s have revealed their roles in modulation of immunity and related disorders. Application of mass spectrometric lipidomics to these mice has enabled to identify target substrates and products of individual sPLA2 s in given tissue microenvironments. sPLA2 s hydrolyze not only phospholipids in the plasma membrane of activated, damaged or dying mammalian cells, but also extracellular phospholipids such as those in extracellular vesicles, microbe membranes, lipoproteins, surfactants, and dietary phospholipids, thereby exacerbating or ameliorating various diseases. The actions of sPLA2 s are dependent on, or independent of, the generation of fatty acid- or lysophospholipid-derived lipid mediators according to the pathophysiological contexts. In this review, we make an overview of our current understanding of the roles of individual sPLA2 s in various immune responses and associated diseases.

MP10-17 OSTEOPONTIN PHOSPHOPEPTIDE MITIGATES UROLITHIASIS IN A DROSOPHILA MELANOGASTER MODEL

You have accessJournal of UrologyCME1 Apr 2023MP10-17 OSTEOPONTIN PHOSPHOPEPTIDE MITIGATES UROLITHIASIS IN A DROSOPHILA MELANOGASTER MODEL Polycronis Akouris, John Chmiel, Gerrit Stuivenberg, Wongsakorn Kiattiburut, Jennifer Bjazevic, Hassan Razvi, Bernd Grohe, Harvey Goldberg, Jeremy Burton, and Kait Al Polycronis AkourisPolycronis Akouris More articles by this author , John ChmielJohn Chmiel More articles by this author , Gerrit StuivenbergGerrit Stuivenberg More articles by this author , Wongsakorn KiattiburutWongsakorn Kiattiburut More articles by this author , Jennifer BjazevicJennifer Bjazevic More articles by this author , Hassan RazviHassan Razvi More articles by this author , Bernd GroheBernd Grohe More articles by this author , Harvey GoldbergHarvey Goldberg More articles by this author , Jeremy BurtonJeremy Burton More articles by this author , and Kait AlKait Al More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003225.17AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Kidney stone disease affects between 7 and 13% of the North American population and continues to burden global healthcare systems. The majority of kidney stones are calcium-based, with approximately 80% composed of calcium oxalate (CaOx). Despite advances in current preventative strategies, first-time symptomatic stone formers have an approximate 39% recurrence rate after 15 years. Thus, it is clear that more effective strategies are needed to prevent disease recurrence. Osteopontin (OPN) is a urinary protein that can inhibit the formation of CaOx monohydrate (COM) crystals in vitro. Numerous studies have confirmed that human and other mammalian OPN isoforms are potent inhibitors of COM crystallization. Despite this, the efficacy of these peptides as a supplement to prevent kidney stones has never been assessed in a living organism. We therefore sought to determine if a 16-residue synthetic OPN phosphopeptide (hereafter denoted as P3) could reduce CaOx stone burden and adhesion to mammalian kidney cells using in vivo and in vitro models, respectively. METHODS: To evaluate the effect of P3 on adhesion of COM crystals in vitro, synthetic crystals were allowed to adhere to Madin-Darby canine kidney (MDCK) cells in the presence of P3. An established Drosophila melanogaster model of CaOx-based urolithiasis was used to test the applicability of P3 to reduce stone burden in vivo. CaOx crystal burden and morphology were assessed using birefringence and confocal microscopy, respectively. RESULTS: P3 directly reduced the adherence of synthetic COM crystals to MDCK cells. Furthermore, oral supplementation of P3 significantly reduced CaOx burden in D. melanogaster. Confocal microscopy of crystals in D. melanogaster Malpighian tubules revealed that P3 was able to interact with COM crystals and alter morphology similar to previous in vitro studies. CONCLUSIONS: Altogether, this study demonstrates that a synthetic OPN phosphopeptide can directly mitigate CaOx-based urolithiasis formation in vivo by modulating crystal morphology. While in vitro evidence from simplistic models supports the inhibitory role of OPN phosphopeptides on CaOx crystal formation, this is the first study to directly illustrate that they can mitigate crystal formation in a complex living system. These findings suggest that OPN supplementation is a promising therapeutic approach and may be clinically useful in the management of recurrent CaOx urolithiasis in humans. Source of Funding: N/A © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e120 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Polycronis Akouris More articles by this author John Chmiel More articles by this author Gerrit Stuivenberg More articles by this author Wongsakorn Kiattiburut More articles by this author Jennifer Bjazevic More articles by this author Hassan Razvi More articles by this author Bernd Grohe More articles by this author Harvey Goldberg More articles by this author Jeremy Burton More articles by this author Kait Al More articles by this author Expand All Advertisement PDF downloadLoading ...

Molecular Aspects Implicated in Dantrolene Selectivity with Respect to Ryanodine Receptor Isoforms

Dantrolene is an intra-cellularly acting skeletal muscle relaxant used for the treatment of the rare genetic disorder, malignant hyperthermia (MH). In most cases, MH susceptibility is caused by dysfunction of the skeletal ryanodine receptor (RyR1) harboring one of nearly 230 single-point MH mutations. The therapeutic effect of dantrolene is the result of a direct inhibitory action on the RyR1 channel, thus suppressing aberrant Ca2+ release from the sarcoplasmic reticulum. Despite the almost identical dantrolene-binding sequence exits in all three mammalian RyR isoforms, dantrolene appears to be an isoform-selective inhibitor. Whereas RyR1 and RyR3 channels are competent to bind dantrolene, the RyR2 channel, predominantly expressed in the heart, is unresponsive. However, a large body of evidence suggests that the RyR2 channel becomes sensitive to dantrolene-mediated inhibition under certain pathological conditions. Although a consistent picture of the dantrolene effect emerges from in vivo studies, in vitro results are often contradictory. Hence, our goal in this perspective is to provide the best possible clues to the molecular mechanism of dantrolene's action on RyR isoforms by identifying and discussing potential sources of conflicting results, mainly coming from cell-free experiments. Moreover, we propose that, specifically in the case of the RyR2 channel, its phosphorylation could be implicated in acquiring the channel responsiveness to dantrolene inhibition, interpreting functional findings in the structural context.

VDAC interactions with lipids and their effect on isoform specificity.

The Voltage-Dependent Anion Channel (VDAC) is a β-barrel, the most abundant protein of the mitochondrial outer membrane, allowing passage of ions and metabolites (such as ATP, ADP, NADH⋯) in and out of the mitochondria. Besides its transport function, VDAC is also implicated in mitochondrial regulation with roles in apoptosis, calcium homeostasis or neurodegeneration. However, its role in those different pathways remains to be understood at the molecular level. VDAC possesses 3 isoforms in mammals (VDAC1, 2 and 3), sharing a high sequence identity, the same architecture and an overlapping tissue distribution. They display very similar transport properties such as conductance, selectivity and voltage-induced closure while their isoform specificity originates from their different interaction with partner proteins. This may determine their unique role in mitochondrial regulation, where each isoform seems to have different functions and a different set of partners: VDAC1 is implicated in neurodegeneration (Alzheimer and Parkinson disease), VDAC2 in apoptosis and calcium homeostasis, and VDAC3 probably in spermatogenesis and oxidative stress. Using a combination of biochemistry, molecular dynamics and electrophysiology, we investigated the lipid organization around each VDAC isoform and their impact on VDAC function. Our data suggest that the three isoforms have different affinities for lipids, and that could contribute to the selection of interacting partners.