Trpv6 channel targeting using monoclonal antibody induces prostate cancer cell apoptosis and tumor regression.

TRPV6 calcium channel is a prospective target in prostate cancer (PCa) since it is not expressed in healthy prostate while its expression increases during cancer progression. Despite the role of TRPV6 in PCa cell survival and apoptotic resistance has been already established, no reliable tool to target TRPV6 channel in vivo and thus to reduce tumor burden is known to date. Here we report the generation of mouse monoclonal antibody mAb82 raised against extracellular epitope of the pore region of the channel. mAb82 inhibited TRPV6 currents by 90% at 24 µg/ml in a dose-dependent manner while decreasing store-operated calcium entry to 56% at only 2.4 µg/ml. mAb82 decreased PCa survival rate in vitro by 71% at 12 µg/ml via inducing cell death through the apoptosis cascade via activation of the protease calpain, following bax activation, mitochondria enlargement, and loss of cristae, Cyt C release, pro-caspase 9 cleavage with the subsequent activation of caspases 3/7. In vivo, mice bearing either PC3Mtrpv6+/+ or PC3Mtrpv6-/-+pTRPV6 tumors were successfully treated with mAb82 at the dose as low as 100 µg/kg resulting in a significant reduction tumor growth by 31% and 90%, respectively. The survival rate was markedly improved by 3.5 times in mice treated with mAb82 in PC3Mtrpv6+/+ tumor group and completely restored in PC3Mtrpv6-/-+pTRPV6 tumor group. mAb82 showed a TRPV6-expression dependent organ distribution and virtually no toxicity in the same way as mAbAU1, a control antibody of the same Ig2a isotype. Overall, our data demonstrate for the first time the use of an anti-TRPV6 monoclonal antibody in vitro and in vivo in the treatment of the TRPV6-expressing PCa tumors.

LIKE EARLY STARVATION 1 interacts with amylopectin during starch biosynthesis.

Starch is the major energy storage compound in plants. Both transient starch and long-lasting storage starch accumulate in the form of insoluble, partly crystalline granules. The structure of these granules is related to the structure of the branched polymer amylopectin: linear chains of glucose units organized in double helices that align to form semicrystalline lamellae, with branching points located in amorphous regions between them. EARLY STARVATION 1 (ESV1) and LIKE EARLY STARVATION 1 (LESV) proteins are involved in the maintenance of starch granule structure and in the phase transition of amylopectin, respectively, in Arabidopsis (Arabidopsis thaliana). These proteins contain a conserved tryptophan-rich C-terminal domain folded into an antiparallel ß-sheet, likely responsible for binding of the proteins to starch, and different N-terminal domains whose structure and function are unknown. In this work, we combined biochemical and biophysical approaches to analyze the structures of LESV and ESV1 and their interactions with the different starch polyglucans. We determined that both proteins interact with amylopectin but not with amylose and that only LESV is capable of interacting with amylopectin during starch biosynthesis. While the C-terminal domain interacts with amylopectin in its semicrystalline form, the N-terminal domain of LESV undergoes induced conformational changes that are probably involved in its specific function of mediating glucan phase transition. These results clarify the specific mechanism of action of these 2 proteins in the biosynthesis of starch granules.

A Step-by-Step Guide for the Production of Recombinant Fluorescent TAT-HA-Tagged Proteins and their Transduction into Mammalian Cells.

Investigating the function of target proteins for functional prospection or therapeutic applications typically requires the production and purification of recombinant proteins. The fusion of these proteins with tag peptides and fluorescently derived proteins allows the monitoring of candidate proteins using SDS-PAGE coupled with western blotting and fluorescent microscopy, respectively. However, protein engineering poses a significant challenge for many researchers. In this protocol, we describe step-by-step the engineering of a recombinant protein with various tags: TAT-HA (trans-activator of transduction-hemagglutinin), 6×His and EGFP (enhanced green fluorescent protein) or mCherry. Fusion proteins are produced in E. coli BL21(DE3) cells and purified by immobilized metal affinity chromatography (IMAC) using a Ni-nitrilotriacetic acid (NTA) column. Then, tagged recombinant proteins are introduced into cultured animal cells by using the penetrating peptide TAT-HA. Here, we present a thorough protocol providing a detailed guide encompassing every critical step from plasmid DNA molecular assembly to protein expression and subsequent purification and outlines the conditions necessary for protein transduction technology into animal cells in a comprehensive manner. We believe that this protocol will be a valuable resource for researchers seeking an exhaustive, step-by-step guide for the successful production and purification of recombinant proteins and their entry by transduction within living cells. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: DNA cloning, molecular assembly strategies, and protein production Basic Protocol 2: Protein purification Basic Protocol 3: Protein transduction in mammalian cells.

NALCN-mediated sodium influx confers metastatic prostate cancer cell invasiveness.

There is growing evidence that ion channels are critically involved in cancer cell invasiveness and metastasis. However, the molecular mechanisms of ion signaling promoting cancer behavior are poorly understood and the complexity of the underlying remodeling during metastasis remains to be explored. Here, using a variety of in vitro and in vivo techniques, we show that metastatic prostate cancer cells acquire a specific Na+ /Ca2+ signature required for persistent invasion. We identify the Na+ leak channel, NALCN, which is overexpressed in metastatic prostate cancer, as a major initiator and regulator of Ca2+ oscillations required for invadopodia formation. Indeed, NALCN-mediated Na+ influx into cancer cells maintains intracellular Ca2+ oscillations via a specific chain of ion transport proteins including plasmalemmal and mitochondrial Na+ /Ca2+ exchangers, SERCA and store-operated channels. This signaling cascade promotes activity of the NACLN-colocalized proto-oncogene Src kinase, actin remodeling and secretion of proteolytic enzymes, thus increasing cancer cell invasive potential and metastatic lesions in vivo. Overall, our findings provide new insights into an ion signaling pathway specific for metastatic cells where NALCN acts as persistent invasion controller.

Further insight into the involvement of PII1 in starch granule initiation in Arabidopsis leaf chloroplasts.

The control of starch granule initiation in plant leaves is a complex process that requires active enzymes like Starch Synthase 4 and 3 (SS4 or SS3) and several noncatalytic proteins such as Protein Involved in starch Initiation 1 (PII1). In Arabidopsis leaves, SS4 is the main enzyme that control starch granule initiation, but in its absence, SS3 partly fulfills this function. How these proteins collectively act to control the initiation of starch granules remains elusive. PII1 and SS4 physically interact, and PII1 is required for SS4 to be fully active. However, Arabidopsis mutants lacking SS4 or PII1 still accumulate starch granules. Combining pii1 KO mutation with either ss3 or ss4 KO mutations provide new insights of how the remaining starch granules are synthesized. The ss3 pii1 line still accumulates starch, while the phenotype of ss4 pii1 is stronger than that of ss4. Our results indicate first that SS4 initiates starch granule synthesis in the absence of PII1 albeit being limited to one large lenticular granule per plastid. Second, that if in the absence of SS4, SS3 is able to initiate starch granules with low efficiency, this ability is further reduced with the additional absence of PII1.

TRPV6 Calcium Channel Targeting by Antibodies Raised against Extracellular Epitopes Induces Prostate Cancer Cell Apoptosis.

The TRPV6 calcium channel is known to be up-regulated in various tumors. The efforts to target the TRPV6 channel in vivo are still ongoing to propose an effective therapy against cancer. Here, we report the generation of two antibodies raised against extracellular epitopes corresponding to the extracellular loop between S1 and S2 (rb79) and the pore region (rb82). These antibodies generated a complex biphasic response with the transient activation of the TRPV6 channel. Store-operated calcium entry was consequently potentiated in the prostate cancer cell line LNCaP upon the treatment. Both rb79 and rb82 antibodies significantly decreased cell survival rate in a dose-dependent manner as compared to the control antibodies of the same isotype. This decrease was due to the enhanced cell death via apoptosis revealed using a sub-G1 peak in a cell cycle assay, TUNEL assay, and a Hoechst staining, having no effects in the PC3Mtrpv6-/- cell line. Moreover, all TUNEL-positive cells had TRPV6 membrane staining as compared to the control antibody treatment where TRPV6-positive cells were all TUNEL negative. These data clearly demonstrate that TRPV6 channel targeting using rb79 and rb82 antibodies is fatal and may be successfully used in the anticancer therapies.

Automated quantification of fluorescence and morphological changes in pretreated wood cells by fluorescence macroscopy.

BACKGROUND: Lignocellulosic biomass is a complex network of polysaccharides and lignin that requires a pretreatment step to overcome recalcitrance and optimize valorisation into biobased products. Pretreatment of biomass induces chemical and morphological changes. Quantification of these changes is critical to understand biomass recalcitrance and to predict lignocellulose reactivity. In this study, we propose an automated method for the quantification of chemical and morphological parameters through fluorescence macroscopy, which was applied on wood samples (spruce, beechwood) pretreated with steam explosion. RESULTS: Results in fluorescence macroscopy highlighted the impact of steam explosion on spruce and beechwood: fluorescence intensity of samples was highly altered, especially for the most severe conditions. Morphological changes were also revealed: shrinkage of cells and deformation of cell walls manifested as the loss of rectangularity or circular shape, for tracheids in spruce and vessels in beechwood respectively. Quantification of fluorescence intensity of cell walls and quantification of morphological parameters related to cell lumens were carried out accurately by applying the automated method onto the macroscopic images. The results showed that lumens area and circularity could be considered as complementary markers of cell deformation, and that fluorescence intensity of the cell walls could be related to morphological changes and to the conditions of pretreatment. CONCLUSIONS: The developed procedure allows simultaneous and effective quantification of morphological parameters and fluorescence intensity of the cell walls. This approach can be applied to fluorescence macroscopy as well as other imaging techniques and provides encouraging results towards the understanding of biomass architecture.

Ratiometric Fluorescent Safranin-O Staining Allows the Quantification of Lignin Contents In Muro.

In some specific vascular plant tissues, lignin can impregnate the entire cell wall to make it more rigid and hydrophobic. Different techniques have been developed in the past years to make possible the quantification of this polyphenolic polymer at the organ or tissue level, but difficulties of access to the cellular level remain. Here we describe an approach based on ratiometric emission measurements using safranin-O and the development of a macro adapted for the FIJI software, which makes it possible to quantify lignin in three different layers of the cell wall on images captured on a fluorescent confocal microscope.

REPRISAL: mapping lignification dynamics using chemistry, data segmentation, and ratiometric analysis.

This article describes a methodology for detailed mapping of the lignification capacity of plant cell walls that we have called "REPRISAL" for REPorter Ratiometrics Integrating Segmentation for Analyzing Lignification. REPRISAL consists of the combination of three separate approaches. In the first approach, H*, G*, and S* monolignol chemical reporters, corresponding to p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol, are used to label the growing lignin polymer in a fluorescent triple labeling strategy based on the sequential use of three main bioorthogonal chemical reactions. In the second step, an automatic parametric and/or artificial intelligence segmentation algorithm is developed that assigns fluorescent image pixels to three distinct cell wall zones corresponding to cell corners, compound middle lamella and secondary cell walls. The last step corresponds to the exploitation of a ratiometric approach enabling statistical analyses of differences in monolignol reporter distribution (ratiometric method [RM] 1) and proportions (RM 2) within the different cell wall zones. We first describe the use of this methodology to map developmentally related changes in the lignification capacity of wild-type Arabidopsis (Arabidopsis thaliana) interfascicular fiber cells. We then apply REPRISAL to analyze the Arabidopsis peroxidase (PRX) mutant prx64 and provide further evidence for the implication of the AtPRX64 protein in floral stem lignification. In addition, we also demonstrate the general applicability of REPRISAL by using it to map lignification capacity in poplar (Populus tremula × Populus alba), flax (Linum usitatissimum), and maize (Zea mays). Finally, we show that the methodology can be used to map the incorporation of a fucose reporter into noncellulosic cell wall polymers.

Comparative Analysis of Hepatitis C Virus NS5A Dynamics and Localization in Assembly-Deficient Mutants.

The hepatitis C virus (HCV) life cycle is a tightly regulated process, during which structural and non-structural proteins cooperate. However, the interplay between HCV proteins during genomic RNA replication and progeny virion assembly is not completely understood. Here, we studied the dynamics and intracellular localization of non-structural 5A protein (NS5A), which is a protein involved both in genome replication and encapsidation. An NS5A-eGFP (enhanced green fluorescent protein) tagged version of the strain JFH-1-derived wild-type HCV was compared to the corresponding assembly-deficient viruses Δcore, NS5A basic cluster 352-533 mutant (BCM), and serine cluster 451 + 454 + 457 mutant (SC). These analyses highlighted an increase of NS5A motility when the viral protein core was lacking. Although to a lesser extent, NS5A motility was also increased in the BCM virus, which is characterized by a lack of interaction of NS5A with the viral RNA, impairing HCV genome encapsidation. This observation suggests that the more static NS5A population is mainly involved in viral assembly rather than in RNA replication. Finally, NS4B exhibited a reduced co-localization with NS5A and lipid droplets for both Δcore and SC mutants, which is characterized by the absence of interaction of NS5A with core. This observation strongly suggests that NS5A is involved in targeting NS4B to lipid droplets (LDs). In summary, this work contributes to a better understanding of the interplay between HCV proteins during the viral life cycle.