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Copy number alterations detected by comparative genomic hybridization (CGH) can lead to the identification of novel cancer‐related genes. We analyzed chromosomal aberrations in a set of 100 human primary colorectal cancers (CRCs) using CGH and found a solute carrier (SLC) 7A1 gene, which encodes cationic amino acid transporter 1 (CAT1) with 14 putative transmembrane domains, in a chromosome region (13q12.3) with a high frequency of gene amplifications. SLC7A1/CAT1 is a transporter responsible for the uptake of cationic amino acids (arginine, lysine, and ornithine) essential for cellular growth. Microarray and PCR analyses have revealed that mRNA transcribed from CAT1 is overexpressed in more than 70% of human CRC samples, and RNA interference–mediated knockdown of CAT1 inhibited the cell growth of CRCs. Rats were immunized with rat hepatoma cells expressing CAT1 tagged with green fluorescent protein (GFP), and rat splenocytes were fused with mouse myeloma cells. Five rat monoclonal antibodies (mAbs) (CA1 ~ CA5) reacting with HEK293 cells expressing CAT1‐GFP in a GFP expression–dependent manner were selected from established hybridoma clones. Novel anti‐CAT1 mAbs selectively reacted with human CRC tumor tissues compared with adjacent normal tissues according to immuno‐histochemical staining and bound strongly to numerous human cancer cell lines by flow cytometry. Anti‐CAT1 mAbs exhibited internalization activity, antibody‐dependent cellular cytotoxicity, and migration inhibition activity against CRC cell lines. Furthermore, CA2 inhibited the in vivo growth of human HT29 and SW‐C4 CRC tumors in nude mice. This study suggested CAT1 to be a promising target for mAb therapy against CRCs. Frequent gene amplification of SLC7A1/CAT1 and increased CAT1 mRNA expression in colorectal cancers (CRC) were demonstrated. Novel anti‐CAT1 monoclonal antibodies (mAbs) were developed, and higher expression of CAT1 protein in CRC and in vivo antitumor effects of mAbs on xenografted CRC cells in nude mice were confirmed. CAT1 may be a promising target for anti‐CRC therapy.

The cationic amino acid transporter 1 (CAT1/SLC7A1) plays a key role in the cellular uptake or export of l-arginine and some of its derivatives. This study investigated the effect of 113 chemically diverse and commonly used drugs (at 20 and 200 µM) on the CAT1-mediated cellular uptake of l-arginine, l-homoarginine, and asymmetric dimethylarginine (ADMA). Twenty-three (20%) of the tested substances showed weak inhibitory or stimulatory effects, but only verapamil showed consistent inhibitory effects on CAT1-mediated transport of all tested substrates.

Background: Since common drug therapies cannot eradicate Candida biofilm, extensive studies are required to develop more effective antifungal compounds and identify their mechanism of action against Candida biofilm. Peganum harmala L. is a traditional medicinal plant, the seeds of which have been used to treat various diseases. Objectives: This study aimed to investigate the anti-biofilm mechanisms of P. harmala extract (PHE) and the expression of CAT1, EFG1, and BCR1 genes involved in oxidative stress response and biofilm formation in Candida albicans. Methods: Anti-biofilm activity of PHE was evaluated by crystal violet assay to determine biofilm formation on 33 C. albicans isolates. Finally, a real-time polymerase chain reaction was performed to analyze the effect of PHE on the expression of CAT1, EFG1, and BCR1 genes in C. albicans. Results: This study determined the minimum biofilm eradication concentration (MBEC) of 15 isolates in concentrations between 0.49 - 3.9 μg/mL of P. harmala extract. Statistical analysis showed that the exposure of C. albicans biofilm to PHE significantly reduced the expression of CAT1 mRNA in C. albicans isolates (P = 0.0068). However, no significant difference was observed in the expression of EFG1 and BCR1 genes. Conclusions: The results demonstrated that PHE significantly decreased CAT1 expression in C. albicans cells treated with the herbal extract. PHE is likely to accumulate hydrogen peroxide (H2O2) by reducing CAT1 expression and disrupting the pro-oxidant/antioxidant balance that leads to the overproduction of reactive oxygen species (ROS) and can cause damage to cellular components and eventually destroy C. albicans biofilm.

The SLC7 family is divided into two subgroups, the cationic amino acid transporters (the CAT family, SLC7A1-4) and the glycoprotein-associated amino acid transporters (the gpaAT family, SLC7A5-11), also called light chains or catalytic chains of the hetero(di)meric amino acid transporters (HAT). The associated glycoproteins (heavy chains) 4F2hc (CD98) or rBAT (D2, NBAT) form the SLC3 family. Members of the CAT family transport essentially cationic amino acids by facilitated diffusion with differential trans-stimulation by intracellular substrates. In some cells, they may regulate the rate of NO synthesis by controlling the uptake of l-arginine as the substrate for nitric oxide synthase (NOS). The heterodimeric amino acid transporters are, in contrast, quite diverse in terms of substrate selectivity and function (mostly) as obligatory exchangers. Their selectivity ranges from large neutral amino acids (system L) to small neutral amino acids (ala, ser, cys-preferring, system asc), negatively charged amino acid (system x(c)(-)) and cationic amino acids plus neutral amino acids (system y(+)L and b(0,+)-like). Cotransport of Na(+) is observed only for the y(+)L transporters when they carry neutral amino acids. Mutations in b(0,+)-like and y(+)L transporters lead to the hereditary diseases cystinuria and lysinuric protein intolerance (LPI), respectively.

Patatin, a prominent food protein derived from potatoes, is renowned for its exceptional nutritional value. Patatin has been characterized for its diverse physiological attributes, including esterase activity, antioxidative properties, cholesterol-lowering effects, and high lysine content, alongside notable physicochemical traits such as foaming, emulsification, and gelation capabilities. Conventional methods for patatin extraction are fraught with inefficiencies, elevated costs, and detrimental impacts on protein structural and functional integrity. Herein, we leveraged an optimized strategy integrating an expression cassette toolbox and regulation of protein secretion to harness Komagataella phaffii as the expression host and achieved an expression level of 3.2 g per litre (g/L) in a 5-Litre bioreactor, which is the highest yield of patatin production using engineered bacteria and funguses that has been reported thus far. In this study, we innovatively refined the endogenous promoter PCAT1, and its efficacy in driving heterologous protein expression under methanol induction surpassed that of the conventional AOX1 promoter. Furthermore, crucial nodes for patatin heterologous expression in yeast were identified, substantially curtailing the production costs associated with patatin synthesis.

Oxidative stress (OS) is generated by the imbalance between reactive oxygen species (ROS) and antioxidant enzyme activities, such as catalases, superoxide dismutases, and glutathione peroxidases. In the Y. lipolytica genome, three genes encoding catalases (CAT1, CAT2, and CAT3) have been identified; all three genes are transcriptionally active in cells grown under OS conditions. This study aimed to analyze whether the CAT1 and CAT2 genes exhibit a compensatory function that allows maintaining the functionality of the antioxidant response in Y. lipolytica cells lacking the CAT3 gene. The construction of the mutant strain (Ylcat3-Δ) was performed using Double-Joint PCR. OS was induced by the addition of H2O2 [5 mM], ROS production was quantified by fluorescence using 2′,7′-dichlorofluorescein diacetate (DCFH-DA), and gene expression was analyzed by semi-quantitative RT-PCR in both parental (P01a) and mutant (Ylcat3-Δ) strains exposed or not to oxidative conditions. ROS production was lower in P01a cells than in Ylcat3-Δ cells when exposed to H2O2 [5 mM]. Also, under OS conditions, CAT1 gene expression levels decreased in both strains, whereas CAT2 gene expression increased in both types of cells. Under OS, both parental and Ylcat3-Δ strains showed similar growth rate, sensitivity to oxidative conditions and gene expression patterns, and it can be concluded that CAT3 gene deletion does not alter the transcriptional activity of CAT1 and CAT2 genes, suggesting that the compensatory function among the CAT genes of Y. lipolytica may not be limited to the presence/absence of CAT3 gene.

TRPV5 and TRPV6 constitute the Ca 2+ influx pathway in a variety of epithelial cells. Here, we identified S100A10 as the first auxiliary protein of these epithelial Ca 2+ channels using yeast two‐hybrid and GST pull‐down assays. This S100 protein forms a heterotetrameric complex with annexin 2 and associates specifically with the conserved sequence VATTV located in the C‐terminal tail of TRPV5 and TRPV6. Of these five amino acids, the first threonine plays a crucial role since the corresponding mutants (TRPV5 T599A and TRPV6 T600A) exhibited a diminished capacity to bind S100A10, were redistributed to a subplasma membrane area and did not display channel activity. Using GST pull‐down and co‐immunoprecipitation assays we demonstrated that annexin 2 is part of the TRPV5–S100A10 complex. Furthermore, the S100A10–annexin 2 pair colocalizes with the Ca 2+ channels in TRPV5‐expressing renal tubules and TRPV6‐expressing duodenal cells. Importantly, downregulation of annexin 2 using annexin 2‐specific small interfering RNA inhibited TRPV5 and TRPV6‐mediated currents in transfected HEK293 cells. In conclusion, the S100A10–annexin 2 complex plays a crucial role in routing of TRPV5 and TRPV6 to plasma membrane.

The classical laboratory mouse strains are genetic mosaics of three Mus musculus subspecies that occupy distinct regions of Eurasia. These strains and subspecies carry infectious and endogenous mouse leukemia viruses (MLVs) that can be pathogenic and mutagenic. MLVs evolved in concert with restrictive host factors with some under positive selection, including the XPR1 receptor for xenotropic/polytropic MLVs (X/P-MLVs) and the post-entry restriction factor Fv1. Since positive selection marks host-pathogen genetic conflicts, we examined MLVs for counter-adaptations at sites that interact with XPR1, Fv1, and the CAT1 receptor for ecotropic MLVs (E-MLVs). Results describe different co-adaptive evolutionary paths within the ranges occupied by these virus-infected subspecies. The interface of CAT1, and the otherwise variable E-MLV envelopes, is highly conserved; antiviral protection is afforded by the Fv4 restriction factor. XPR1 and X/P-MLVs variants show coordinate geographic distributions, with receptor critical sites in envelope, under positive selection but with little variation in envelope and XPR1 in mice carrying P-ERVs. The major Fv1 target in the viral capsid is under positive selection, and the distribution of Fv1 alleles is subspecies-correlated. These data document adaptive, spatial and temporal, co-evolutionary trajectories at the critical interfaces of MLVs and the host factors that restrict their replication.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis, the most common neoplastic disease in cattle. We previously reported the development and protocol of the luminescence syncytium induction assay (LuSIA), a method for evaluating BLV infectivity based on CC81-GREMG cells. These cells form syncytia expressing enhanced green fluorescent protein when co-cultured with BLV-infected cells. Recently, we confirmed CAT1/SLC7A1 functions as a receptor of BLV. Here, we focused on CAT1/SLC7A1 to increase the sensitivity of LuSIA. We constructed a bovine CAT1-expressing plasmid and established a new CC81-GREMG-derived reporter cell line highly expressing bovine CAT1 (CC81-GREMG-CAT1). The new LuSIA protocol using CC81-GREMG-CAT1 cells measures cell-to-cell infectivity and cell-free infectivity of BLV faster and with greater sensitivity than the previous protocol using CC81-GREMG. The new LuSIA protocol is quantitative and more sensitive than the previous assay based on CC81-GREMG cells and will facilitate the development of several new BLV assays.