Molecular Mechanism of SLC5A8 Inactivation in Breast Cancer

SLC5A8 is a putative tumor suppressor that is inactivated in more than 10 different types of cancer, but neither the oncogenic signaling responsible for SLC5A8 inactivation nor the functional relevance of SLC5A8 loss to tumor growth has been elucidated. Here, we identify oncogenic HRAS (HRAS^G12V) as a potent mediator of SLC5A8 silencing in human nontransformed normal mammary epithelial cell lines and in mouse mammary tumors through DNMT1. Further, we demonstrate that loss of Slc5a8 increases cancer-initiating stem cell formation and promotes mammary tumorigenesis and lung metastasis in an HRAS-driven murine model of mammary tumors. Mammary-gland-specific overexpression of Slc5a8 (mouse mammary tumor virus-Slc5a8 transgenic mice), as well as induction of endogenous Slc5a8 in mice with inhibitors of DNA methylation, protects against HRAS-driven mammary tumors. Collectively, our results provide the tumor-suppressive role of SLC5A8 and identify the oncogenic HRAS as a mediator of tumor-associated silencing of this tumor suppressor in mammary glands. These findings suggest that pharmacological approaches to reactivate SLC5A8 expression in tumor cells have potential as a novel therapeutic strategy for breast cancer treatment.     

Absence of Siglec-H in MCMV Infection Elevates Interferon Alpha Production but Does Not Enhance Viral Clearance

Plasmacytoid dendritic cells (pDCs) express the I-type lectin receptor Siglec-H and produce interferon α (IFNα), a critical anti-viral cytokine during the acute phase of murine cytomegalovirus (MCMV) infection. The ligands and biological functions of Siglec-H still remain incompletely defined in vivo. Thus, we generated a novel bacterial artificial chromosome (BAC)-transgenic “pDCre” mouse which expresses Cre recombinase under the control of the Siglec-H promoter. By crossing these mice with a Rosa26 reporter strain, a representative fraction of Siglec-H⁺ pDCs is terminally labeled with red fluorescent protein (RFP). Interestingly, systemic MCMV infection of these mice causes the downregulation of Siglec-H surface expression. This decline occurs in a TLR9- and MyD88-dependent manner. To elucidate the functional role of Siglec-H during MCMV infection, we utilized a novel Siglec-H deficient mouse strain. In the absence of Siglec-H, the low infection rate of pDCs with MCMV remained unchanged, and pDC activation was still intact. Strikingly, Siglec-H deficiency induced a significant increase in serum IFNα levels following systemic MCMV infection. Although Siglec-H modulates anti-viral IFNα production, the control of viral replication was unchanged in vivo. The novel mouse models will be valuable to shed further light on pDC biology in future studies.     

Endoplasmic reticulum targeted Bcl2 confers long term cell survival through phosphorylation of heat shock protein 27

Overexpression of anti-apoptotic Bcl2 family proteins is often seen in cancers rendering them insensitive to apoptosis inducing anticancer strategies. Anti-apoptotic Bcl2 family proteins are associated with different organelles like mitochondria and endoplasmic reticulum (ER) and exert their anti-apoptotic activity by inhibiting the release of Cyt.C from mitochondria irrespective of its localization. Here, we have identified a long term survival function for Bcl2 targeted at ER in mammalian system compared to wild type Bcl2 that is mediated by enhanced phosphorylation of heat shock protein 27 at ser 15, 78 and 82 sites with inhibition of caspase9 activity. Phosphorylation of hsp27 was prevented and the survival of ER-Bcl2 cells was reversed by inhibiting p38 and MEK suggesting that these kinases can act as the upstream targets for hsp27 phosphorylation. The results suggest that Bcl2 possess additional survival function in the regulation of apoptosis which is primarily regulated by its association with the ER in an hsp27 dependent manner. The interplay of both hsp27 and ER-Bcl2 in providing long term survival to cancer cells is interesting since both of these proteins are overexpressed in tumors with aggressive phenotype. The results suggest that spatial localization of Bcl2 family proteins also play a key role in long term survival of cancers indicating another level of functional regulation of Bcl2 in cancer cell survival.     

Hydroimidazolone modification of the conserved Arg12 in small heat shock proteins: studies on the structure and chaperone function using mutant mimics

Methylglyoxal (MGO) is an α-dicarbonyl compound present ubiquitously in the human body. MGO reacts with arginine residues in proteins and forms adducts such as hydroimidazolone and argpyrimidine in vivo. Previously, we showed that MGO-mediated modification of αA-crystallin increased its chaperone function. We identified MGO-modified arginine residues in αA-crystallin and found that replacing such arginine residues with alanine residues mimicked the effects of MGO on the chaperone function. Arginine 12 (R12) is a conserved amino acid residue in Hsp27 as well as αA- and αB-crystallin. When treated with MGO at or near physiological concentrations (2-10 μM), R12 was modified to hydroimidazolone in all three small heat shock proteins. In this study, we determined the effect of arginine substitution with alanine at position 12 (R12A to mimic MGO modification) on the structure and chaperone function of these proteins. Among the three proteins, the R12A mutation improved the chaperone function of only αA-crystallin. This enhancement in the chaperone function was accompanied by subtle changes in the tertiary structure, which increased the thermodynamic stability of αA-crystallin. This mutation induced the exposure of additional client protein binding sites on αA-crystallin. Altogether, our data suggest that MGO-modification of the conserved R12 in αA-crystallin to hydroimidazolone may play an important role in reducing protein aggregation in the lens during aging and cataract formation.     

Acetylation of αA-crystallin in the human lens: effects on structure and chaperone function

α-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including αA-crystallin are acetylated in vivo. We found that K70 and K99 in αA-crystallin and, K92 and K166 in αB-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, αA-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N(ε)-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated αA-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in αA-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against γ-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of αA-crystallin occurs in the human lens and that it affects the chaperone function of the protein.     

Evaluation of stem aqueous extract and synthesized silver nanoparticles using Cissus quadrangularis against Hippobosca maculata and Rhipicephalus (Boophilus) microplus

The present study was to determine the efficacies of anti-parasitic activities of synthesized silver nanoparticles (Ag NPs) using stem aqueous extract of Cissus quadrangularis against the adult of hematophagous fly, Hippobosca maculata (Diptera: Hippoboscidae), and the larvae of cattle tick, Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Contact toxicity method was followed to determine the potential of parasitic activity. Twelve milliliters of stem aqueous extract of C. quadrangularis was treated with 88ml of 1mM silver nitrate (AgNO(3)) solution at room temperature for 30min and the resulting solution was yellow-brown color indicating the formation extracellular synthesis of Ag NPs. The synthesized Ag NPs were characterized with UV-visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Field emission scanning electron microscope (FESEM) and energy dispersive X-ray (EDX) spectroscopy. The synthesized Ag NPs were recorded by UV-visible spectrum at 420nm and XRD patterns showed the nanoparticles crystalline in nature. FTIR analysis confirmed that the bioreduction of Ag((+)) ions to Ag NPs were due to the reduction by capping material of plant extract. FESEM image of Ag NPs showed spherical and oval in shape. By using the Bragg's Law and Scherrer's constant, the average mean size of synthesized Ag NPs was 42.46nm. The spot EDX analysis showed the complete chemical composition of the synthesized Ag NPs. The mortality obtained by the synthesized Ag NPs from the C. quadrangularis was more effective than the aqueous extract of C. quadrangularis and AgNO(3) solution (1mM). The adulticidal activity was observed in the aqueous extract, AgNO(3) solution and synthesized Ag NPs against the adult of H. maculata with LC(50) values of 37.08, 40.35 and 6.30mg/L; LC(90) values of 175.46, 192.17 and 18.14mg/L and r(2) values of 0.970, 0.992 and 0.969, respectively. The maximum efficacy showed in the aqueous extract, AgNO(3) solution and synthesized Ag NPs against the larvae of R. (B.) microplus with LC(50) values of 50.00, 21.72 and 7.61mg/L; LC(90) values of 205.12, 82.99 and 22.68mg/L and r(2) values of 0.968, 0.945and 0.994, respectively. The present study is the first report on antiparasitic activity of the experimental plant extract and synthesized Ag NPs. This is an ideal eco-friendly and inexpensive approach for the control of H. maculata and R. (B.) microplus.     

Phe71 is essential for chaperone-like function in alpha A-crystallin

Experiments with mini-alphaA-crystallin (KFVIFLDVKHFSPEDLTVK) showed that Phe(71) in alphaA-crystallin could be essential for the chaperone-like action of the protein (Sharma, K. K., Kumar, R. S., Kumar, G. S., and Quinn, P. T. (2000) J. Biol. Chem. 275, 3767-3771). In the present study we replaced Phe(71) in rat alphaA-crystallin with Gly by site-directed mutagenesis and then compared the structural and functional properties of the mutant protein with the wild-type protein. There were no differences in molecular size or intrinsic tryptophan fluorescence between the proteins. However, 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid interaction indicated a higher hydrophobicity for the mutant protein. Both wild-type and mutant proteins displayed similar secondary structure during far UV CD experiments. Near UV CD signal showed a slight difference in the tertiary structure around the 285-295 region for the two proteins. The mutant protein was totally inactive in suppressing the aggregation of reduced insulin, heat-denatured citrate synthase, and alcohol dehydrogenase. However, a marginal suppression of beta(L)-crystallin aggregation was observed when mutant alphaA-crystallin was included. These results suggest that Phe(71) contributes to the chaperone-like action of alphaA-crystallin. Therefore we conclude that the 70-88-region in alphaA-crystallin, identified by us earlier, is the functional chaperone site in alphaA-crystallin.     

Upregulation of the amino acid transporter ATB0,+ (SLC6A14) in colorectal cancer and metastasis in humans

ATB(0,+) (SLC6A14) is a Na(+)/Cl(-)-coupled arginine transporter expressed at low levels in normal colon. Arginine is an essential amino acid for tumor cells. Arginine is also the substrate for nitric oxide synthases (NOSs). Since arginine and arginine-derived nitric oxide (NO) play a critical role in cancer, we examined the expression of ATB(0,+) in colorectal cancer. Paired normal and cancer tissues from colectomy specimens of 10 patients with colorectal cancer and from the liver tissue of one patient with hepatic metastasis from a colonic primary were used for the analysis of the levels of ATB(0,+) mRNA, inducible NOS (iNOS) mRNA and the corresponding proteins. Tissues samples from the colon, liver, and lymph nodes of an additional patient with metastatic colon cancer were analyzed for ATB(0,+) protein alone. We also examined the levels of nitrotyrosylated proteins. The ATB(0,+) mRNA increased 22.9+/-3.0-fold in colorectal cancer compared to normal tissue and the increase was evident in each of the 10 cases examined. iNOS mRNA increased 5.2+/-1.1-fold in cancer specimens. The changes in mRNA levels were associated with an increase in ATB(0,+), iNOS, and nitrotyrosylated proteins. The increased expression of ATB(0,+) and iNOS was also demonstrated in liver and lymph node specimens with metastases from colonic primaries. This study strongly suggests that the upregulation of ATB(0,+) may have a pathogenic role in colorectal cancer. Since ATB(0,+) is a versatile transporter not only for arginine but also for several drugs including NOS inhibitors, these findings have significant clinical and therapeutic relevance.     

Cloning and functional characterization of a new subtype of the amino acid transport system N

We have cloned a new subtype of theamino acid transport system N2 (SN2 or second subtype of system N) fromrat brain. Rat SN2 consists of 471 amino acids and belongs to therecently identified glutamine transporter gene family that consists ofsystem N and system A. Rat SN2 exhibits 63% identity with rat SN1. Italso shows considerable sequence identity (50-56%) with themembers of the amino acid transporter A subfamily. In the rat, SN2 mRNA is most abundant in the liver but is detectable in the brain, lung,stomach, kidney, testis, and spleen. When expressed in Xenopus laevis oocytes and in mammalian cells, rat SN2 mediatesNa⁺-dependent transport of several neutral amino acids,including glycine, asparagine, alanine, serine, glutamine, andhistidine. The transport process is electrogenic, Li⁺tolerant, and pH sensitive. The transport mechanism involves the influxof Na⁺ and amino acids coupled to the efflux ofH⁺, resulting in intracellular alkalization. Proline,-(methylamino)isobutyric acid, and anionic and cationic amino acidsare not recognized by rat SN2.