Background: CHAC1 is associated with the stress response in atherosclerosis. Results: ATF4, ATF3, and CEBP␤ regulate CHAC1 transcription. Human CHAC1 protein overexpression depletes glutathione. Conclusion: CHAC1 is induced following multiple cell stress signals and leads to depletion of glutathione. Significance: CHAC1 may be an essential link between stress signaling and the oxidative status of the cell, contributing to multiple diseases.

A rapid method for isolation of a major surface membrane glycoprotein from whole, unfractionated cultured human B lymphoblasts is described. After detergent solubilization the method uses gel filtration followed by affinity chromatography on Sepharose Con A and then alkaline acrylamide gel electrophoresis. Specific high-titre, rabbit antisera to the isolated protein reacted with cultured and normal peripheral blood B lymphocytes, as well as peritoneal macrophages from a renal dialysis patient. The antisera selectively inhibited the mixed lymphocyte reaction at high dilution. The protein reacted with a heterologous antiserum to HL-B antigens and contained subunits of MW 33 000 and 27 000. Resolution of the subunits, however, required a discontinuous SDS gel system. These properties indicate its similarity to murine Ia antigens. The protein was not associated with beta 2 microglobulin and showed no structural or antigenic similarity to the major erythocyte glycoprotein, glycophorin. Antisera to the protein failed to precipitate surface-radiodinated components from similarily treated extracts of cultured human T lymphoblasts. This method now makes available a reference membrane glycoprotein from a differentiated, nucleated human cell in sufficient purity and quantity for kinetic and biosynthetic studies.

During their parasitic life cycle, through sandflies and vertebrate hosts, Leishmania parasites confront strikingly different environments, including abrupt changes in pH and temperature, to which they must rapidly adapt. These adaptations include alterations in Leishmania gene expression, metabolism, and morphology, allowing them to thrive as promastigotes in the sandfly and as intracellular amastigotes in the vertebrate host. A critical aspect of Leishmania metabolic adaptation to these changes is maintenance of efficient mitochondrial function in the hostile vertebrate environment. Such functions, including generation of ATP, depend upon the expression of many mitochondrial proteins, including subunits of cytochrome c oxidase (COX). Significantly, under mammalian temperature conditions, expression of Leishmania major COX subunit IV (LmCOX4) and virulence are dependent upon two copies of LACK, a gene that encodes the ribosome-associated scaffold protein, LACK (Leishmania ortholog of RACK1 [receptor for activated C kinase]). Targeted replacement of an endogenous LACK copy with a putative ribosome-binding motif-disrupted variant (LACKR34D35G36→LACKD34D35E36) resulted in thermosensitive parasites that showed diminished LmCOX4 expression, mitochondrial fitness, and replication in macrophages. Surprisingly, despite these phenotypes, LACKD34D35E36 associated with monosomes and polysomes and showed no major impairment of global protein synthesis. Collectively, these data suggest that wild-type (WT) LACK orchestrates robust LmCOX4 expression and mitochondrial fitness to ensure parasite virulence, via optimized functional interactions with the ribosome. IMPORTANCE Leishmania parasites are trypanosomatid protozoans that persist in infected human hosts to cause a spectrum of pathologies, from cutaneous and mucocutaneous manifestations to visceral leishmaniasis caused by Leishmania donovani. The latter is usually fatal if not treated. Persistence of L. major in the mammalian host depends upon maintaining gene-regulatory programs to support essential parasite metabolic functions. These include expression and assembly of mitochondrial L. major cytochrome c oxidase (LmCOX) subunits, important for Leishmania ATP production. Significantly, under mammalian conditions, WT levels of LmCOX subunits require threshold levels of the Leishmania ribosome-associated scaffold protein, LACK. Unexpectedly, we find that although disruption of LACK’s putative ribosome-binding motif does not grossly perturb ribosome association or global protein synthesis, it nonetheless impairs COX subunit expression, mitochondrial function, and virulence. Our data indicate that the quality of LACK’s interaction with Leishmania ribosomes is critical for LmCOX subunit expression and parasite mitochondrial function in the mammalian host. Collectively, these findings validate LACK’s ribosomal interactions as a potential therapeutic target.

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Renal cell carcinoma (RCC) is a cancer that is hard to treat because of its evasive nature, and its resistance to chemotherapy, radiotherapy, and immunotherapy. There is only a 10% survival rate in humans if RCC is not caught at early stages. L-Arginine metabolism is a highly regulated process that can produce global effects in tumors and in the immune system. Arginase 1 (ARG1) and ARG2 can metabolize L-arginine to induce the synthesis of polyamines necessary for tumor growth, whereas inducible nitric oxide synthase (NOS2) can metabolize L-arginine to produce nitric oxide (NO) which has been shown to possess antitumor activity. In addition, L-citrulline, another metabolite of NOS2, may play an important role in cancer, since it acts as a substrate for the de novo synthesis of L-arginine. Previous data have shown that certain RCC cell lines respond to IFNy or IFNa treatments by activating the NOS2 protein to produce NO, which inhibits tumor growth. However, in several other RCC cell lines there is a lack of NOS2 protein induction, suggesting that these RCC cells have developed a mechanism that blocks NOS2 expression. Therefore, treatments that could activate NOS2 expression to induce tumor regression are much needed. We believe that the lack of NOS2 is due to L-arginine deprivation, which is highly depleted in ARG2-RCC tumors. It is possible that decreased extracellular L-arginine limits its intracellular availability, deactivating downstream translational proteins, then blocking NOS2 expression. We hypothesize that in some RCC, the competition of ARG and NOS2 for L-arginine regulates the expression of NOS2 as a mechanism to increase tumor growth. To test our hypothesis, increasing concentrations of L-arginine of 1,000, 2,000 and 4,000 uM were added to RPMI media and cells were cultured for 24 and 48 hours. At each time point cell lysates were tested for ARG2, NOS2, GCN2 and eIF2a proteins, as well as for intracellular levels of L-arginine. Supernatants were tested for L-arginine, L-glutamine, L-citrulline and nitrite production. Our results show that after 48 hours in culture, there was a decrease in ARG activity as compared to untreated controls (p=0.004) in cells treated with 4,000 uM of L-arginine; a slight increase in NOS2 protein was observed with no significant increases in nitrites. More experiments using closer L-arginine concentration increments are under way in lieu to determine the expression of GNC2 and eIF2a. The levels of L-citrulline were significantly higher at 48 hours in cells cultured in 4,000 uM of L-arginine. One striking observation was that the cells consumed L-glutamine at higher rates as early as 24 hours. This finding is very important because these cells are possibly using L-glutamine as a source for polyamine synthesis, bypassing the L-arginine-ARG pathway. Understanding the mechanisms by which RCC tumors grow could help us to develop new and more effective immune-therapeutic strategies to overcome RCC growth and resistance. Citation Format: Charity F. Sylvester, Paula Datri, Arnold Zea. L-Arginine in the regulation of NOS2 expression to overcome renal cell carcinoma tumor growth [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B067.