Early Alteration of Nucleocytoplasmic Traffic Induced by Some RNA Viruses

AbstractA HeLa cell line expressing the green fluorescent protein fused to the SV40 T-antigen nuclear localization signal (EGFP-NLS) was established. Fluorescence in these cells was confined to the nuclei. After poliovirus infection, cytoplasmic fluorescence in a proportion of cells could be detected by 1 h postinfection (p.i.) and in virtually all of the fluorescent cells by 2 h p.i. The relocation could be prevented by cycloheximide but not by inhibition of poliovirus replication by guanidine · HCl. Nuclear exit of a protein composed of three copies of GFP fused to the NLS also occurred upon poliovirus infection. A similar redistribution of EGFP-NLS took place upon infection with coxsakievirus B3 and, to a lesser extent, with vesicular stomatitis virus. The EGFP-NLS efflux was not due to the loss of NLS. Thus, some positive-strand and negative-strand RNA viruses trigger a rapid nonspecific relocation of nuclear proteins

Prothymosin α fragmentation in apoptosis

AbstractWe observed fragmentation of an essential proliferation-related human nuclear protein prothymosin α in the course of apoptosis induced by various stimuli. Prothymosin α cleavage occurred at the DDVD99 motif. In vitro, prothymosin α could be cleaved at D99 by caspase-3 and -7. Caspase hydrolysis disrupted the nuclear localization signal of prothymosin α and abrogated the ability of the truncated protein to accumulate inside the nucleus. Prothymosin α fragmentation may therefore be proposed to disable intranuclear proliferation-related function of prothymosin α in two ways: by cleaving off a short peptide containing important determinants, and by preventing active nuclear uptake of the truncated protein

Mitochondrial localization of SESN2

SESN2 is a member of the evolutionarily conserved sestrin protein family found in most of the Metazoa species. The SESN2 gene is transcriptionally activated by many stress factors, including metabolic derangements, reactive oxygen species (ROS), and DNA-damage. As a result, SESN2 controls ROS accumulation, metabolism, and cell viability. The best-known function of SESN2 is the inhibition of the mechanistic target of rapamycin complex 1 kinase (mTORC1) that plays a central role in support of cell growth and suppression of autophagy. SESN2 inhibits mTORC1 activity through interaction with the GATOR2 protein complex preventing an inhibitory effect of GATOR2 on the GATOR1 protein complex. GATOR1 stimulates GTPase activity of the RagA/B small GTPase, the component of RagA/B:RagC/D complex, preventing mTORC1 translocation to the lysosomes and its activation by the small GTPase Rheb. Despite the well-established role of SESN2 in mTORC1 inhibition, other SESN2 activities are not well-characterized. We recently showed that SESN2 could control mitochondrial function and cell death via mTORC1-independent mechanisms, and these activities might be explained by direct effects of SESN2 on mitochondria. In this work, we examined mitochondrial localization of SESN2 and demonstrated that SESN2 is located on mitochondria and can be directly involved in the regulation of mitochondrial functions

Impact of ER-stress on <i>ATF4</i>, <i>KRT16</i>, <i>FAM129A</i> and <i>HKDC1</i> genes expression in HaCaT cells.

<p>Fold changes of KRT16, FAM129A and HKDC1 transcripts in HaCaT cells treated with Brefeldin A or Tunicamycin for 15 h. The data was obtained by RT-qPCR and processed as described in Materials and Methods.</p

Implication of <i>KRT16</i>, <i>FAM129A</i> and <i>HKDC1</i> genes as ATF4 regulated components of the integrated stress response

<div><p>The ATF4 transcription factor is a key regulator of the adaptive integrated stress response (ISR) induced by various stresses and pathologies. Identification of novel transcription targets of ATF4 during ISR would contribute to the understanding of adaptive networks and help to identify novel therapeutic targets. We were previously searching for genes that display an inverse regulation mode by the transcription factors ATF4 and p53 in response to mitochondrial respiration chain complex III inhibition. Among the selected candidates the human genes for cytokeratine 16 (<i>KRT16</i>), anti-apoptotic protein Niban (<i>FAM129A</i>) and hexokinase <i>HKDC1</i> have been found highly responsive to ATF4 overexpression. Here we explored potential roles of the induction of <i>KRT16</i>, <i>FAM129A</i> and <i>HKDC1</i> genes in ISR. As verified by RT-qPCR, a dysfunction of mitochondrial respiration chain and ER stress resulted in a partially ATF4-dependent stimulation of <i>KRT16</i>, <i>FAM129A</i> and <i>HKDC1</i> expression in the HCT116 colon carcinoma cell line. ISRIB, a specific inhibitor of ISR, was able to downregulate the ER stress-induced levels of <i>KRT16</i>, <i>FAM129A</i> and <i>HKDC1</i> transcripts. An inhibition of ATF4 by RNAi attenuated the induction of <i>KRT16</i>, <i>FAM129A</i> and <i>HKDC1</i> mRNAs in response to ER stress or to a dysfunctional mitochondrial respiration. The similar induction of the three genes was observed in another tumor-derived cervical carcinoma cell line HeLa. However, in HaCaT and HEK293T cells that display transformed phenotypes, but do not originate from patient-derived tumors, the ER stress-inducing treatments resulted in an upregulation of <i>FAM129A</i> and <i>HKDC1</i>, but not <i>KRT16</i> transcripts, By a luciferase reporter approach we identified a highly active ATF4-responsive element within the upstream region of the <i>KRT16</i> gene. The results suggest a conditional regulation of <i>KRT16</i> gene by ATF4 that may be inhibited in normal cells, but engaged during cancer progression. Potential roles of <i>KRT16</i>, <i>FAM129A</i> and <i>HKDC1</i> genes upregulation in adaptive stress responses and pathologies are discussed.</p></div

Induction of KRT16, FAM129A and HKDC1 transcripts by the inhibition of mitochondrial respiratory chain complexes I or III.

<p>Fold changes of KRT16, FAM129A and HKDC1 transcripts in HCT116 cells treated with complex III inhibitor Myxothiazol for 5 h (a, b, c) or with complex I inhibitor Piericidin A for 13 h (d, e, f). The data was obtained by RT-qPCR and processed as described in Materials and Methods.</p

ATF4-specific RNAi leads to a suppression of <i>FAM129A</i>, <i>KRT16</i> and <i>HKDC1</i> induction in response to inhibition of the mitochondrial respiratory chain.

<p>Fold changes of ATF4 (a), FAM129A (b), KRT16 (c) and HKDC1 (d) transcripts in HCT116 cells expressing ATF4 shRNA (ATF4) or control scrambled shRNA (Scr) in response to treatment with Piericidin A for 8 h. The data was obtained by RT-qPCR and processed as described in Materials and Methods.</p

Induction of KRT16, FAM129A and HKDC1 transcripts by the UPR to ER stress.

<p>Fold changes of KRT16, FAM129A and HKDC1 transcripts in HCT116 cells treated with Brefeldin A or Tunicamycin for 14 h. The data was obtained by RT-qPCR and processed as described in Materials and Methods.</p

Ectopic overexpression of ATF4 and ER stress increase levels of Niban protein.

<p>Western analysis of ATF4 and Niban proteins in HCT116 cells overexpressing ATF4 from a plasmid construct (1, 2), or treated with Brifeldin A (BFA) for 16 hours (3, 4). Probing with actin antibodies was carried out as a loading control.</p