Protein synthesis inhibitors such as cycloheximide (CHX) are known to suppress protein degradation including autophagy. The fact that CHX inhibits autophagy has been generally interpreted to indicate that newly synthesized protein is indispensable for autophagy. However, CHX is also known to increase the intracellular level of amino acids and activate mTORC1 activity, a master negative regulator of autophagy. Accordingly, CHX can affect autophagic activity through inhibition of de novo protein synthesis and/or modulation of mTORC1 signaling. In this study, we investigated the effects of CHX on autophagy using specific autophagy markers. We found that CHX inhibited starvation-induced autophagy but not Torin1-induced autophagy. CHX also suppressed starvation-induced puncta formation of GFP-ULK1, an early-step marker of the autophagic process which is regulated by mTORC1. CHX activated mTORC1 even under autophagy-inducible starvation conditions. Finally, the inhibitory effect of CHX on starvation-induced autophagy was cancelled by the mTOR inhibitor Torin1. These results suggest that CHX inhibits starvation-induced autophagy through mTORC1 activation and also that autophagy does not require new protein synthesis at least in the acute phase of starvation.

Phosphate-starved plants reduce phosphatidylcholine content presumably to provide an internal phosphate source while replacing membrane phospholipids by galactolipids, a process termed membrane lipid remodeling. However, whether the metabolic fate of released phosphocholine is a phosphate source remains elusive because primary phosphocholine phosphatases in vivo are unknown in seed plants. Here, we show that PECP1 and PS2 are the primary phosphocholine phosphatases in Arabidopsis and function redundantly under phosphate starvation. Under phosphate starvation, the double knockout mutant of PECP1 and PS2 showed reduced content of choline but no severe growth phenotype, which suggests that phosphocholine dephosphorylation is not likely a major source of internal phosphate reserve. We identified primary phosphocholine phosphatases, demonstrated their involvement under phosphate starvation, and updated the metabolic map of membrane lipid remodeling.

Upon nutrient starvation, eukaryotic cells exploit autophagy to reconstruct cellular components. Although autophagy is induced by depletion of various nutrients such as nitrogen, carbon, amino acids, and sulfur in yeast, it was previously ambiguous whether phosphate depletion could trigger the induction of autophagy. Here, we showed that phosphate depletion induced autophagy in Saccharomyces cerevisiae, albeit to a lesser extent than nitrogen starvation. It is known that rapid inactivation of the target of rapamycin complex 1 (TORC1) signaling pathway contributes to Atg13 dephosphorylation, which is one of the cues for autophagy induction. We found that phosphate starvation caused Atg13 dephosphorylation with slower kinetics than nitrogen starvation, suggesting that poor autophagic activity during phosphate starvation was associated with slower inactivation of the TORC1 pathway. Phosphate starvation-induced autophagy requires Atg11, an adaptor protein for selective autophagy, but not its cargo recognition domain. These results suggested that Atg11 plays important roles in low-level nonselective autophagy.

Presence of unperfused regions containing cells under hypoxia and nutrient starvation; contributes to radioresistance in solid human tumors. We have previously reported that cultured cells; under nutrient starvation show resistance to ionizing radiation compare with cells under normal; condition, and that nutrient starvation increases ATM activity, which causes cellular resistance to; ionizing radiation (Murata et al., BBRC2018). For further investigation of molecular mechanisms; underlying radioresistance of cells under nutrient starvation, effects of nutrient starvation on activity; of DNA-PKcs have been investigated because both DNA-PKcs and ATM belong to the PIKK family; and are required for DNA DSBs repair. In addition to DNA-PKcs, effects of nutrient starvation on; activities of FoxO3a and its regulators Akt, MST1 and AMPK have been investigated because FoxO3a; mediates cellular responses to stress and is activated under nutrient starvation.

Autophagy is a conserved mechanism for controlling the degradation of misfolded proteins and damaged organelles in eukaryotes and can be induced by nutrient withdrawal, including serum starvation. Although differential acetylation of autophagy-related proteins has been reported to be involved in autophagic flux, the regulation of acetylated microtubule-associated protein 1 light chain 3 (LC3) is incompletely understood. In this study, we found that the acetylation levels of phosphotidylethanolamine (PE)-conjugated LC3B (LC3B-II), which is a critical component of double-membrane autophagosome, were profoundly decreased in HeLa cells upon autophagy induction by serum starvation. Pretreatment with lysosomal inhibitor chloroquine did not attenuate such deacetylation. Under normal culture medium, we observed increased levels of acetylated LC3B-II in cells treated with tubacin, a specific inhibitor of histone deacetylase 6 (HDAC6). However, tubacin only partially suppressed serum-starvation-induced LC3B-II deacetylation, suggesting that HDAC6 is not the only deacetylase acting on LC3B-II during serum-starvation-induced autophagy. Interestingly, tubacin-induced increase in LC3B-II acetylation was associated with p62/SQSTM1 accumulation upon serum starvation. HDAC6 knockdown did not influence autophagosome formation but resulted in impaired degradation of p62/SQSTM1 during serum starvation. Collectively, our data indicated that LC3B-II deacetylation, which was partly mediated by HDAC6, is involved in autophagic degradation during serum starvation.

GOLPH3, an oncoprotein, plays crucial roles in tumor etiology. Compelling evidences have demonstrated that GOLPH3 contributes to regulate tumor cell growth, migration and invasion under normal nutrient condition. However, the oncogenic activity of GOLPH3 under serum starvation remains largely unknown. In this study, we reported that GOLPH3 depletion led to marked reduction in adhesion of glioma U251 cells, particularly under serum deprivation. We found that silencing of GOLPH3 expression reduced the protein amount of ITGB1 only in serum-free medium. Further insights into the mechanism between GOLPH3 and ITGB1, we applied proteasome or lysosome inhibitor to block the degradation of ITGB1, and identified GOLPH3 silencing can prompt ITGB1 lysosomal degradation under serum starvation. Finally, we found the reductions in glioma cell adhesion and ITGB1 protein amount could be rescued by ITGB1 overexpression. Taken together, these results show that GOLPH3 contributes to the adhesion of glioma cells by regulating the lysosomal degradation of ITGB1 under serum starvation.

The deprivation of myocardial nutrition causes cardiomyocyte death and disturbance of energy metabolism. IKKε plays an important regulatory role in many biological events such as inflammation, redox reaction, cell death, etc. However, the more in-depth mechanism by which IKKε contributes to cardiomyocytes death in nutrition deprivation remains poorly understood. IKKε expression was knocked down by siRNA in H9c2 cells, and cells were cultured under starvation conditions to simulate ischemic conditions. Starvation triggered greater NLRP3 activation, accompanied by more IL-1β, IL-18 and caspase-1 release in the siIKKε H9c2 cells compared with the control H9c2 cells. Western blot and immunofluorescence showed that the IKKε konckdown promoted NLRP3 expressions and ROS release under starvation conditions. Furthermore, electron micrography and JC-1 analysis revealed that IKKε konckdown resulted in aggravated mitochondrial damage and more mitochondrial ROS (mtROS) released in vitro. Notably, Western blot analysis showed that IKKε deficiency activated the TBK1 and IRF3 signaling pathways to promote pyroptosis in vitro. Collectively, our results indicate that IKKε protects against cardiomyocyte injury by reducing mitochondrial damage and NLRP3 expression following nutrition deprivation via regulation of the TBK1/IRF3 signaling pathway. This study further revealed the mechanism of IKKε in inflammation and myocardial nutrition deprivation.

The proteasome is one of the main catalytic machineries of eukaryotic cells responsible for protein degradation, and is known to be regulated during several cellular stress conditions. Recent studies suggest that the activity of the proteasome is modulated following mTOR inhibition. However, it is not clear how this process affects the proteome. In the present study, we investigated the role of the proteasome in the modulation of the proteome of HeLa cells following amino acid starvation, a stress known to inhibit mTOR activity. We used label-free quantitative proteomics to identify proteins regulated by the proteasome in starved cells. We found that nearly 50% of the proteins the level of which decreased significantly during starvation stress, could be rescued by addition of the proteasome inhibitor MG132. This suggests a key role for the proteasome in reshaping the proteome under starvation. Importantly, the expression of several of these proteins is known to be dependent on the transcription factor E2F1. Further investigation of E2F1 level showed that this transcription factor along with several other proteins involved in its pathway are regulated by the proteasome upon amino acids starvation.

LRRK2 is one of the most important genetic contributors to Parkinson's disease (PD). Point mutations in this gene cause an autosomal dominant form of PD, but to date no cellular phenotype has been consistently linked with mutations in each of the functional domains (ROC, COR and Kinase) of the protein product of this gene. In this study, primary fibroblasts from individuals carrying pathogenic mutations in the three central domains of LRRK2 were assessed for alterations in the autophagy/lysosomal pathway using a combination of biochemical and cellular approaches. Mutations in all three domains resulted in alterations in markers for autophagy/lysosomal function compared to wild type cells. These data highlight the autophagy and lysosomal pathways as read outs for pathogenic LRRK2 function and as a marker for disease, and provide insight into the mechanisms linking LRRK2 function and mutations.

The presence of unperfused regions containing cells under hypoxic and nutrient starvation conditions contributes to radioresistance in solid human tumors. It is well known that the hypoxia causes cellular radioresistance. However, the effects of nutrient starvation conditions on cellular radiosensitivity remain unclear.

Bacteria commonly exhibit social activities through acyl-homoserine lactones (AHLs)-based quorum sensing (QS) systems to form their unique social network. The sigma factor RpoS is an important regulator that controls QS system in different bacteria. However, the upstream of RpoS involving regulation on QS system remains unclear. In Escherichia coli RpoS is regulated by stringent starvation protein A (SspA), which is dependent of histone-like nucleoid structuring protein (H-NS). To date, the connection between SspA and QS system is essentially unknown. Here, we characterized a typical LuxI/LuxR-type QS system in marine bacterium Pseudoalteromonas sp. T1lg65 which can produce four types of AHLs. The luxI encoding AHLs synthase and luxR encoding AHLs-responsive receptor are co-transcribed, providing advantages in rapidly amplifying QS signaling. Notably, SspA positively regulated luxI/luxR transcription by activating RpoS expression, which is mediated by H-NS. Interestingly, LuxR in turn positively regulated SspA expression. Therefore, SspA and QS system constitute a mutual positive regulation loop in T1lg65. In view of the crucial roles of SspA and QS system in environmental adaption, we believe that the improvement of bacterial tolerance to marine environments could be related to rapidly tuning SspA-involved QS programming.

The hepatitis B virus X protein (HBx) has been implicated in the development of hepatocellular carcinoma (HCC) associated with chronic infection. As a multifunctional protein, HBx regulates numerous cellular pathways, including autophagy. Although autophagy has been shown to participate in viral DNA replication and envelopment, it remains unclear whether HBx-activated autophagy affects host cell death, which is relevant to both viral pathogenicity and the development of HCC. Here, we showed that enforced expression of HBx can inhibit starvation-induced cell death in hepatic (L02 and Chang) or hepatoma (HepG2 and BEL-7404) cell lines. Starvation-induced cell death was greatly increased in HBX-expressing cell lines treated either with the autophagy inhibitor 3-methyladenine (3-MA) or with an siRNA directed against an autophagy gene, beclin 1. In contrast, treatment of cells with the apoptosis inhibitor Z-Vad-fmk significantly reduced cell death. Our results demonstrate that HBx-mediated cell survival during starvation is dependent on autophagy. We then further investigated the mechanisms of cell death inhibition by HBx. We found that HBx inhibited the activation of caspase-3, an execution caspase, blocked the release of mitochondrial apoptogenic factors, such as cytochrome c and apoptosis-inducing factor (AIF), and inhibited the activation of caspase-9 during starvation. These results demonstrate that HBx reduces cell death through inhibition of mitochondrial apoptotic pathways. Moreover, increased cell viability was also observed in HepG2.2.15 cells that replicate HBV and in cells transfected with HBV genomic DNA. Our findings demonstrate that HBx promotes cell survival during nutrient deprivation through inhibition of apoptosis and activation of autophagy. This highlights an important potential role of autophagy in HBV-infected hepatocytes growing under nutrient-deficient conditions.

We demonstrated that exogenous pyruvate promotes survival under glucose depletion in aerobic mutant p53 (R175H) human melanoma cells. Others subsequently indicated that mutant p53 tumor cells undergo p53 degradation and cell death under aerobic glucose-free conditions. Since glucose starvation occurs in hypoxic gradients of poorly vascularized tumors, we investigated the role of p53 siRNA under hypoxia in wt p53 C8161 melanoma using glucose starvation or 5mM physiological glucose. p53 Silencing decreased survival of glucose-starved C8161 melanoma with pyruvate supplementation under hypoxia (≤1% oxygen), but increased resistance to glycolytic inhibitors oxamate and 2-deoxyglucose in 5mM glucose, preferentially under normoxia. Aiming to counteract hypoxic tumor cell survival irrespective of p53 status, genetically-matched human C8161 melanoma harboring wt p53 or mutant p53 (R175H) were used combining true hypoxia (≤1% oxygen) and hypoxia mimetic CoCl2. No significant decrease in metabolic activity was evidenced in C8161 melanoma irrespective of p53 status in 2.5mM glucose after 48h of physical hypoxia. However, combining the latter with 100μM CoCl2 was preferentially toxic for mutant p53 C8161 melanoma, and was enhanced by catalase in wt p53 C8161 cells. Downregulation of MnSOD and LDHA accompanied the toxicity induced by hypoxia and CoCl2 in 5mM glucose, and these changes were enhanced by oxamate or 2-deoxyglucose. Our results show for the first time that survival of malignant cells in a hypoxic microenvironment can be counteracted by hypoxia mimetic co-treatment in a p53 dependent manner.

Presence of unperfused regions containing cells under hypoxia and nutrient starvation contributes to radioresistance in solid human tumors. It is well known that hypoxia causes cellular radioresistance, but little is known about the effects of nutrient starvation on radiosensitivity. We have reported that nutrient starvation induced decrease of mTORC1 activity and decrease of radiosensitivity in an SV40-transformed human fibroblast cell line, LM217, and that nutrient starvation induced increase of mTORC1 activity and increase of radiosensitivity in human liver cancer cell lines, HepG2 and HuH6 (Murata et al., BBRC 2015). Knockdown of mTOR using small interfering RNA (siRNA) for mTOR suppressed radiosensitivity under nutrient starvation alone in HepG2 cells, which suggests that mTORC1 pathway regulates radiosensitivity under nutrient starvation alone. In the present study, effects of hypoxia and nutrient starvation on radiosensitivity were investigated using the same cell lines.

The yeast RSC, an ATP-dependent chromatin-remodeling complex, is essential for mitotic and meiotic growth. There are two distinct isoforms of this complex defined by the presence of either Rsc1 or Rsc2; however, the functional differences between these complexes are unclear. Here we show that the RSC complex containing Rsc1, but not Rsc2, functions in autophagy induction. Rsc1 was required not only for full expression of ATG8 mRNA but also for maintenance of Atg8 protein stability. Interestingly, decreased autophagic activity and Atg8 protein stability in rsc1Δ cells, but not the defect in ATG8 mRNA expression, were partially suppressed by deletion of TOR1. In addition, we found that rsc1Δ impaired the binding between the Rho GTPase Rho1 and the TORC1-specific component Kog1, which is required for down-regulation of TORC1 activity. These results suggest that the Rsc1-containing RSC complex plays dual roles in the proper induction of autophagy: 1) the transcriptional activation of autophagy-related genes independent of the TORC1 pathway and 2) the inactivation of TORC1, possibly through enhancement of Rho1-Kog1 binding.

Macroautophagy is involved in the bulk degradation of long-lived cytosolic proteins and subcellular organelles, which is important for the survival of cells during starvation. To identify potential players of the autophagy process, we subjected HCT116 cells cultured in complete medium and in Earle's balanced salt solution to proteomics analysis. In approximately 1500 protein spots detected, we characterized 52 unique proteins, whose expression levels were significantly changed following starvation. Notably, we found that Annexin A1 was significantly upregulated following starvation at both mRNA and protein levels. Inhibition of Annexin A1 expression with specific siRNA did not alter starvation-induced autophagy as measured by the level of lipidated LC3, but significantly reversed autophagy degradation as measured by the level of p62/SQSTM 1. Thus Annexin A1 seemed to be positively upregulated during starvation to promote autophagic degradation. Overall, the data presented in this study established a expression profile of the proteome in starved cells, which allowed the identification of proteins with potential significance in starvation-induced autophagy.

CDC-48 is a AAA (ATPases associated with diverse cellular activities) chaperone and participates in a wide range of cellular activities. Its functional diversity is determined by differential binding of a variety of cofactors. In this study, we analyzed the physiological role of a CDC-48 cofactor UBXN-6 in Caenorhabditis elegans. The amount of UBXN-6 was markedly increased upon starvation, but not with the treatment of tunicamycin and rapamycin. The induction upon starvation is a unique characteristic for UBXN-6 among C-terminal cofactors of CDC-48. During starvation, lysosomal activity is triggered for rapid clearance of cellular materials. We observed the lysosomal activity by monitoring GLO-1::GFP, a marker for lysosome-related organelles. We found that more puncta of GLO-1::GFP were observed in the ubxn-6 deletion mutant after 12 h starvation compared with the wild-type strain. Taken together, we propose that UBXN-6 is involved in clearance of cellular materials upon starvation in C. elegans.

Insulin-like peptide 5 (INSL5) is a member of the insulin superfamily, and is a potent agonist for RXFP4. We have shown that INSL5 is expressed in enteroendocrine cells (EECs) along the colorectum with a gradient increase toward the rectum. RXFP4 is ubiquitously expressed along the digestive tract. INSL5-positive EECs have little immunoreactivity to chromogranin A (CgA) and might be a unique marker of colorectal EECs. CgA-positive EECs were distributed normally along the colorectum in INSL5 null mice, suggesting that INSL5 is not required for the development of CgA-positive EECs. Exogenous INSL5 did not affect the proliferation of human colon cancer cell lines, and chemically-induced colitis in INSL5 null mice did not show any significant changes in inflammation or mucosal healing compared to wild-type mice. In contrast, all of the rectal neuroendocrine tumors examined co-expressed INSL5 and RXFP4. INSL5 may be a unique marker of colorectal EECs, and INSL5-RXFP4 signaling might play a role in an autocrine/paracrine fashion in the colorectal epithelium and rectal neuroendocrine tumors.

Groucho related genes encode transcriptional repressor proteins critical for normal developmental processes. The bone morphogenetic proteins belong to the transforming growth factor-β (TGF-β) superfamily and play important signaling roles in development and disease. However, the regulation of BMP signaling, especially within cells, is largely unknown. In this report, we show that expression of the Groucho related gene Grg4 robustly activates the expression of a BMP reporter gene, as well as enhancing and sustaining the upregulation of the endogenous Id1 gene induced by BMP7. BMP7 administration did not affect the endogenous level of Grg4 nor did it enhance the phosphorylation of receptor activated Smad proteins. Rather, Grg4 expression reduced the levels of the endogenous inhibitory Smad7, thus increasing the transcriptional responses mediated by BMP responsive sequences. The data point to a novel mechanisms for attenuating BMP signaling through altering the ratio of activating versus inhibitory Smad proteins.

Prostaglandins (PGs) play important roles in diverse physiological processes in the central nervous system. PGD2 is the most abundant PG in the brain and acts through specific receptors, DP1 and CRTH2. We investigated the effects of PGD2 on the morphology of the hypothalamic cell line mHypoE-N37 (N37). In N37 cells, serum starvation induced neurite outgrowth and PGD2 elicited neurite retraction, although we failed to detect transcripts for DP1 and CRTH2. Such an effect of PGD2 was efficiently mimicked by its metabolite, 15-deoxy-Δ(12,14)-prostaglandin J2. N-acetyl cysteine completely abolished the effect of PGD2, and reactive oxygen species (ROS) were considered to be important. Notably, neurite outgrowth was restored by PGD2 removal. These results suggest that PGD2 induces reversible neurite retraction in a ROS-mediated mechanism that does not involve any known receptor.