IBMPFD disease-causing mutant VCP/p97 proteins are targets of autophagic-lysosomal degradation

The ubiquitin-proteasome system (UPS) degrades soluble proteins and small aggregates, whereas macroautophagy (autophagy herein) eliminates larger protein aggregates, tangles and even whole organelles in a lysosome-dependent manner. VCP/p97 was implicated in both pathways. VCP/p97 mutations cause a rare multisystem disease called IBMPFD (Inclusion Body Myopathy with Paget's Disease and Frontotemporal Dementia). Here, we studied the role IBMPFD-related mutants of VCP/p97 in autophagy. In contrast with the wild-type VCP/p97 protein or R155C or R191Q mutants, the P137L mutant was aggregate-prone. We showed that, unlike commonly studied R155C or R191Q mutants, the P137L mutant protein stimulated both autophagosome and autolysosome formation. Moreover, P137L mutant protein itself was a substrate of autophagy. Starvation- and mTOR inhibition-induced autophagy led to the degradation of the P137L mutant protein, while preserving the wild-type and functional VCP/p97. Strikingly, similar to the P137L mutant, other IBMPFD-related VCP/p97 mutants, namely R93C and G157R mutants induced autophagosome and autolysosome formation; and G157R mutant formed aggregates that could be cleared by autophagy. Therefore, cellular phenotypes caused by P137L mutant expression were not isolated observations, and some other IBMPFD disease-related VCP/p97 mutations could lead to similar outcomes. Our results indicate that cellular mechanisms leading to IBMPFD disease may be various, and underline the importance of studying different disease-associated mutations in order to better understand human pathologies and tailor mutation-specific treatment strategies

Analyzing Hotel Customer Experience through Big Data Method: Samples from Hotels in Antalya and Istanbul

In order to ensure customer satisfaction in hotel business, it is required to have a good understanding of customers’ preferences, to foresee customer expectations, to find factors affecting customer experience. In this way, with increasing use of social media and user generated content over internet, there has been a tendency to use big data tools in service sector research studies. This study deals with the use of big data for analyzing 4 and 5 star hotel’s customer experiences in Antalya and İstanbul. Non-structural data, namely customer reviews, were analyzed and most used words in hotel comments have been taken. After filtering and elimination process, it is revealed that accommodation and staff are the factors that impact hotel customer experience

Analyzing Hotel Customer Experience through Big Data Method: Samples from Hotels in Antalya and Istanbul

In order to ensure customer satisfaction in the hotel business, it is required to have a good understanding of customers’ preferences to force customer expectations, to find factors affecting customer experience. In this way with the increasing use of social media and user generated content over the internet, there has been a tendency to use big data tools in service sector studies. This study deals with the use of big data for analyzing 4 and 5 star hotel’s customer experiences in Antalya and Istanbul. Nonstructural data, namely customer reviews were analyzed and most used words in hotel comments have been taken. After filtering and elimination process it is revealed that accommodation and staff are the factors that impact hotel customer experienc

Physiological and pathological significance of the molecular cross-talk between autophagy and apoptosis

y. Autophagy and apoptosis are two important molecular mechanisms that maintain cellular homeostasis under stress conditions. Autophagy represents an intracellular mechanism responsible for turnover of organelles and long-lived proteins through a lysosome-dependent degradation pathway. Cell death signals or sustained stress might trigger programmed cell death pathways, and among them, apoptosis is the most extensively studied one. Recent studies indicate the presence of a complex interplay between autophagy and apoptosis. Physiological relevance of autophagyapoptosis crosstalk was mainly shown in vitro. However, in vivo consequences possibly exist both during health and disease. In this review, we will summarize the current knowledge about molecular mechanisms connecting autophagy and apoptosis, and about the significance of this crosstalk for human health

Physiological and pathological significance of the molecular cross-talk between autophagy and apoptosis

Autophagy and apoptosis are two important molecular mechanisms that maintain cellular homeostasis under stress conditions. Autophagy represents an intracellular mechanism responsible for turnover of organelles and long-lived proteins through a lysosome-dependent degradation pathway. Cell death signals or sustained stress might trigger programmed cell death pathways, and among them, apoptosis is the most extensively studied one. Recent studies indicate the presence of a complex interplay between autophagy and apoptosis. Physiological relevance of autophagy-apoptosis crosstalk was mainly shown in vitro. However, in vivo consequences possibly exist both during health and disease. In this review, we will summarize the current knowledge about molecular mechanisms connecting autophagy and apoptosis, and about the significance of this crosstalk for human health

Autophagy as a molecular target for cancer treatment

Autophagy is an evolutionarily conserved catabolic mechanism, by which eukaryotic cells recycle or degrades internal constituents through membrane-trafficking pathway. Thus, autophagy provides the cells with a sustainable source of biomolecules and energy for the maintenance of homeostasis under stressful conditions such as tumor microenvironment. Recent findings revealed a close relationship between autophagy and malignant transformation. However, due to the complex dual role of autophagy in tumor survival or cell death, efforts to develop efficient treatment strategies targeting the autophagy/cancer relation have largely been unsuccessful. Here we review the two-faced role of autophagy in cancer as a tumor suppressor or as a pro-oncogenic mechanism. In this sense, we also review the shared regulatory pathways that play a role in autophagy and malignant transformation. Finally, anti-cancer therapeutic agents used as either inhibitors or inducers of autophagy have been discussed

Autophagy as a molecular target for cancer treatment

Autophagy is an evolutionarily conserved catabolic mechanism, by which eukaryotic cells recycle or degrades internal constituents through membrane-trafficking pathway. Thus, autophagy provides the cells with a sus-tainable source of biomolecules and energy for the maintenance of homeostasis under stressful conditions suchas tumor microenvironment. Recent findings revealed a close relationship between autophagy and malignant transformation. However, due to the complex dual role of autophagy in tumor survival or cell death, efforts to develop efficient treatment strategies targeting the autophagy/cancer relation have largely been unsuccessful.Here we review the two-faced role of autophagy in cancer as a tumor suppressor or as a pro-oncogenic mechanism. In this sense, we also review the shared regulatory pathways that play a role in autophagy and malignant transformation. Finally, anti-cancer therapeutic agents used as either inhibitors or inducers of autophagy have been discussed

Role of Autophagy in Breast Cancer Development and Progression: Opposite Sides of the Same Coin

The term "autophagy", which means "self (auto) - eating (phagy)", describes a catabolic process that is evolutionarially conserved among all eukaryotes. Although autophagy is mainly accepted as a cell survival mechanism, it also modulates the process known as "type II cell death". AKT/mTOR pathway is an upstream activator of autophagy and it is tightly regulated by the ATG (autophagy-related genes) signaling cascade. In addition, wide ranging cell signaling pathways and non-coding RNAs played essential roles in the control of autophagy. Autophagy is closely related to pathological processes such as neurodegenerative diseases and cancer as well as physiological conditions. After the Nobel Prize in Physiology or Medicine 2016 was awarded to Yoshinori Ohsumi "for his discoveries of mechanisms for autophagy", there was an explosion in the field of autophagy and molecular biologists started to pay considerable attention to the mechanistic insights related to autophagy in different diseases. Since autophagy behaved dualistically, both as a cell death and a cell survival mechanism, it opened new horizons for a deeper -analysis of cell type and context dependent behavior of autophagy in different types of cancers. There are numerous studies showing that the induction of autophagy mechanism will promote survival of cancer cells. Since autophagy is mainly a mechanism to keep the cells alive, it may protect breast cancer cells against stress conditions such as starvation and hypoxia. For these reasons, autophagy was noted to be instrumental in metastasis and drug resistance. In this chapter we have emphasized on role of role of autophagy in breast cancer. Additionally we have partitioned this chapter into exciting role of microRNAs in modulation of autophagy in breast cancer. We have also comprehensively summarized how TRAIL-mediated signaling and autophagy operated in breast cancer cells

VCP/p97 P137L mutant induced both autophagosome and autolysosome formation in HEK293T cells.

<p>(A) Cells expressing empty vector (CNT), wild type (WT) or mutant VCP/p97 (P137L) were co-transfected with RFP-GFP-LC3 fusion construct and dot formation was visualized under fluorescent microscope. Arrow heads indicate autophagosomes and arrows indicate autolysosomes. (B) The graph shows the number of autophagosomes and autolysosomes in WT and P137L mutant expressing cells. Data were shown as mean ± SD of independent experiments (n = 3). **, p<0.05; ***, p<0.01 (C) Cells were transfected with CNT, and MYC-tagged WT or P137L mutant in the absence or presence of lysosomal inhibitors E64d and Pepstatin A (PepA) and western blot analysis was performed. ACTIN was used as loading control. ImageJ software was used for the quantification of band intensities.</p

P137L mutant VCP/p97, but not the WT VCP/p97 or R155C and R191Q mutants, was a target of starvation-induced autophagy.

<p>(A) HEK293T cells were transfected with empty vector (CNT), MYC-tagged wild type (WT) or mutant VCP/p97 (P137L) and then starved for indicated time points. (B and C) HEK293T cells and (D) U2OS cells expressing empty vector (CNT), wild type (WT) or mutant (P137L) VCP/p97 were starved for 40 min in the absence or presence of lysosomal inhibitors E64d/Pepstatin A (E64D/PEPA) or chloroquine (CQ). (E) HEK293T cells and (F) U2OS cells expressing other VCP/p97 mutants (R155C) and (R191Q) were starved for 40 min in the absence or presence of lysosomal inhibitor chloroquine (CQ). (G) U2OS cells expressing wild type (WT) or mutant VCP/p97 (P137L) and (R155C) were starved for 40 min in the absence or presence of lysosomal inhibitor chloroquine (CQ). (H) U2OS cells were treated with carrier (DMSO) or Torin-1 (TRN) and western blot analysis was performed by using LC3 antibody. (I) Cells were transfected with wild type (WT) and mutant (P137L or R155C) VCP/p97 and treated with TRN in the presence or absence of CQ. ACTIN was used as loading control. Image J software was used for the quantification of band intensities. Western blot analysis was performed by using MYC and ACTIN antibodies. ACTIN was used as loading control. Image J software was used for the quantification of band intensities.</p