The Enigma of Tripeptidyl-Peptidase II: Dual Roles in Housekeeping and Stress

The tripeptidyl-peptidase II complex consists of repeated 138 kDa subunits, assembled into two twisted strands that form a high molecular weight complex (>5 MDa). TPPII, like many other cytosolic peptidases, plays a role in the ubiquitin-proteasome pathway downstream of the proteasome as well as in the production and destruction of MHC class I antigens and degradation of neuropeptides. Tripeptidyl-peptidase II activity is increased in cells with an increased demand for protein degradation, but whether degradation of cytosolic peptides is the only cell biological role for TPPII has remained unclear. Recent data indicated that TPPII translocates into the nucleus to control DNA damage responses in malignant cells, supporting that cytosolic “housekeeping peptidases” may have additional roles in cell biology, besides their contribution to protein turnover. Overall, TPPII has an emerging importance in several cancer-related fields, such as metabolism, cell death control, and control of genome integrity; roles that are not understood in detail. The present paper reviews the cell biology of TPPII and discusses distinct roles for TPPII in the nucleus and cytosol

Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes

The virulence of many Gram-positive bacteria depends on cholesterol-dependent cytolysins (CDCs), which form pores in eukaryotic cell plasma membranes. Pyolysin (PLO) from Trueperella pyogenes provided a unique opportunity to explore cellular responses to CDCs because it does not require thiol activation. Sublytic concentrations of PLO stimulated phosphorylation of MAPK ERK and p38 in primary stromal cells, and induced autophagy as determined by protein light-chain 3B cleavage. Although, inhibitors of MAPK or autophagy did not affect PLO-induced cytolysis. However, 10 μM 3-hydroxynaphthalene-2-carboxylic acid-(3,4-dihydroxybenzylidene)-hydrazide (Dynasore), a dynamin guanosine 5′-triphosphatase inhibitor, protected stromal cells against PLO-induced cytolysis as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (85 ± 17% versus 50 ± 9% cell viability), measuring extracellular ATP, and kinetic assays. This was a generalized mechanism because Dynasore also protected HeLa cells against streptolysin O. Furthermore, the effect was reversible, with stromal cell sensitivity to PLO restored within 30 minutes of Dynasore removal. The protective effect of Dynasore was not conferred by dynamin inhibition, induction of ERK phosphorylation, or Dynasore binding to PLO. Rather, Dynasore reduced cellular cholesterol and disrupted plasma membrane lipid rafts, similar to positive control methyl-β-cyclodextrin. Dynasore is a tractable tool to explore the complexity of cholesterol homeostasis in eukaryotic cells and to develop strategies to counter CDCs.—Preta, G., Lotti, V., Cronin, J. G., and Sheldon, I. M. Protective role of the dynamin inhibitor Dynasore against the cholesterol-dependent cytolysin of Trueperella pyogenes

MAML1 acts cooperatively with EGR1 to activate EGR1-regulated promoters: implications for nephrogenesis and the development of renal cancer.

Mastermind-like 1 (MAML1) is a transcriptional coregulator of activators in various signaling pathways, such as Notch, p53, myocyte enhancer factor 2C (MEF2C) and beta-catenin. In earlier studies, we demonstrated that MAML1 enhanced p300 acetyltransferase activity, which increased the acetylation of Notch by p300. In this study, we show that MAML1 strongly induced acetylation of the transcription factor early growth response-1 (EGR1) by p300, and increased EGR1 protein expression in embryonic kidney cells. EGR1 mRNA transcripts were also upregulated in the presence of MAML1. We show that MAML1 physically interacted with, and acted cooperatively with EGR1 to increase transcriptional activity of the EGR1 and p300 promoters, which both contain EGR1 binding sites. Bioinformatics assessment revealed a correlation between p300, EGR1 and MAML1 copy number and mRNA alterations in renal clear cell carcinoma and p300, EGR1 and MAML1 gene alterations were associated with increased overall survival. Our findings suggest MAML1 may be a component of the transcriptional networks which regulate EGR1 target genes during nephrogenesis and could also have implications for the development of renal cell carcinoma

Role of Lactone and Acid Forms in the Pleiotropic Effects of Statins

Statins are a class of drugs used worldwide to lower low-density lipoprotein cholesterol [...

Understanding the Dr. Jekyll and Mr. Hyde nature of apoptosis-inducing factor: future perspectives

Apoptosis-inducing factor (AIF) is emerging as a key protein in regulation of basic physiological processes including phagocytosis, mitophagy and regulation of the redox state. Recent evidences suggest that the enzymatic activity of AIF may play an active role in tumor progression controlling energy metabolism and redox balance. The present manuscript briefly describes the story of this protein from its initial discovery as caspase-independent apoptotic protein, throughout its role in oxidative phosphorylation and lately involvement in tumor progression. Understanding the dualistic nature of AIF is a critical starting point to clarify its contribution in tumor metabolic balance and to develop new AIF-specific therapeutic strategies

Development of new genome editing tools for the treatment of hyperlipidemia /

Hyperlipidemia is a medical condition characterized by high levels of lipids in the blood. It is often associated with an increased risk of cardiovascular diseases such as heart attacks and strokes. Traditional treatment approaches for hyperlipidemia involve lifestyle modifications, dietary changes, and the use of medications like statins. Recent advancements in genome editing technologies, including CRISPR-Cas9, have opened up new possibilities for the treatment of this condition. This review provides a general overview of the main target genes involved in lipid metabolism and highlights the progress made during recent years towards the development of new treatments for dyslipidemia

AIF and Scythe (Bat3) Regulate Phosphatidylserine Exposure and Macrophage Clearance of Cells Undergoing Fas (APO-1)-Mediated Apoptosis

<div><p>Phosphatidylserine (PS) exposure on the cell surface has been considered a characteristic feature of apoptosis and serves as a molecular cue for engulfment of dying cells by phagocytes. However, the mechanism of PS exposure is still not fully elucidated. Here we show that the cytosolic release from mitochondria of apoptosis-inducing factor (AIF) is required for PS exposure during death receptor-induced apoptosis and for efficient clearance of cell corpses by primary human macrophages. Fas-triggered PS exposure was significantly reduced upon siRNA-mediated silencing of AIF expression and by inhibition of the cytosolic translocation of AIF. In addition, AIF localizes to the plasma membrane upon Fas ligation and promotes activation of phospholipid scrambling activity. Finally, cytosolic stabilization of AIF through interaction with Scythe is shown to be involved in apoptotic PS exposure. Taken together, our results suggest an essential role for AIF and its binding partner Scythe in the pathway leading to apoptotic corpse clearance.</p> </div

Cytosolic interaction between AIF and Scythe is required for macrophage clearance of Fas-triggered Jurkat cells.

<p>A. Immunocytochemistry analysis of Scythe and AIF in Jurkat cells un-treated and treated for 3 h with Fas mAb (250 ng/ml) in the presence or absence of the pan-caspase inhibitor, Z-VAD-FMK (20 µM). Nuclei are stained in blue (DAPI). B. Jurkat cells treated like in A were lysed and AIF was then immunoprecipitated. Western blot is 10% of the input and is derived from both control and Fas mAb treated cells. Negative control (NC) consists of glass beads incubated with normal rabbit serum. C. Jurkat cells were transfected with siRNA for AIF, Scythe and non-targeting siRNA (NC). After 48 h cells were collected, lysed in RIPA buffer and Western blotting analysis was performed to detect the levels of AIF. GAPDH was used as loading control. Quantification was made using the program Image J and the values of optical density (OD) were normalized to the levels of GAPDH. D. Jurkat cells, treated with siRNA for AIF, Scythe or NC were induced to undergo apoptosis by Fas mAb (50 ng/ml) or staurosporine (0.5 µM). After 3 h cells were co-cultivated with human monocyte-derived macrophages (HMDM) for 1 h. Examples of phagocytosis of TAMRA-labelled Jurkat cells (red) are depicted. Nuclei of macrophages and engulfed target cells are stained with DAPI (blue). E. Phagocytosis is reported as the percentages of macrophages positive for uptake of target cells. Data are presented as the mean ± S.D of six values derived from two different experiments performed in triplicate. *P<0.05; **P<0.01 (one-way ANOVA <i>Dunnett’s</i> test). Caspase-3 activation in WT versus AIF or Scythe siRNA-transfected cells determined by immunoblotting is shown in the panel below. GAPDH is used as loading control.</p