The RNA exosome processes a wide variety of RNA and mediates RNA maturation, quality control and decay. In marked contrast to its high processivity in vivo, the purified exosome exhibits only weak activity on RNA substrates in vitro. Its activity is regulated by several auxiliary proteins, and protein complexes. In budding yeast, the activity of exosome is enhanced by the polyadenylation complex referred to as TRAMP. TRAMP oligoadenylates precursors and aberrant forms of RNAs to promote their trimming or complete degradation by exosomes. This chapter provides protocols for the purification of TRAMP and exosome complexes from yeast and the in vitro evaluation of exosome activation by the TRAMP complex. The protocols can be used for different purposes, such as the assessment of the role of individual subunits, protein domains or particular mutations in TRAMP-exosome RNA processing in vitro.

• Klíčová slova
• Air1, Air2, Degradation assay, Mtr4, Noncanonical poly(A) polymerase, Noncoding RNAs, Polyadenylation assay, RNA exosome, RNA quality control, Rrp6, TAP purification, TRAMP4, Trf4,
• MeSH
• buněčné jádro metabolismus   MeSH
• exozom metabolismus   MeSH
• exozómy metabolismus   MeSH
• polyadenylace fyziologie   MeSH
• RNA metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• serinové endopeptidasy metabolismus   MeSH
• stabilita RNA fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• exozom MeSH
• RNA MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• serinové endopeptidasy MeSH
• tunicate retinoic acid-inducible modular protease MeSH Prohlížeč

RNA metabolism is altered following DNA damage, but the underlying mechanisms are not well understood. Through a 14-3-3 interaction screen for DNA damage-induced protein interactions in human cells, we identified protein complexes connected to RNA biology. These include the nuclear exosome targeting (NEXT) complex that regulates turnover of noncoding RNAs termed promoter upstream transcripts (PROMPTs). We show that the NEXT subunit RBM7 is phosphorylated upon DNA damage by the MAPKAPK2 kinase and establish that this mediates 14-3-3 binding and decreases PROMPT binding. These findings and our observation that cells lacking RBM7 display DNA damage hypersensitivity link PROMPT turnover to the DNA damage response.

• Klíčová slova
• 14-3-3, DNA damage response, MAPKAPK2, UV, nuclear exosome,
• MeSH
• exozom metabolismus   MeSH
• fosforylace MeSH
• intracelulární signální peptidy a proteiny metabolismus   MeSH
• lidé MeSH
• MAP kinasový signální systém MeSH
• nekódující RNA metabolismus   MeSH
• poškození DNA fyziologie   MeSH
• protein-serin-threoninkinasy metabolismus   MeSH
• proteiny 14-3-3 metabolismus   MeSH
• ultrafialové záření MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• exozom MeSH
• intracelulární signální peptidy a proteiny MeSH
• MAP-kinase-activated kinase 2 MeSH Prohlížeč
• nekódující RNA MeSH
• protein-serin-threoninkinasy MeSH
• proteiny 14-3-3 MeSH

Cancer-associated fibroblasts (CAFs) are an essential component of the tumour microenvironment. They represent a heterogeneous group of cells that are under the control of cancer cells and can reversely influence the cancer cell population. They affect the cancer cell differentiation status, and the migration and formation of metastases. This is achieved through the production of the extracellular matrix and numerous bioactive factors. IL-6 seems to play the central role in the communication of noncancerous and cancer cells in the tumour. This review outlines the role of exosomes in cancer cells and cancer-associated fibroblasts. Available data on the exosomal cargo, which can significantly intensify interactions in the tumour, are summarised. The role of exosomes as mediators of the dialogue between cancer cells and cancer-associated fibroblasts is discussed together with their therapeutic relevance. The functional unity of the paracrine- and exosome-mediated communication of cancer cells with the tumour microenvironment represented by CAFs is worthy of attention.

• Klíčová slova
• IL-6, cancer ecosystem, cancer microenvironment, cancer-associated fibroblast, exosome,
• MeSH
• exozómy metabolismus   MeSH
• fibroblasty asociované s nádorem metabolismus   MeSH
• interleukin-6 metabolismus   MeSH
• lidé MeSH
• nádorové mikroprostředí MeSH
• nádory metabolismus   MeSH
• parakrinní signalizace MeSH
• pohyb buněk MeSH
• proliferace buněk MeSH
• regulace genové exprese u nádorů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• IL6 protein, human MeSH Prohlížeč
• interleukin-6 MeSH

BACKGROUND: Exosomes arise from viable cancer cells and may reflect a different biology than circulating cell-free DNA (cfDNA) shed from dying tissues. We compare exosome-derived DNA (exoDNA) to cfDNA in liquid biopsies of patients with pancreatic ductal adenocarcinoma (PDAC). PATIENTS AND METHODS: Patient samples were obtained between 2003 and 2010, with clinically annotated follow up to 2015. Droplet digital PCR was performed on exoDNA and cfDNA for sensitive detection of KRAS mutants at codons 12/13. A cumulative series of 263 individuals were studied, including a discovery cohort of 142 individuals: 68 PDAC patients of all stages; 20 PDAC patients initially staged with localized disease, with blood drawn after resection for curative intent; and 54 age-matched healthy controls. A validation cohort of 121 individuals (39 cancer patients and 82 healthy controls) was studied to validate KRAS detection rates in early-stage PDAC patients. Primary outcome was circulating KRAS status as detected by droplet digital PCR. Secondary outcomes were disease-free and overall survival. RESULTS: KRAS mutations in exoDNA, were identified in 7.4%, 66.7%, 80%, and 85% of age-matched controls, localized, locally advanced, and metastatic PDAC patients, respectively. Comparatively, mutant KRAS cfDNA was detected in 14.8%, 45.5%, 30.8%, and 57.9% of these individuals. Higher exoKRAS MAFs were associated with decreased disease-free survival in patients with localized disease. In the validation cohort, mutant KRAS exoDNA was detected in 43.6% of early-stage PDAC patients and 20% of healthy controls. CONCLUSIONS: Exosomes are a distinct source of tumor DNA that may be complementary to other liquid biopsy DNA sources. A higher percentage of patients with localized PDAC exhibited detectable KRAS mutations in exoDNA than previously reported for cfDNA. A substantial minority of healthy samples demonstrated mutant KRAS in circulation, dictating careful consideration and application of liquid biopsy findings, which may limit its utility as a broad cancer-screening method.

• Klíčová slova
• KRAS, circulating tumor DNA, exosome, liquid biopsy, pancreatic cancer,
• MeSH
• časná detekce nádoru metody   MeSH
• DNA nádorová krev   genetika   MeSH
• dospělí MeSH
• duktální karcinom pankreatu krev   genetika   patologie   MeSH
• exozómy genetika   MeSH
• Kaplanův-Meierův odhad MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidé středního věku MeSH
• lidé MeSH
• mutace MeSH
• nádorové biomarkery krev   genetika   MeSH
• nádory slinivky břišní krev   genetika   patologie   MeSH
• přežití po terapii bez příznaků nemoci MeSH
• proporcionální rizikové modely MeSH
• protoonkogenní proteiny p21(ras) genetika   MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA nádorová MeSH
• KRAS protein, human MeSH Prohlížeč
• nádorové biomarkery MeSH
• protoonkogenní proteiny p21(ras) MeSH

Recent molecular and cellular studies have highlighted a potentially important role for tick exosomes in parasite transmission. Here we summarize evolving hypotheses about the largely unknown cellular events that may take place at the tick-host-pathogen interface, focusing on a potential role for arthropod exosomes in this tripartite interaction.

• Klíčová slova
• disease vectors, exosomes, pathogen transmission, tick–host interaction, vector-borne diseases, viruses,
• MeSH
• členovci - vektory parazitologie   MeSH
• exozómy fyziologie   MeSH
• interakce hostitele a parazita * MeSH
• klíšťata parazitologie   MeSH
• lidé MeSH
• parazitární nemoci přenos   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In order to control and/or enhance the specificity and activity of nuclear surveillance and degradation, exosomes cooperate with the polyadenylation complex called TRAMP. Two forms of TRAMP operate in budding yeast, TRAMP4 and TRAMP5. They oligoadenylate defective or precursor forms of RNAs and promote trimming or complete degradation by exosomes. TRAMPs target a wide variety of nuclear transcripts. The known substrates include the noncoding RNAs originating from pervasive transcription from diverse parts of the yeast genome. Although TRAMP and exosomes can be triggered to a subset of their targets via the RNA-binding complex Nrd1, it is still not completely understood how TRAMP recognizes other aberrant RNAs. The existence of TRAMP-like complexes in other organisms indicates the importance of nuclear surveillance for general cell biology. In this chapter, we review the current understanding of TRAMP function and substrate repertoire. We discuss the advances in TRAMP biochemistry with respect to its catalytic activities and RNA recognition. Finally, we speculate about the possible mechanisms by which TRAMP activates exosomes.

• Klíčová slova
• Air1, Mtr4, Noncanonical polyadenylation, Noncoding RNAs, RNA degradation, RNA surveillance, TRAMP, Trf4, Zinc knuckle proteins,
• Publikační typ
• časopisecké články MeSH

Exosomes are small vesicles that are secreted by cells and act as mediators of cell to cell communication. Because of their potential therapeutic significance, important efforts are being made towards characterizing exosomal contents. However, little is known about the mechanisms that govern exosome biogenesis. We have recently shown that the exosomal protein syntenin supports exosome production. Here we identify the small GTPase ADP ribosylation factor 6 (ARF6) and its effector phospholipase D2 (PLD2) as regulators of syntenin exosomes. ARF6 and PLD2 affect exosomes by controlling the budding of intraluminal vesicles (ILVs) into multivesicular bodies (MVBs). ARF6 also controls epidermal growth factor receptor degradation, suggesting a role in degradative MVBs. Yet ARF6 does not affect HIV-1 budding, excluding general effects on Endosomal Sorting Complexes Required for Transport. Our study highlights a novel pathway controlling ILV budding and exosome biogenesis and identifies an unexpected role for ARF6 in late endosomal trafficking.

• MeSH
• ADP-ribosylační faktor 6 MeSH
• ADP-ribosylační faktory genetika   metabolismus   MeSH
• buněčné linie MeSH
• endozomální třídící komplexy pro transport genetika   metabolismus   MeSH
• erbB receptory metabolismus   MeSH
• exozómy enzymologie   genetika   metabolismus   MeSH
• fosfolipasa D genetika   metabolismus   MeSH
• HIV infekce genetika   metabolismus   virologie   MeSH
• HIV-1 fyziologie   MeSH
• lidé MeSH
• multivezikulární tělíska enzymologie   genetika   metabolismus   MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• proteiny vázající vápník genetika   metabolismus   MeSH
• synteniny genetika   metabolismus   MeSH
• transport proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ADP-ribosylační faktor 6 MeSH
• ADP-ribosylační faktory MeSH
• ARF6 protein, human MeSH Prohlížeč
• endozomální třídící komplexy pro transport MeSH
• erbB receptory MeSH
• fosfolipasa D MeSH
• PDCD6IP protein, human MeSH Prohlížeč
• phospholipase D2 MeSH Prohlížeč
• proteiny buněčného cyklu MeSH
• proteiny vázající vápník MeSH
• synteniny MeSH

Malignant progression is greatly affected by dynamic cross-talk between stromal and cancer cells. Exosomes are secreted nanovesicles that have key roles in cell-cell communication by transferring nucleic acids and proteins to target cells and tissues. Recently, MicroRNAs (miRs) and their delivery in exosomes have been implicated in physiological and pathological processes. Tumor-delivered miRs, interacting with stromal cells in the tumor microenvironment, modulate tumor progression, angiogenesis, metastasis and immune escape. Altered cell metabolism is one of the hallmarks of cancer. A number of different types of tumor rely on mitochondrial metabolism by triggering adaptive mechanisms to optimize their oxidative phosphorylation in relation to their substrate supply and energy demands. Exogenous exosomes can induce metabolic reprogramming by restoring the respiration of cancer cells and supress tumor growth. The exosomal miRs involved in the modulation of cancer metabolism may be potentially utilized for better diagnostics and therapy.

• MeSH
• biologický transport MeSH
• buňky stromatu metabolismus   MeSH
• energetický metabolismus MeSH
• exozómy genetika   MeSH
• genetická terapie MeSH
• lidé MeSH
• mezibuněčná komunikace MeSH
• mikro RNA genetika   MeSH
• nádorové buněčné linie MeSH
• nádorové mikroprostředí genetika   MeSH
• nádory genetika   metabolismus   patologie   terapie   MeSH
• regulace genové exprese u nádorů MeSH
• signální transdukce MeSH
• technika přenosu genů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• mikro RNA MeSH

Although cisplatin is an effective chemotherapy drug for the treatment of various cancers, its clinical use is limited due to its side effects, especially nephrotoxicity. Unfortunately, acute kidney injury (AKI) caused by cisplatin remains one of the main challenges in effective cancer treatment. Evidence increasingly suggests that renal inflammation and pyroptotic inflammatory cell death of renal tubular epithelial cells (RTECs) mainly determine the progression and outcome of cisplatin-induced AKI. However, it is not clear how cisplatin regulates the pyroptosis of RTECs cells in AKI. The current study aimed to determine the regulation mechanism of AKI induced by cisplatin. We used cisplatin to induce AKI in vivo. We performed H&E staining of mouse kidney tissue sections and evaluated serological indicators of kidney injury (including blood urea nitrogen (BUN), serum creatinine, and tumor necrosis factor-alpha (TNF-alpha)). We used immunohistochemistry and western blot to detect the important substrate protein gasdermin D (GSDMD) and key target caspase-1 of pyroptosis, respectively. Cisplatin induced mouse AKI and RTECs pyroptosis. HK2 cell-derived exosomes treated with cisplatin influenced pyroptosis of the surrounding HK2 cells. Cisplatin-treated HK2 cells exosome-derived miR-122 regulated pyroptosis in the surrounding cells. Exosome-derived miR-122 affected cisplatin-induced AKI and HK2 cells pyroptosis by regulating the expression of embryonic lethal abnormal vision (ELAVL1). These results suggest that exosome miR-122 inhibited pyroptosis and AKI by targeting ELAVL1 under cisplatin treatment, and this offers a potential target for the treatment of AKI.

• MeSH
• akutní poškození ledvin * chemicky indukované   MeSH
• cisplatina toxicita   MeSH
• exozómy * metabolismus   patologie   MeSH
• mikro RNA * metabolismus   MeSH
• myši MeSH
• pyroptóza MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cisplatina MeSH
• mikro RNA * MeSH
• Mirn122 microRNA, mouse MeSH Prohlížeč

The original version of this book was inadvertently published with the hyperlinks deleted from the reference section in the Chapters 1,5,8,11,13,15,16 and 24. These are corrected now.

• Publikační typ
• časopisecké články MeSH
• tisková chyba MeSH