Showing papers by "Vojo Deretic published in 2008"

Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes

Abstract: Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms Recent reviews have described the range of assays that have been used for this purpose(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi) Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response

Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking

Abstract: Autophagic and endocytic pathways are tightly regulated membrane rearrangement processes that are crucial for homeostasis, development and disease. Autophagic cargo is delivered from autophagosomes to lysosomes for degradation through a complex process that topologically resembles endosomal maturation. Here, we report that a Beclin1-binding autophagic tumour suppressor, UVRAG, interacts with the class C Vps complex, a key component of the endosomal fusion machinery. This interaction stimulates Rab7 GTPase activity and autophagosome fusion with late endosomes/lysosomes, thereby enhancing delivery and degradation of autophagic cargo. Furthermore, the UVRAG-class-C-Vps complex accelerates endosome–endosome fusion, resulting in rapid degradation of endocytic cargo. Remarkably, autophagosome/endosome maturation mediated by the UVRAG-class-C-Vps complex is genetically separable from UVRAG–Beclin1-mediated autophagosome formation. This result indicates that UVRAG functions as a multivalent trafficking effector that regulates not only two important steps of autophagy — autophagosome formation and maturation — but also endosomal fusion, which concomitantly promotes transport of autophagic and endocytic cargo to the degradative compartments.

Toll-like receptors control autophagy

Abstract: Autophagy is a newly recognized innate defense mechanism, acting as a cell-autonomous system for elimination of intracellular pathogens. The signals and signalling pathways inducing autophagy in response to pathogen invasion are presently not known. Here we show that autophagy is controlled by recognizing conserved pathogen-associated molecular patterns (PAMPs). We screened a PAMP library for effects on autophagy in RAW 264.7 macrophages and found that several prototype Toll-like receptor (TLR) ligands induced autophagy. Single-stranded RNA and TLR7 generated the most potent effects. Induction of autophagy via TLR7 depended on MyD88 expression. Stimulation of autophagy with TLR7 ligands was functional in eliminating intracellular microbes, even when the target pathogen was normally not associated with TLR7 signalling. These findings link two innate immunity defense systems, TLR signalling and autophagy, provide a potential molecular mechanism for induction of autophagy in response to pathogen invasion, and show that the newly recognized ability of TLR ligands to stimulate autophagy can be used to treat intracellular pathogens.

Autophagosome and Phagosome

Abstract: Autophagy and phagocytosis are evolutionarily ancient processes functioning in capture and digestion of material found in the cellular interior and exterior, respectively. In their most primordial form, both processes are involved in cellular metabolism and feeding, supplying cells with externally obtained particulate nutrients or using portions of cell's own cytoplasm to generate essential nutrients and energy at times of starvation. Although autophagy and phagocytosis are commonly treated as completely separate biological phenomena, they are topologically similar and can be, at least morphologically, viewed as different manifestations of a spectrum of related processes. Autophagy is the process of sequestering portions of cellular interior (cytosol and intracellular organelles) into a membranous organelle (autophagosome), whereas phagocystosis is its topological equivalent engaged in sequestering cellular exterior. Both autophagosomes and phagosomes mature into acidified, degradative organelles, termed autolysosomes and phagolysosomes, respectively. The basic role of autophagy as a nutritional process, and that of phagocytosis where applicable, has survived in present-day organisms ranging from yeast to man. It has in addition evolved into a variety of specialized processes in metazoans, with a major role in cellular/cytoplasmic homeostasis. In humans, autophagy has been implicated in many health and disease states, including cancer, neurodegeneration, aging and immunity, while phagocytosis plays a role in immunity and tissue homeostasis. Autophagy and phagocytosis cooperate in the latter two processes. In this chapter, we briefly review the regulatory and execution stages of both autophagy and phagocytosis.

Autophagy, an immunologic magic bullet: Mycobacterium tuberculosis phagosome maturation block and how to bypass it

Abstract: Mycobacterium tuberculosis is a facultative intracellular pathogen that parasitizes host macrophages where it persists in immature phagosomes by avoiding their maturation into phagolysosomes. The mechanisms of how M. tuberculosis inhibits phagolysosome biogenesis have been researched in detail and the maturation block at least partially depends on the manipulation of host phosphoinositide interconversions, with phosphatidylinositol 3-phosphate (PI3P) being a central target since it has been shown to be required for phagolysosome biogenesis. PI3P earmarks intracellular organelles for binding and assembly of effector molecules that interact with PI3P or its derivatives, including Class E Vps proteins such as Hrs and ESCRT components, early endosome antigen 1, which are required for sequential protein and membrane sorting within the endosomal and, by extension, phagosomal systems. In a search of a cellular mechanism that can bypass the tubercule bacillus-imposed PI3P block, researchers have uncovered a new g...

Autophagic proteolysis of long-lived proteins in nonliver cells.

Abstract: Autophagy is a cellular homeostasis pathway used to sustain cellular anabolic needs during times of nutrient or energy deprivation. Autophagosomes sequester cytoplasmic constituents, including macromolecules such as long-lived proteins. Upon fusion of autophagosomes with lysosomes, the engulfed cargo is degraded. The proteolysis of longlived proteins by macroautophagy is a standard, specific measure of autophagic degradation and represents an end-point assay for the pathway. The assay is based on a pulse-chase approach, whereby cellular proteins are radiolabeled by an isotopically marked amino acid, the short-lived, rapidly turned over, proteins are allowed to be degraded during a long chase period, and then the remaining, stable radiolabeled proteins are subjected to autophagic degradation. The classical application of this method has been in hepatocytes, but the recent growth of interest in autophagy has necessitated adaptation of this method in nonliver cells. Here we describe a protocol to quantify autophagic degradation of longlived proteins in macrophages. This chapter details the method of analyzing autophagic proteolysis in RAW264.7 mouse macrophages.

How Cells Clean House

Abstract: Inside the cytoplasm of a living cell, organelles called autophagosomes continually engulf bits of cytoplasm, along with damaged cell parts and invading bacteria and viruses. The "sweepings" are carried to digestive organelles for breakup and recycling. The process is called autophagy. Cell biologists are learning about autophagy in great detail by tracing the protein signals that drive and control the process. A fuller understanding of autophagy is opening up new options for treating cancer, infectious disease, immune disorders and dementia, and it may one day even help to slow down aging.

Rationally improved isoniazid and ethionamide derivatives and activity through selective isotopic substitution

Abstract: The present invention relates to the use of isotopically labeled derivatives of isoniazid, ethionamide and related compounds as effective therapy for the treatment of mycobacterial diseases, including Mycobacterium tuberculosis.

Analysis of p. aeruginosa infection in patients

Abstract: The present invention relates to methods for detecting P aeruginosa infection and bacterial burden in the lungs of patients who are at risk for P. aeruginosa infections, especially including patients with Cystic Fibrosis (CF). The present method provides numerous tests (breath, blood, urine) which are readily administered to a patient that will sensitively and specifically detect the presence and extent of lung infection P. aeruginosa (both mucoid and non-mucoid), and allow monitoring of bacterial load as a parameter in monitoring treatment.

Diagnosis of P. aeruginosa infection in the lungs of patients

Abstract: The present invention relates to methods for detecting P aeruginosa infection and bacterial burden in the lungs of patients who are at risk for P. aeruginosa infections, especially including patients with Cystic Fibrosis (CF). The present method provides numerous tests (breath, blood, urine) which are readily administered to a patient that will sensitively and specifically detect the presence and extent of lung infection P. aeruginosa (both mucoid and non-mucoid), and allow monitoring of bacterial load as a parameter in monitoring treatment.

In vitro phagosome-endosome fusion.

Abstract: Phagolysosome biogenesis plays a pivotal role in elimination of foreign particles and pathogens by leukocytes. This process is achieved by multiple cycles of membrane fusion between the phagosome and the endosomal system. In vitro reconstitution of phagosome fusion with endosomes is instrumental in studying this intricate process. Such an assay is also invaluable for the identification of effectors produced by intracellular pathogens that may hamper the pathway. In this chapter we describe a highly sensitive and relatively rapid method to measure fusion between phagosomes and early, as well as late, endosomal compartments. The assay is based on the formation of a stable biotin-streptavidin complex upon fusion between biotinylated-peroxidase loaded endosomes and magnetic streptavidin conjugated bead-containing phagosomes. Fusion is quantified using a fluorescence-based detection method that measures the peroxidase activity associated with the beads.