Autophagy pathway: Cellular and molecular mechanisms

Autophagy is an essential homeostatic process involving degradation of intracellular components, including soluble proteins, aggregated proteins, organelles, macromolecular complexes and foreign bodies. Several components of the autophagy pathway have been linked to allergic asthma and macrophage phenotypes, however the contribution of autophagy to these processes is unclear. We previously showed that house dust mite (HDM) increased the number of fluorescent foci (autophagosomes) in bone-marrow derived macrophages (BMM) in a manner similar to the autophagy inducer Torin 1. Further, electron microscopy revealed an increase in double membraned vesicles in response to HDM and Torin 1. While Torin induced a specific increase in lipidated LC3, treatment with HDM caused equal increases in both non-lipidated and lipidated LC3. Torin 1 stimulated autophagic degradation as measured by reduction of SQSTM1/p62. In contrast, SQSTM1/p62 levels increased dramatically in response to HDM. The increase was blocked by actinomycin D and cycloheximide indicating that new gene transcription and protein synthesis is required for the HDM-induced effect. HDM-stimulated increase in SQSTM1/p62 was also observed in alveolar macrophages treated in vitro. An increase in SQSTM1/p62 is often interpreted as a block in autophagic degradation. Indeed, we observed that in the presence of HDM, Torin 1 treatment did not lead to the loss of SQSTM1/p62 below baseline; however, the protein did not accumulate to the high level observed in the HDM-alone treatment group. Our results suggest that HDM engages autophagy machinery at multiple points, both inducing expression of SQSTM1/p62 and suppressing its degradation.

Autophagy is known as a conserved self-eating mechanism that contributes to cells to degrade different intracellular components (i.e., macromolecular complexes, aggregated proteins, soluble proteins, organelles, and foreign bodies). Autophagy needs formation of a double-membrane structure, which is composed of the sequestered cytoplasmic contents, called autophagosome. There are a variety of internal and external factors involved in initiation and progression of autophagy process. Viruses as external factors are one of the particles that could be associated with different stages of this process. Viruses exert their functions via activation and/or inhibition of a wide range of cellular and molecular targets, which are involved in autophagy process. Besides viruses, a variety of cellular and molecular pathways that are activated and inhibited by several factors (e.g., genetics, epigenetics, and environment factors) are related to beginning and developing of autophagy mechanism. Exosomes and microRNAs have been emerged as novel and effective players anticipated in various stages of autophagy. More knowledge in these pathways and identification of accurate roles of them could help to provide better therapeutic approaches in several diseases such as cancer. We highlighted the roles of viruses, exosomes, and microRNAs in the autophagy processes.

Asthma is a chronic inflammatory disease that results in narrowing of the airways. Inhalation of house dust mites (HDM) is a main cause of allergic asthma. Macrophages (Mf) are abundant in the lung and are one of the first cells exposed to HDM, however little is known about the effects of HDM on Mf. Autophagy has been linked to Mf phenotypes and asthma but the mechanism by which autophagy contributes to these processes is unclear. Autophagy is a homeostatic process involving degradation of intracellular components, including proteins, organelles, and foreign bodies. To determine whether HDM stimulates autophagy, we treated bone marrow-derived Mf (BMM) with HDM and evaluated autophagosome foci formation. HDM induced foci formation that was similar to treatment with the autophagy inducer Torin 1. Further, electron microscopy revealed an increase in double membraned vesicles in response to HDM and Torin 1, which could be due to an increase in autophagosome formation or inhibition of their fusion with lysosomes. To differentiate these hypotheses, we investigated markers of autophagy. During canonical autophagy, LC3-I is converted to membrane-associated LC3-II by lipidation, while the cargo adapter protein SQSTM1/p62 is degraded. Surprisingly, treatment with HDM caused a general increase in LC3-I and LC3-II without a substantial change in the LC3-II to LC3-I ratio. As expected, Torin 1 induced autophagic degradation demonstrated by reduction of SQSTM1/p62. However, SQSTM1/p62 increased dramatically in response to HDM, indicating a block in autophagic degradation. We observed a similar trend in THP-1 and human CD14+ Mf. These results suggest that HDM engages autophagy machinery but does not stimulate degradative autophagy.

The term autophagy, (Type II Apoptosis) is derived from the Greek roots “auto” (self) and “phagy” (eat) and was first coined by De Duve in 1967 to epitomise this type of cell death. The mechanism of organelle autophagy in cells undergoing macro-autophagy (from here on termed autophagy) is poorly understood. Cytoplasm, misfolded protein aggregates, dysfunc‐ tional mitochondria and stressed endoplasmic reticulum (ER) are engulfed by the formation of a double membrane forming an autophagosome [1,2]. The formation of the autophagosome double membrane structure within the cytoplasm is thought to be formed from pre-existing membranes within the cell, although it is unknown whether the Golgi apparatus, endoplasmic reticulum (ER) or mitochondria are used preferentially to form the autophagosome structure [1,2]. During the formation of the autophagosome structure, organelles such as mitochondria, parts of the ER and Golgi apparatus are engulfed by the autophagosome with the final closure of the double membrane structure occurring next. This then fuses with nearby lysosomes, giving rise to an autolysosome, where the intracellular components are degraded by hydrolytic enzymes [1,3-5]. This process generates ATP, which may delay cell death if the cell is under nutrient depleted conditions leading to the survival of the cell. Thus it is unclear whether the process protects or causes diseases such as cancer and neurodegenerative disorders [6,7].

The functional units within cells are often macromolecular complexes rather than single species. Production of these complexes as assembled homogenous samples is a prerequisite for their biophysical and structural characterization and hence an understanding of their function in molecular terms. Co-expression in Escherichia coli has been used routinely to decipher the subunit composition, assembly, and production of whole protein complexes. Such complexes can then be used to reconstitute protein/nucleic acid complexes in vitro. In this chapter we present protocols for the widely utilized ACEMBL and pET-MCN/pET-MCP vector series which enable the rapid and automated co-expression of protein complexes in Escherichia coli.

The development of protein bodies in proteinoplasts of tobacco (Nicotiana tabacum L. var. Wis. 38) roots was investigated with TEM, HVEM, and enzyme cytochemistry. These plastids contain a three-dimensional network of fenestrated tubules which originate from invaginations of the inner membrane of the plastid envelope. Elaboration of the network occurs in parallel with cell differentiation: slender tubules common to plastids in meristematic cells undergo dilation as protein accumulates during cell differentiation; proteinoplasts of vacuolate and root cap cells usually contain a large protein body. The contents of the peripheral tubules, originating from the inner membrane, are less electron dense than the tubules making up the central network. Localized dilations within the tubular network result in the formation of dense spheroidal structures, protein bodies, apparently as a result of continued protein accumulation via tubules connecting to the central network. Protein might be imported from segments of rough ER attached to or apposed to the outer membrane of the proteinoplast envelope. The presence of catalase (E.C. 1.11.1.6), peroxidase (E.C. 1.11.1.7), and cytochrome oxidase (E.C. 1.9.3.1) was demonstrated by cytochemistry with diaminobenzidine (DAB) as substrate. Oxidized DAB was found in protein bodies after incubation in each of the specific reaction media. While aminotriazole and sodium azide inhibited oxidation of DAB by catalase and peroxidase, respectively, only potassium cyanide completely inhibited oxidation of DAB in protein bodies. We conclude that protein bodies of proteinoplasts in tobacco roots are not sites for storage of protein, rather protein bodies contain heme protein(s) with strong oxidase activity that may convey a specific function to proteinoplasts.

The seed storage proteins of maize (Zea mays L.) are synthesized during endosperm development on membrane-bound polyribosomes. Protein body formation in normal genotypes occurs via a sequential deposition of the various types of zeins, and leads to the formation of spherical structures with a diameter of about l μm. In the endosperm mutantopaque-2 the level of one zein class is reduced; these kernels exhibit an opaque phenotype instead of the vitreous phenotype displayed in normal genotypes, presumably due to the decrease in total zein protein at the time of desiccation. Previous microscopic examination ofopaque-2 protein bodies at 22 DAP (days after pollination) showed that the protein bodies were morphologically similar to those of normal genotypes. However, the endosperm ofopaque-2 maize at 14 DAP contains tubular arrays within the rough endoplasmic reticulum. These tubular arrays are tightly associated with the developing protein bodies. Long strands of tubules, sometimes 10 μm in length, are observed in the endosperm, and partially formed protein bodies often seem to be forming directly from these tubular arrays. No immunostaining is associated with this tubular material when any of the anti-zein antibodies are used.

Comparison between hydrophilic and hydrophobic metal nanoparticles on the phase separation phenomena during formation of asymmetric polyethersulphone membranes

Foreign Body Giant Cell Formation Is Preceded by Lamellipodia Formation and Can Be Attenuated by Inhibition of Rac1 Activation

Stable accumulation of storage proteins, lipids and carbohydrates is a hallmark of the plant seed, and is a characteristic that is typically deficient in existing platforms for recombinant protein manufacture. One of the biological sequestration mechanisms that facilitate the folding, assembly and stabilization of plant seed storage proteins involve the de novo formation of unique intracellular organelles, the endoplasmic reticulum (ER)-derived protein bodies (PBs). In cereals, such as maize, PBs are formed directly in the lumen of the ER of endosperm cells and contain zeins, a group of polypeptides, which account for more than half of the total seed protein mass. The 27 kD gamma zein protein localizes to the periphery of the PBs surrounding aggregates of other zeins (including a zein and delta zein). Heterologous expression of gamma zein has been shown to result in the formation of PB-like structures, and the N-terminal proline-rich domain of gamma zein (Zera), containing eight PPPVHL repeats and a Pro-X sequence is by itself capable of directing ER retention and PB formation in non-seed tissues. We present a novel approach to produce recombinant proteins in plants based on the ability of gamma zein-Zera domain to store recombinant proteins inside PBs. Zera domain fused to several proteins, including a enhanced cyan fluorescent protein (ECFP), calcitonin (Ct) and epidermal growth factor (EGF), were cloned into vectors for transient or stable transformation of tobacco plants. In tobacco leaves, we observed the formation of dense, ER-localized structures containing high concentrations of the respective target proteins. The intact synthetic organelles containing Zera fusions were readily isolated from cellular material using density-based separation methods.

Autophagy refers to a group of processes that involve degradation of cytoplasmic components including cytosol, macromolecular complexes, and organelles, within the vacuole or the lysosome of higher eukaryotes. The various types of autophagy have attracted increasing attention for at least two reasons. First, autophagy provides a compelling example of dynamic rearrangements of subcellular membranes involving issues of protein trafficking and organelle identity, and thus it is fascinating for researchers interested in questions pertinent to basic cell biology. Second, autophagy plays a central role in normal development and cell homeostasis, and, as a result, autophagic dysfunctions are associated with a range of illnesses including cancer, diabetes, myopathies, some types of neurodegeneration, and liver and heart diseases. That said, this review focuses on autophagy in yeast. Many aspects of autophagy are conserved from yeast to human; in particular, this applies to the gene products mediating these pathways as well as some of the signaling cascades regulating it, so that the information we relate is relevant to higher eukaryotes. Indeed, as with many cellular pathways, the initial molecular insights were made possible due to genetic studies in Saccharomyces cerevisiae and other fungi.

Protein kinase CK2 (formerly known as casein kinase II) has been viewed traditionally as a stable heterotetrameric complex, but new analytical techniques are bringing a different picture into focus. The transient nature of this complex has been highlighted by the elucidation of its structure. Furthermore, analysis of the spatiotemporal organization of individual CK2 subunits in living cells has shown that they are dynamic and that they integrate into different multimolecular assemblies. These new studies give an additional dimension to the challenge of determining the cellular regulation of this protein kinase.

A developmental study at the electron microscopic level was conducted of the fat body cells of Hyalophora cecropia (L.). During the last larval instar the fat body increases in volume and the cells exhibit a well developed rough endoplasmic reticulum and protein bodies of diverse sizes. In the pupal fat body, the protein bodies appear to be enclosed by a double membrane and contain glycogen granules, ribosomes and mitochondrion-like structures. In addition, there are large lipid globules, cytolysomes and rough endoplasmic reticulum. The ultrastructure of the protein bodies suggests the development of large bodies by fusion of smaller protein bodies. Changes in fat body cell ultrastructure were followed during adult development and cytological evidence was obtained for the depletion of protein, glycogen and lipid in the female during this period. The female adult fat body cell contains free ribosomes, protein bodies, many mitochondria, a few lipid globules and glycogen granules. The male moth fat body cells have many mitochondria, a few glycogen granules, essentially no protein bodies, but an abundance of large lipid globules.

G protein-coupled receptor (GPCR) heteromers are macromolecular complexes with unique functional properties different from those of its individual protomers. Little is known about what determines the quaternary structure of GPCR heteromers resulting in their unique functional properties. In this study, using resonance energy transfer techniques in experiments with mutated receptors, we provide for the first time clear evidence for a key role of intracellular domains in the determination of the quaternary structure of GPCR heteromers between adenosine A(2A), cannabinoid CB(1), and dopamine D(2) receptors. In these interactions, arginine-rich epitopes form salt bridges with phosphorylated serine or threonine residues from CK1/2 consensus sites. Each receptor (A(2A), CB(1), and D(2)) was found to include two evolutionarily conserved intracellular domains to establish selective electrostatic interactions with intracellular domains of the other two receptors, indicating that these particular electrostatic interactions constitute a general mechanism for receptor heteromerization. Mutation experiments indicated that the interactions of the intracellular domains of the CB(1) receptor with A(2A) and D(2) receptors are fundamental for the correct formation of the quaternary structure needed for the function (MAPK signaling) of the A(2A)-CB(1)-D(2) receptor heteromers. Analysis of MAPK signaling in striatal slices of CB(1) receptor KO mice and wild-type littermates supported the existence of A(1)-CB(1)-D(2) receptor heteromer in the brain. These findings allowed us to propose the first molecular model of the quaternary structure of a receptor heteromultimer.

Protein bodies (PBs) are endoplasmic reticulum (ER) derived organelles originally found in seeds whose function is to accumulate seed storage proteins. It has been shown that PB formation is not limited to seeds and green fluorescent protein (GFP) fused to either elastin-like polypeptide (ELP) or hydrophobin (HFBI) fusion tags induce the formation of PBs in leaves of N.benthamiana. In this study, we compared the ELP- and HFBI-induced PBs and showed that ELP-induced PBs are larger than HFBI-induced PBs. The size of ELP- and HFBI-induced PBs increased over time along with the accumulation levels of their fused protein. Our results show that PB formation is a concentration-dependent mechanism in which proteins accumulating at levels higher than 0.2% of total soluble protein are capable of inducing PBs in vivo. Our results show that the presence of fusion tags is not necessary for the formation of PBs, but affects the distribution pattern and sizeof PBs. This was confirmed by PBs induced by fluorescent proteins as well as fungal xylanases. We noticed that in the process of PB formation, secretory and ER-resident molecules are passively sequestered into the lumen of PBs. We propose to use this property of PBs as a tool to increase the accumulation levels of erythropoietin and human interleukin-10 by co-expression with PB-inducing proteins.