Featured Article
Monday February 08, 2010
by Caitlin Smith
Autophagy—literally, eating oneself—is a crucial recycling function in cells whereby they rid themselves of intracellular matter such as protein and organelles. Though it may sound like a routine housekeeping function, autophagy also plays a role in mechanisms of cell death, stress responses, and even embryogenesis and postnatal development. “Autophagy is an evolutionarily conserved, fundamental intracellular degradative process operating as a homeostatic mechanism in all eukaryotic cells,” says Paul Townsend, a professor in molecular cell biology at the University of Southampton School of Medicine. “Using lower order eukaryotic organisms and mutants thereof has allowed rapid breakthroughs in the autophagy arena. These include adaptation to stress such as genotoxic insults. In man, autophagic impairment has been associated with neurodegeneration, cancer and inflammatory diseases.”
Indeed, as we learn more about how autophagy works, we also learn more about the close links between its dysfunction and our diseases. For example, mice lacking the key autophagy protein Beclin-1 show greater tumorigenesis, suggesting that normal Beclin-1 levels may help to prevent tumor growth. It is thought that the Beclin-1 protein helps to initiate the formation of the autophagosome by binding to the pre-autophagosomal structure. Autophagosomes are membrane-bound organelles that envelop and transport components to lysosomes, where they fuse to become autolysosomes. The enzymes contained within the autolysosomes degrade the engulfed components into basic materials that can be recycled and used again by the cell. “Research continues to show the involvement of autophagy in an ever-increasing number of diseases, including various cancers and neurodegenerative diseases,” says Bryan Tinsley, VP of marketing at Novus Biologicals. “As more is learned about the basic biological controls which guide and control autophagy, new areas of research will require an ever-evolving suite of tools.”
Autophagy tools
Townsend notes that “the development of specific and easy to use reagents for monitoring autophagy has been crucial in our understanding of physiology.” The autophagic pathway and the endocytic recycling pathway overlap in their vesicular-mediated transport of membrane-bound materials to lysosomes. Luckily, new tools are still evolving to facilitate our understanding of autophagy. The antibody manufacturer Abcam offers a wide range of primary antibody tools for researchers. “But more importantly, [Abcam also offers] an ever-expanding range of secondary antibodies, proteins, peptides and lysates as well as ELISA kits for autophagy research,” says Candy Smellie, CFA marketing coordinator at Abcam. “An increasing number of cardiovascular researchers are performing ChIP (chromatin immunoprecipitation) as part of their research, and we have an extensive range of some of the best ChIP grade antibodies alongside an excellent ChIP kit.”
In addition to a wide range of autophagy-related antibodies, Novus Biologicals offers a new bioinformatics tool called Explorer. According to Tinsley, Explorer “was designed to assist researchers studying emerging areas such as autophagy. For instance, by typing the word ‘autophagy’ into the pathway section of the Explorer you can see the most relevant gene, disease, pathway or post-translational modification data, and, more importantly, the references that describe how this relevancy was determined and direct links to the abstract. The tool can keep expanding and is not limited to the initial starting point. It can also be started from the disease or gene level. In addition, it is not based on static data but is continually updating its relevancy using Biolab Experiment Assistant licensed from Biovista.”
Researchers are also devising their own tools at the bench to access the angle of autophagy that fits their research. For example, Antonio Zorzano, head of the molecular medicine program and professor in the department of biochemistry and molecular biology at the University of Barcelona, recently published a paper1 in which he and his colleagues describe how the DOR protein is implicated in the initial—so far, the least understood—stages of autophagy. The DOR protein is thought to help with the formation of autophagosomes.
“In the laboratory we have set up an automatized fluorescent microscopy-based method to quantify LC3 positive autophagosomes in cells,” says Zorzano. “This permits a high content screening of modulators of autophagy. In addition, we have set up a precise and trustworthy methodology for the analysis of proteins that colocalize in autophagosomes.” Zorzano believes that protocols that incorporate proteomics, such as the purification of autophagosomes, would be an exciting next step, as well as “the development of tools to analyze different types of autophagic pathways that are relevant for the repair of specific cell organelles such as ER, mitochondria, and endosomes.”
Autophagy and disease
As researchers identify more molecular components in autophagic pathways, and as our understanding of their interactions grows, links between autophagy and human diseases are emerging. “Many autophagic proteins have significant tumor suppressor activity (these include beclin-1 and Atg5, to name just two),” says Townsend. “Moreover, and possibly more convincing, is that some genetic mutations of autophagy genes, shown and detected in man or animal models, can lead to DNA damage accumulation and genome instability.”
Researchers still have a long road ahead in understanding autophagy. Though it can be tricky to distinguish normal versus autophagic processes, antibody tools are helping. “It is difficult to differentiate normo- and patho- physiology,” Townsend says. “However, recently, beclin-1 and LCIII antibodies for Western blotting or immunostaining have relieved that pressure. We can now, and rapidly, characterize cell death/stress between key, fundamental processes such as autophagy, necrosis, apoptosis and inflammation.”
A complicating factor to tease out from the roles of autophagic processes is the idea that autophagy is involved in homeostatic mechanisms. “Autophagy may help the cell [to] signal its homeostatic condition to the surrounding milieu, or hastily modify its metabolic state to better counter detrimental, extrinsic stimuli,” says Townsend. Mark Parker, senior scientist and head of product development at Novus Biologicals, cites new developments in our understanding of autophagy and mTOR signaling as an exciting recent advance. “mTOR regulates many cell signaling pathways that involve many aspects of cellular metabolism,” says Parker. “Autophagy is also regulated by many environmental stimuli such as nutrients and stress and is a downstream target of mTOR. The involvement of both pathways in tumorigenesis and cancer maintenance is very intriguing, and it would be really exciting to learn more about how these are coordinated and further lead to novel therapies for treating cancer.”
Challenges on the horizon
One of the main challenges to overcome is how to take the individual components now being discovered in autophagic pathways, and to put them into a framework in which the interactions between them are better understood. This is a tall order for any biochemical pathway, but even more so for one that seems to intersect with so many other crucial cell functions. James Wang, stem cell product developer at Novus Biologicals, notes that, “another challenge will be studying autophagy in the context of an entire multicellular organism. Emerging animal models with autophagy-related genetic alterations are powerful tools to accelerate our learning process.” Townsend suggests that, “we should also look at early developmental stages of life—what impact does autophagy modulation have on early developmental processes?” Surely the results of autophagy research will be felt across many disciplines in the years to come—and if we’re lucky, will produce some cancer or neurodegenerative disease therapies as well.
References
1Mauvezin C, et al. "The nuclear cofactor DOR regulates autophagy in mammalian and Drosophila cells," EMBO Rep. 11: 37-44, 2010.