I am a Master's student in Biochemistry at the Karolinska Institutet in Stockholm, Sweden. I have a range of notes from courses that I undertook as part of my Bachelor's studies. These notes are detailed and in depth that focus on many aspects of how Autophagy works and how it is implicated in heal...
Test Bank - Lehninger Principles of Biochemistry, 8th Edition (Nelson, 2022), Chapter 1-28 | All Chapters
TEST BANK FOR LEHNINGER PRINCIPLES OF BIOCHEMISTRY NELSON 6TH EDITION
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Voorbeeld van de inhoud
Autophagy:
1) General:
Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome.
This process is quite distinct from endocytosis-mediated lysosomal degradation of extracellular and plasma membrane
proteins. There are three types of autophagy—macroautophagy, microautophagy, and chaperone-mediated autophagy.
Autophagy is mediated by a unique organelle called the autophagosome. The autophagosome is transient and only
exists for ~10-20 minutes before fusing with the lysosome. In contrast to the ubiquitin–proteasome system (UPS),
which targets individual short-lived proteins, autophagy functions as a bulk process with the capacity to degrade
longlived proteins and organelles such as the endoplasmic reticulum, mitochondria, peroxisomes, the nucleus and
ribosomes.
Macroautophagy (“self-eating”, hereafter referred to as autophagy) is an evolutionarily conserved process whereby the
eukaryotic cell can recycle part of its own content by sequestering a portion of the cytoplasm in a double-membrane
vesicle that is delivered to the lysosome for digestion. Unlike other cellular degradation machineries, autophagy
removes long-lived proteins, large macro-molecular complexes and organelles that have become obsolete or damaged.
Autophagy mediates the digestion and recycling of non-essential parts of the cell during starvation and participates in
a variety of physiological processes where cellular components must be removed to leave space for new ones. In
addition, autophagy is a key cellular process capable of clearing invading microorganisms and toxic protein
aggregates, and therefore plays an important role during infection, in ageing and in the pathogenesis of many human
diseases.
Macroautophagy involves the formation of double membrane-bound vesicles called autophagosomes that engulf
cytoplasmic proteins and organelles; these autophagosomes are trafficked to lysosomes, at which point the sequestered
cargo is degraded.
Microautophagy refers to the invagination of the lysosomal or endosomal membrane, resulting in the direct
engulfment of substrates that are subsequently degraded by lysosomal proteases7 . Chaperone-mediated autophagy is
distinct from macroautophagy and microautophagy because cargo is not sequestered within a membrane delimited
vesicle. Instead, proteins targeted by chaperonemediated autophagy contain a KFERQ-like pentapetide motif that is
recognized by the cytosolic chaperone heat shock cognate 70 kDa protein (HSC70); HSC70 promotes the
translocation of these targets across lysosomal membranes into the lysosomal lumen via the lysosomalassociated
membrane protein 2A (LAMP2A) receptor
Autophagy, along with the UPS, is also a key mechanism for protein homeostasis and quality control. Indeed,
basal autophagy within cells is important for the degradation of damaged and dysfunctional proteins and organelles;
autophagy-deficient mice exhibit a build-up of misfolded, damaged proteins.
Although protein degradation is a salient feature of autophagy, studies over the past decade have revealed that
autophagy plays a key part in mobilizing diverse cellular energy and nutrient stores, including carbohydrates, lipids
and minerals. Hence, a growing appreciation of the role of autophagy in controlling cellular metabolism in both
normal and diseased cells has fuelled immense interest in elucidating how dysfunctional autophagy influences
metabolic disorders and metabolic adaptation in diseases such as cancer.
Nobel prize 2016:
, In the early 1990’s Yoshinori Ohsumi, then an Assistant Professor at Tokyo University, decided to study autophagy
using the budding yeast Saccharomyces cerevisae as a model system. The first question he addressed was whether
autophagy exists in this unicellular organism. The yeast vacuole is the functional equivalent of the mammalian
lysosome. Ohsumi reasoned that, if autophagy existed in yeast, inhibition of vacuolar enzymes would result in the
accumulation of engulfed cytoplasmic components in the vacuole. To test this hypothesis, he developed yeast strains
that lacked the vacuolar proteases proteinase A, proteinase B and carboxy-peptidase19. He found that autophagic
bodies accumulated in the vacuole when the engineered yeast were grown in nutrient deprived medium19, producing
an abnormal vacuole that was visible under a light microscope. He had now identified a unique phenotype that could
be used to discover genes that control the induction of autophagy. By inducing random mutations in yeast cells lacking
vacuolar proteases, Ohsumi identified the first mutant that could not accumulate autophagic bodies in the vacuole20;
he named this gene autophagy 1 (APG1). He then found that the APG1 mutant lost viability much quicker than wild-
type yeast cells in nitrogendeprived medium. As a second screen he used this more convenient phenotype and
additional characterization to identify 75 recessive mutants that could be categorized into different complementation
groups. In an article published in FEBS Letters in 1993, Ohsumi reported his discovery of as many as 15 genes that
are essential for the activation of autophagy in eukaryotic cells20. He named the genes APG1-15. As new autophagy
genes were identified in yeast and other species, a unified system of gene nomenclature using the ATG abbreviation
was adopted21. This nomenclature will be used henceforth in the text.
During the following years, Ohsumi cloned several ATG genes22-24 and characterized the function of their protein
products. Cloning of the ATG1 gene revealed that it encodes a serine/threonine kinase, demonstrating a role for
protein phosphorylation in autophagy24. Additional studies showed that Atg1 forms a complex with the product of the
ATG13 gene, and that this interaction is regulated by the target of rapamycin (TOR) kinase23,25. TOR is active in
cells grown under nutrient-rich conditions and hyper-phosphorylates Atg13, which prevents the formation of the
Atg13:Atg1 complex. Conversely, when TOR is inactivated by starvation, dephosphorylated Atg13 binds Atg1 and
autophagy is activated25. Subsequently, the active kinase was shown to be a pentameric complex26 that includes, in
addition to Atg1 and Atg13, Atg17, Atg29 and Atg31. The assembly of this complex is a first step in a cascade of
events needed for formation of the autophagosome.
The formation of the autophagosome involves the integral membrane protein Atg9, as well as a phosphatidylinositol-3
kinase (PI3K) complex26 composed of vacuolar protein sorting-associated protein 34 (Vps34), Vps15, Atg6, and
Atg14. This complex generates phosphatidylinositol-3 phosphate and additional Atg proteins are recruited to the
membrane of the phagophore. Extension of the phagophore to form the mature autophagosome involves two
ubiquitin-like protein conjugation cascades.
Studies on the localization of Atg8 showed that, while the protein was evenly distributed throughout the cytoplasm of
growing yeast cells, in starved cells, Atg8 formed large aggregates that colocalized with autophagosomes and
autophagic bodies27. Ohsumi made the surprising discovery that the membrane localization of Atg8 is dependent on
two ubiquitin-like conjugation systems that act sequentially to promote the covalent binding of Atg8 to the membrane
lipid phosphatidylethanolamine. The two systems share the same activating enzyme, Atg7.
In the first conjugation event, Atg12 is activated by forming a thioester bond with a cysteine residue of Atg7, and then
transferred to the conjugating enzyme Atg10 that catalyzes its covalent binding to the Atg5 protein. Further work
showed that the Atg12:Atg5 conjugate recruits Atg16 to form a trimolecular complex that plays an essential role in
autophagy by acting as the ligase of the second ubiquitin-like conjugation system.
In this second unique reaction, the C-terminal arginine of Atg8 is removed by Atg4, and mature Atg8 is subsequently
activated by Atg7 for transfer to the Atg3 conjugating enzyme. Finally, the two conjugation systems converge as the
Atg12:Atg5:Atg16 ligase promotes the conjugation of Atg8 to phosphatidylethanolamine. Lipidated Atg8 is a key
driver of autophagosome elongation and fusion. The two conjugation systems are highly conserved between yeast and
mammals. A fluorescently tagged version of the mammalian homologue of yeast Atg8, called light chain 3 (LC3), is
extensively used as a marker of autophagosome formation in mammalian systems.
The most extensively studied form of autophagy, macroautophagy, degrades large portions of the cytoplasm and
cellular organelles. Non-selective autophagy occurs continuously, and is efficiently induced in response to stress, e.g.
starvation. In addition, the selective autophagy of specific classes of substrates - protein aggregates, cytoplasmic
organelles or invading viruses and bacteria - involves specific adaptors that recognize the cargo and targets it to
Atg8/LC3 on the autophagosomal membrane. Other forms of autophagy include microautophagy, which involves the
direct engulfment of cytoplasmic material via inward folding of the lysosomal membrane, and chaperone-mediated
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