分子细胞生物学：7蛋白质降解相关亚细胞结构

1、12I. Cytoplasm (chapter 6,7,8)Structure and Function of OrganellesII. Cytoplasm (chapter 9)Cytoskeletonchapter 6 Structure and Function of Organelles involved in protein synthesis, processing and transportchapter 7 Structure and Function of Organelles involved in protein degradationchapter 8 Structu

2、re and Function of mitochondrion and peroxisome33 for their discoveries concerning the structural and functional organization of the cell. MLA style: The Nobel Prize in Physiology or Medicine 1974. N. Nobel Media AB 2014. Web. 18 Mar 2015. Albert Claude Christian de Duve George E. Palad

3、e The Nobel Prize in Physiology and medicine, 1974“method”Lysosome, peroxisome“ribosome”4To study the process of protein secretion with pancreatic epithelial cells.George PaladeCell Biology finally makes possible a century old dream: that of analysis of diseases, at the cellular level - the first st

4、ep towards their final control.We never truly touch or see these wonderful tiny devices that keep every cell and every being alive - since they are far beyond what our senses can perceive unaided. But for us they are alive in our minds, close to our hearts, very much parts of the real world, just li

5、ke the galaxies with their neutron stars and their pulsars are at the other end of the spectrum of dimensions of matter for our colleagues, the radio astronomers.5Albert Claude, working during the 1930s and 40s at the Rockefeller Institute had a dominating role both for the application of the electr

6、on microscope for the study of animal cells and for the development of the differential centrifugation. In particular Palade and coworker studied a network of submicroscopic membranes, called the endoplasmic reticulum, originally discovered by Claude and Porter. They showed that the reticulum can be

7、 described as a multiply folded, more or less deflated sack occupying most of the cytoplasm. Palade discovered and described small granular components now known under the name of ribosomes covering the outside of the membranes and he showed, with other groups, that the ribosomes carry out the protei

8、n synthesis in the cell. Lysosomes have now been shown, by de Duve and others, to be engaged in a series of cellular activities during which biological material must be degraded. de Duve has not only a highly dominating role in lysosome research, he is also the discoverer of another cell component,

9、the peroxisome, the function of which is still enigmatic but which may very well offer a story as fascinating as that of the lysosomes in the future.6The Nobel Prize in Chemistry 2004“for the discovery of ubiquitin-mediated protein degradation 7How do Proteins function?(1)Quantity: synthesis (gene e

10、xpression from DNA-RNA to protein)(2)Quality: three-dimensional structure(conformation) (corrected folding and assembly)(3)Right Localization ：transportation (protein sorting and other mechanisms) proper activity* Quantity *Quality *Right Localization8How are protein regulated?（3）Translocation regul

11、ation:（4）Activity Regulation： (alterations of post-translational modifications alterations of and interaction with other molecules) other way? （2）Quality control:（1）Quantity regulation： * up-regulation of gene expression and protein synthesis* ER export misfolded protein and then * down-regulation o

12、f gene expression and protein synthesisHow? 9What is protein degradation?by which protein is digested to short peptides or amino acid by a series of proteases.Degradation of protein is a proteolytic process Removal of proteinBreakdown of proteinDigestion of ProteinTurnover of protein: degradation an

13、d generationGVYVECHS-L-Y-Qproteases10ribosomeERGolgi ApparatuslysosomeproteasomeCellular organelles work for protein11Level A1.Concept: protein degradation; ubiquitin-proteasome system. 2.The structure and function of the lysosome. Level B1.The biogenesis of autophagosome 2. The formation of lysosom

14、e.3.The working principle of the proteasome.Level C1.The functional connection of lysosome, endoplasmic reticulum and Golgi apparatus.2.The biological implication of autophagy. SYLLABUS12Degradation of ProteinsTwo ways*Proteasomal degradation via ubiquitin-proteasome syetem *Lysosomal degradationvia

15、 autophagy and endocytosis13I、Chapter 7.Structure and function of organelles involved in degradation of proteins14*single membrane enclosed*heterogeneity in shape and size*high electron density Classical morphology: dense,round,and small compartment.Shape and structure of lysosomeEM*0.2-0.5M15acid h

16、ydrolases40types hydrolytic enzymes* Membraneglucosylated membrane proteins protect themselves from protease in the lumenH+ pumpATP hydrolysis and pump H+ into the lysosomes*Lumenacid environment, pH=4-5 16*Sorting of newly synthesized lysosomal hydrolases from the trans Golgi network (TGN) to lysos

17、ome via endosome.*Delivering digested materials from extracellular fluid by endocytosis to lysosme via endosome.Multivesicular bodyLysosomal enzymesendocytosed materialsFormation of lysosome (misterious)1717a.PhagocytosisEndocytosis(胞吞） in lysosome formationb.Pinocytosis Receptor-mediated endocytosi

18、sSpecial form of endocytosis in which a cell uses large endocytic vesicles to ingest large particles such as microorganisms and dead cells. *In professional phagocytes-macrophages and neutrophils.A form of endocytosis in which a cell continually ingests bits of their plasma membrane and extracellula

19、r fluid in the form of small pinocytic vesicles. *In all eucaryotic cells.A form of endocytosis in which a cell takes up specific macromolecules bound with plasma membrane receptors from the extracellular fluid such as cholesterol and some kinds of signaling molecules. *In most of animal cells.18mul

20、tivesicular bodylate endosomeTransport vesiclesEndosome（内体）Early endosome Small,irregularly shaped organells, containing endocytosed molecules. The sites where the lysosomal enzymes transport to from TGN.Late endosomeMildly acidic interior. The sites where the hydrolytic digestion of endocytosed mol

21、ecules begin.Endosome is a series of special organelles correlated with endocytosis and extracellular protein and plasma membrane protein degradation. Maturation19Multivesicular bodyLysosomal enzymesendocytosed materialsendocytosisLysosomal protein sorting+2020multivesicular bodylate endosomeTranspo

22、rt vesicles Degration of proteins from endocytosis in lysosomedegraded protein:*extracellular materials;*plasma membrane proteins Function of lysosome21The process begins with the enclosure of the organelles such as mitochondrion, ER by a double membrane of unknown origin, called autophagosome.Autop

23、hagy is one of the major intracellular degradation process in which cytoplasmic materials are delivered to and degraded in lysosome to get cellular renovation and homeostasis. Degration of intracellular proteins by autophagy in lysosome22Mitochondrion damagedOxidative stressCell fateExample1: degrad

24、ation of damaged mitochondrionExample2: degradation of cytosolic protein p62P62 degradationUbiquitously expressed cellular proteinAutophagy impairmentDiseaseExample3: Autophagy in nutrient-starved cellsautolysosomeautophagosomeER fragmentsmouse embryonic fibroblastautophagyapoptosisnecrosis23The fun

25、ctional implications of autophagyElimination of cytoplasmic materials to get cellular renovation and homeostasis.a.Remove of senescent and damaged organells;b.Produce new building blocks and energy for turnover of cytoplasmic components.Steady state of the cell (basal level)c. Save energy and get nu

26、trients from itself for cell survival under starvation.24Autophagy in health and diseaseStress state of the cellImpairment or activation of autophagy contributes to pathogenesis of diverse diseases. from neuron-degenerative disease such as Parkinson disease to inflammatory disorders such as Crohns d

27、isease.The functional implications of autophagy2525Ref: Physiological and pathological of Autophagy26Take-home messages:/pmc/articles/PMC3252826/2.Autophagic cell death?1.How can we interfere autophagy to treat disease?Potential inference method: small molecules, genetic me

28、thods or anything else?27* cytoplasmic components* the organelles proteinWhich kind of proteins are degraded in lysosome by autophagy?Which kind of proteins are degraded in lysosome by endocytosis?* extracellular proteins* plasma membrane proteins28Elimination of extracellular materials;Production o

29、f nutrients;Production of new building blocks for cell renovation;Regulation of hormone secretion（crinophagy）The functional implications of lysosomal degradation system for cell 29Pathology of Lysosome*Pneumoconiosis(尘肺）、 Rheumatoid Arthritis（类风湿）*Lysosome storage disease( LSD ） esp. Inclusion cell

30、disease（I细胞病） ; Gaucher diseasehttp:/ diseaseEnlarged liver and spleen/wiki/Gaucher%27s_diseasehttp:/ Lysosomes are specialized for the intracellular digestion of macromolecules. 2. Lysosomes contain unique membrane proteins and a wide variety of soluble hydrolytic enzymes that

31、 operate best at pH 5,which is the internal pH of lysosomes. An ATP-driven H+ pump in the lysosomal membrane maintains this low pH. 3. Newly synthesized lysosomal hydrolases are transferred into the lumen of the ER, transported through the Golgi apparatus, and then from the trans Golgi network to ea

32、rly endosomes by transport vesicles. 4. Autophagy is the major intracellular degradation system by which cytoplasmic materials are delivered to and degraded in lysosome to get cellular renovation and homeostasis.Much of the material that is endocytosed is delivered to endosomes and then to lysosomes

33、, where it is degraded by hydrolytic enzymes.Essential Concepts31II、Chapter 7.Structure and function of organelles involved in degradation of proteins3232Q1.Why protein degradation is ATP-dependent? The Cell - a teeming mini-workshopIn the cell, proteins are being built up and broken down all the ti

34、me. For everything to function optimally, the cell also has an integral checkpoint where the composition of various proteins is controlled. Unlike in the spontaneous protein breakdown that food undergoes in our intestines, breaking down proteins inside cells requires energy. This was long a research

35、 mystery. 3333* size：20S、26S invisible under LM or EM* non-membrane enclosedComparasion: Ribosome(EM)Ribosome：80S，15-20nmMorphology of proteasome* in both of the nucleus and cytoplasmStaining with proteasome fluoresence marker（Sun X，Yan S et al. unpublished）3434Chemical composition：protein complexes

36、 Structure：barrel-like(cylinder)20S core core particles“proteases active sites”19S cap regulatory particles “recognization sites”Structure of proteasome3535Example 1：Cyclin G1 cyclinSynthesis in G1,Degradation in S phaseExample 2 ：p53 p53p53* non-stress (Resting) : low level P53 (unneede ,degradatio

37、n) MDM2MDM2MDM2To rapidly degrade unneeded or damaged proteinsFunctions of proteasomes36Example 3 ：damaged protein misfolded proteinsProtein quality control in ER and proteasomeTo rapidly degrade unneeded or damaged proteinsFunctions of proteasomesER lumenproteasome37The Cell - a teeming mini-worksh

38、op. Thanks to this years Nobel Laureates, however, we know that this form of breakdown is an extremely detailed control process in which the protein to be destroyed is marked with a special label. This happens through a series of chemical reactions, as shown below.The Nobel Prize in Chemistry 2004ht

39、tp://nobel_prizes/chemistry/laureates/2004/illpres/2_cell.htmlproteins degradation in proteasomeQ2.Who decide certain protein need to be degraded?38ubiquitin:a small peptide of 76 aa, ubiquitous and conserved.Ubiquitin-linked protein: ubiquitinated protein, ubiquitination modificat

40、ion of proteinAs a tag, at least four ubiquitin peptides covalently bind to the target(substrate) proteins for teins degradation in proteasome(ubiquitin-proteasome system, UPS) proteinubiquitin-linked proteins 39391.Proteins are linked by polyubiquitin (polyubiquitinated) E1:Activatin

41、g enzyme E2:Conjugating enzyme E3:ubiquitin Ligase2.Recognization by “cap”3.Translocation into the core4.Digestion to short peptides or amino acid by teins degradation in proteasomeATP-dependentATP-dependent40* ATP consumption * Ubiquitin recognization * A1(why). ATP dependent protein d

42、egradation* A2(who). Ubiquitin-mediated protein degradationproteins degradation in proteasome4141To rapidly degrade unneeded or damaged proteins/nobel_prizes/chemistry/laureates/2004/animation.htmlproteins degradation in proteasome for cellsBiological function4242Resting: p53

43、 degradation, low level P53Stress: decrease of p53 degradation, high level p53 DNA damageExample 2 ：p53, “stress sensor”p53-UbSubstrate protein：MDM2, p53E3 ligase for p53 MDM2P53 accumulationCell cycle arrestDNA repairGene expressionp53p53MDM2MDM2MDM2P53 degradationMDM2 degradationUbUb4343Bortezomib

44、 (Velcade), inhibitor of proteasome becomes anticancer drug. 1.Clinical treatment of multiple myeloma. (approved by FDA in 2003) 2.Preclinical trial: B-cell cancers 3.Animal trial: pancreatic cancerLight Blue: Bortezomib，Binding to subunits of 20S coreproteins degradation in proteasome in disease4444The Nobel Prize in Chemistry 2004“Thanks to the work of the three Laureates it is now possible to understand at molecular level how the cell controls a number of central processes by breaking down certain proteins and not others. Examples of processes