Intracellular Protein Degradation Lecture PDF

Intracellular Protein Degradation Dr. Deeba S. Jairajpuri, Ph.D Overview Intracellular Protein Degradation: ● Permits the cell a constant supply of amino acids ● Prevents the accumulation of abnormal proteins Protein Degradation Turnover of protein is NOT constant or same for all proteins means...

Intracellular Protein Degradation Dr. Deeba S. Jairajpuri, Ph.D Overview Intracellular Protein Degradation: ● Permits the cell a constant supply of amino acids ● Prevents the accumulation of abnormal proteins Protein Degradation Turnover of protein is NOT constant or same for all proteins means, there is an huge variation in the half-life of individual proteins and it is a direct reflection of their role within the cell. “Normally” proteins – 100-200 hrs Short-lived proteins Loading… regulatory proteins enzymes that catalyze committed steps transcription factors Long-lived proteins Special cases (structural proteins, crystallins) Protein Degradation • May depend on tissue distribution Example: Lactic Acid Dehydrogenase Tissue Half-life Heart 1.6 days Muscle 31 days Liver 16 days • Protein degradation is a regulated process Example: Acetyl CoA carboxylase Nutritional state Half-life Fed 48 hours Fasted 18 hours Protein Degradation Occurs in several compartments: Does not & play a big role -Lysosomes(Cell organelles)-acidified compartment:10-20%; Extracellular proteins and some intracellular proteins requires a Loading… certain Protein - Cytosol-ubiquitin dependent proteosome 2 machinery system: 80-90%; Most intracellular proteins & Structural proteins are long lived Lysosomal protein degradation ● Lysosomes are organelles that contain digestive enzymes such lipases, nucleases, and proteases ●Their pH is acidic (pH=4.8) ●It is a process by which vesicles are formed autophagosomes that engulf small amounts of cytoplasm or specific organelles using portions of Endoplasmic Reticulum membranes. ●Autophagosomes fuse with lysosomes results in the release of lysosomal hydrolytic enzymes causing the degradation of the macromolecules. Proteosomal degradation -This ATP-dependent pathway is responsible for the breakdown of most short lived proteins in human cells. For example, in skeletal muscle, the proteosomal system is responsible for the breakdown of the major contractile proteins, actin and myosins. - In addition, the proteosomal pathway also controls various major biological events: cell cycle progression, transcriptional control, cell development and differentiation, signal transduction…, via the breakdown of specific proteins (p53, Rb, cyclins, CDK inhibitors, transcription factors…..) How are proteins selected for degradation? Ubiquitin: proteins destined for degradation (called as substrate) are tagged by covalent attachment of ubiquitin and then degraded by proteosome ➢ ➢ ➢ ➢ ➢ UBIQUITIN Ubiquitin is a highly conserved protein 76 amino acids C-terminal glycine of ubiquitin bond with the lysine residue on the substrate (protein destined for degradation) It can get Attached to the substrate as monoubiquitin or polyubiquitin chains Attachment is performed by array of enzymes (E1, E2, E3) G K ➢ Ubiquitination of proteins is a Three-step process First, Ubiquitin is activated by forming a link to “enzyme 1” (E1: ubiquitin activating enzyme). ➢ Then, ubiquitin is transferred to the “enzyme 2” (E2: ubiquitin conjugating enzyme). ➢ Then, “enzyme 3” (E3: ubiquitin ligase) catalyzes the transfer of ubiquitin from E2 to a Lysine amino group of the “condemned” protein. ➢ Condemned protein also mean substrate i.e. protein destined for degradation. AMP STEPS IN PROTEIN UBIQUITINATION Basic features of proteasome Degrades most of cytoplasmic, nuclear and membrane proteins (> 90 %) Ubiquitin is recycled, not cleaved Loading… Eukaryotic proteosomes are large protein complexes of ~ 2000 kDa, consisting of a “core” and a “cap” region PROTEASOME COMPONENTS ●The 26S proteosome is a large complex of proteins. ●Structurally resembles a large cylinder: it contain a central core (20S) and 19S regulatory particle at either end. The central core is barrel shaped formed of four rings. ●The 20S is formed of 4 rings: the outer are formed from 7 alpha subunits and the inner ring is formed from 7 beta subunits. ●The 19S regulatory particle is important for recognition and binding of polyubiquitinated proteins, removal of ubiquitin, unfolding the protein substrate, and translocation(movement) into the central core. ●The unfolded proteins are then hydrolyzed within the central core into smaller peptides. These peptides emerge from the opposite end of the 20S particle and are further degraded by cytosolic peptidases 20S Proteasome 19S Particle ATP 26S Proteasome Degradation of proteins in proteosomes Regulation of ubiquitination Ubiquitination is a highly regulated process which is important for protein degradation. While several ubiquitin molecules have to be added to the protein destined for degradation, some proteins appear to be monoubiquitinated and this type of regulation determines protein function. The addition of ubiquitin to some proteins serves other functions. For example, the addition of single ubiquitin molecules to some proteins is involved in regulation of DNA repair, transcription and endocytosis. A minimum of four ubiquitin molecules on the target protein are critical for efficient degradation of the protein. Deubiquitination enzymes (DUBs) Eukaryotic cells also contain DUBs (DeUBiquitinating enzymes), which are encoded by the UCH (Ubiquitin Carboxyl-terminal Hydrolases) and the UBP (UBiquitin-specific Processing proteases) gene families. UCHs are relatively small proteins (< 40-kDa);in contrast, UBPs are 50-250kDa 8 proteins and constitute a large family. DUBs can reverse the degradation effects by cleaving the peptide or isopeptide bond between ubiquitin and its substrate protein. Deubiquitinating