Protein Post-Translational Modifications Summary Case 6 PDF

**How are proteins modified (post-translation) ?** Protein posttranslational modifications (PTMs) refer to the breaking or generation of covalent bonds on the backbones or amino acid side chains of proteins and are also called covalent modifications *[Folding (ER, molecular chaperones)]* - Prim...

**How are proteins modified (post-translation) ?** Protein posttranslational modifications (PTMs) refer to the breaking or generation of covalent bonds on the backbones or amino acid side chains of proteins and are also called covalent modifications *[Folding (ER, molecular chaperones)]* - Primary structure -\>chain of amino acids - Secondary structure -\>proteins can fold=\>repeating patterns occur, reactions in the backbone =\>2 most common secondary structures - Alpha helix 1 protein chain twists=\>shape resembles a right-handed coil =\>maintained by numerous hydrogen bonds (carboxyl- and nitrogen-groups) in the backbone - Beta pleated cheat Can occur when polypeptide chains run parallel =\>maintained by intermolecular and intramolecular hydrogen bonds - Tertiary structure 3D arrangement, interaction of side chains and not only the backbone =\>stabilized in 5 ways 1. Covalent bonds 2. Hydrogen bonding 3. Salt bridges 4. Hydrophobic interactions 5. Metal-ion coordination - Quaternary structure =\>for proteins with \>1 polypeptide chain =\>genes have specific binding sites and bind to each other =\>determines how the different sub-units fit into an organized whole =\>subunits-\>packed+ held together by.... - Hydrogen bonds - Salt bridges - Hydrophobic interactions =\>10^78^ different proteins with 20 amino acids -\>folding done by chaperones (heat-shock proteins, BIP's,....) =\>protein folded correctly by chaperones=\>in vesicle transported to Golgi =\>NOT folded correctly by chaperones=\>chaperones try to fold again=\>successful? = still transported to Golgi, NOT successful?= degraded by proteosome and amino acids are reused. =\>proteins are not able to be degraded =\> accumulation, protein aggregation= degenerative diseases like Alzheimer occur *[Other ptm's ]* - Glycosylation (ER, Golgi) - In ER=\>sugar tree placed on protein (different sugars) - In Golgi-\>protein with sugar is refined - ! Protein stability ! - Underpins ABO blood group system - Defects in glycosylation cause congenital disorders - Glycosylation= used by viruses to hide viral protein for immune recognition by host. - (de-)phosphorylation (deactivation, activation of proteins) - Adding a phosphate group (from ATP) by **kinases** to protein (switched on ) - Removing a phosphate group by **phosphatase** to protein (switched off) - !!! process can work the other way around !!! (removing = protein switched on and adding is protein switched off) - Example= insuline (glucose in cell), insuline binds to receptor=\> activated and glucose in plasma membrane - Proteolysis (removal of methionine, peptide,....) Example with insulin: [pre-form] of insulin, held together by disulfide bridges -\>proteolysis: removing the bigger part=\>form insulin =\> hydrolysis reaction of peptide bonds =\>proteins break down in smaller peptides or amino acids - Hydroxylation (structural integrity) *[Signal sequence ]* Based on amino acid sequence-\>proteins directed to their destination  *[Intracellular protein transport/protein sorting ]* *overview of where the different kinds of transport take place\ * - Gated transport =transport between 'equal' compartments, same environment (e.g. from nucleus to cytosol) -\>transport via [pores (part of nuclear envelope) ] =\> transport of protein through nucleopore depends on size \-\-- small molecules: free diffusion \-\-- larger molecules (macromolecules) : enter by *active transport* - Active transport regulation - Transmembrane transport =transport via protein translocators -\>transport between compartment with [different environment] (e.g. cytosol-\>mitochondria,ER,...) - Cytosol-\>mitochondria =\>import from cytosol to mitochondria-\> use of TIM- and TOM-complex 1\) TOM-complex = helps transport through outer membrane 2)TIM-complex = helps transport through inner membrane  Afbeelding met tekst, schermopname, Lettertype, lijn Automatisch gegenereerde beschrijving -\>protein binds to import receptor in TOM-complex =\>translocation through first membrane -\>TIM-complex and signal peptide take over =\>translocation protein into matrix mitochondria -\> release of signal peptide (cleavage by signal peptidase) - Cytosol-\>ER -\>ribosomes bound to ER =\>translation in ribosomes=\>signal sequence appears  Afbeelding met kaart, tekst, diagram Automatisch gegenereerde beschrijving -\>signal sequence pops up, SRP (signal recognition particle), binds to ribosome=\> translation stops and complex is brought to SRP (signal recognition particle) receptor protein on the membrane of ER -\>SRP disconnects from ribosome as it connects to SRP receptor-\>translation continues, translocation trough membrane starts, done by protein translocator, signal peptide is cut of by signal peptidase, protein stuffed in the lumen of the ER =\>secretory proteins: proteins that's not attached to membranes, should be generated and sent out of the cell. =\>protein in ER-lumen= packed in vesicle, sent to plasma membrane, release and secretion of protein. =\>membrane proteins: same steps until it reaches stop-transfer sequence, translation continues, signal peptidase is cut off =\> result= half of the protein in the membrane and other half not= transmembrane protein. ====transmembrane protein= alternating stop- and start-transfer sequences Result= sort of like sewing machine, proteins woven trough membrane  !!! difference between ER-bound ribosomes and free ribosomes -\>transport protein the same way, free ribosomes= only dragged to ER-membrane if signal sequence for ER-specific proteins is found. Afbeelding met tekst, schermopname, diagram, Lettertype Automatisch gegenereerde beschrijving  - Vesicular transport Different type of vesicles: vesicles coated with clatherin, COPI, COPII Overview roles: Afbeelding met tekst, diagram, kaart, Lettertype Automatisch gegenereerde beschrijving - Budding **How does the protein transport influence these diseases?** **Gaucher's- and I-cell disease** 1\) receptor in plasma membrane -\>captures substrate that should be brought into cell 2\) use of clathrin to make clathrin proteins (adapter proteins) 3\) adapter proteins surround the plasma membrane that includes the substrates 4\) everything is brought into the cell 5\) release of clathrin and is reused 6\) result= naked transport vesicle (contains receptors and substrate) [= import from something to the inside from the outside ] - How do the vesicles know where to go? =\>special phospholipids in membrane =\>PIP's (Phosphatidylinositol or inositol phospholipid) -\>modifying the different inositol molecules with different phosphates =\>all different types are part of membrane -\>used by proteins -\>together with vesicle sent to the right direction Coating vesicle + composition of vesicle membrane = important for directing to right direction. Afbeelding met tekst, schermopname, clipart, tekenfilm Automatisch gegenereerde beschrijvingdifferent type of cytosis use different types of PIP's -\>RAB-proteins different type of RAB-proteins for different types of organelles e.g. vesicles with RAB-protein 1 travel between ER and Golgi - General vesicular transport =\>V-snare on vesicle should make connection with t-snare on target membrane 1\) specific RAB-effector proteins capture vesicles with a specific RAB-protein 2\) V-snare makes connection with t-snare 3\) fusion of vesicle - Golgi-apparatus Different processes in the different parts of Golgi  Function in vesicular process= sorting station - Consecutive secretory pathway and regulated secretory pathway -\>consecutive= randomly dumping the proteins -\>regulated= pathway also depends on signal of hormone or neurotransmitter (=additional level of control instead of only coating, PIP's and RAB) **Function of proteins in the body** - Structure (hair, nails etc.) - Enzymes - Movement (muscles) - Transport (e.g. O~2~ membrane transport) - Hormones (e.g. insulin) - Nutrition (e.g. casein milk) - Protection ( blood clothing, antibodies,...) - Cell regulation (signal from outside cell-\>receptors , signal transduction-\> gene expression) **Death of protein** =\>Use of proteases (protein degradation systems) - Ubiquitin= death sentence protein -\>degradation signal is recognized by ubiquitin ligase and binds to protein - Proteosome -\>target protein [with ubiquitin chain] goes to proteasome =\>proteasome shreds the protein to amino acids -\>amino acids are reused Afbeelding met tekst, diagram, Lettertype, kaart Automatisch gegenereerde beschrijving =\>20 amino acids -\>we receive them from proteasome and nutrition **How does the protein transport influence these diseases?** **Gaucher's- and I-cell disease** *[Gaucher's Disease]* Gaucher's disease is a lysosomal storage disorder (LSD) caused by a deficiency of the enzyme β-glucocerebrosidase (also called glucocerebrosidase or G Case). This enzyme is responsible for breaking down a specific lipid called [glucocerebroside]. When β-glucocerebrosidase is defective or absent, glucocerebroside accumulates in lysosomes, particularly within macrophages, leading to the characteristic symptoms of the disease, including enlarged liver and spleen,bone abnormalities, and neurological issues. *[Protein Transport Influence]* \- In Gaucher's disease, mutations in the GBA gene encoding β-glucocerebrosidase affect the enzyme's folding, stability, and transport. \- Misfolded β-glucocerebrosidase is recognized by the quality control mechanisms in the ER and may be retained in the ER or degraded via the proteasome (ER-associated degradation, or ERAD), preventing its proper delivery to lysosomes. \- Even when β-glucocerebrosidase reaches the lysosome, its reduced functionality can hinder lipid breakdown, leading to lysosomal dysfunction and substrate accumulation. \- Some therapies, such as enzyme replacement therapy (ERT), involve providing recombinant β-glucocerebrosidase that is efficiently transported to lysosomes to compensate for the defective endogenous enzyme. *[I-Cell Disease ]* I-cell disease is another lysosomal storage disorder, but it stems from a different defect in protein transport. It is caused by mutations in the GNPTA gene, which encodes the enzyme N-acetylglucosamine-1-phosphotransferase. This enzyme is crucial for tagging lysosomal enzymes with a mannose-6-phosphate (M6P) marker in the Golgi apparatus, a signal that directs these enzymes to lysosomes. *[Protein Transport Influence]* \- In I-cell disease, the defective N-acetylglucosamine-1-phosphotransferase enzyme prevents the M6P tagging of lysosomal enzymes. \- Without the M6P tag, lysosomal enzymes are mistakenly secreted outside the cell instead of being transported to the lysosome. \- The absence of these crucial enzymes in lysosomes leads to the accumulation of undigested substrates (such as lipids and carbohydrates), resulting in the formation of \"inclusion cells\" (I-cells) and a range of severe symptoms, including developmental delay, skeletal abnormalities, and organ enlargement. \- This misrouting of enzymes represents a global defect in lysosomal function since nearly all lysosomal hydrolases are affected. *[Comparison of Gaucher's and I-Cell Disease]* While both diseases involve lysosomal dysfunction, the key difference lies in the stage of protein transport: \- In Gaucher's disease, the issue is with a specific enzyme (β-glucocerebrosidase) being misfolded and not reaching the lysosome, leading to substrate accumulation within macrophages. =\>early degradation (improper folding) \- In I-cell disease, a broader defect in the lysosomal targeting mechanism prevents many enzymes from reaching the lysosome, causing widespread substrate accumulation.