Protein Modifications Lecture Notes PDF

Protein modifications (ID# 7264) 1 10/3/2022 WLO Describe the mechanism and significance of protein modification in relation to protein secretion and targeting to different intracellular sites and clinical significance of these processes. (WLO 5044) 2 10/3/2022 References 1. Biochemistry by...

Protein modifications (ID# 7264) 1 10/3/2022 WLO Describe the mechanism and significance of protein modification in relation to protein secretion and targeting to different intracellular sites and clinical significance of these processes. (WLO 5044) 2 10/3/2022 References 1. Biochemistry by Stryer 2. Handouts 3 10/3/2022 Significance Due to genetic mutations, defective proteins / enzymes are produced which are mis-targeted to wrong sites as they cannot reach their target site. Mistargeting leads to deficiency of enzyme or protein. Many diseases happen due to such defects. Why? 4 10/3/2022 Some common protein modifications mRNA (Translation) 1o protein (Not active, needs to undergo modification) Proteins are activated by one of these modifications 1. Cleavage Digestive enzymes, Preprohormones 2. Hydroxylation Collagen = Strength 3. Phosphorylation (>30% of all proteins are phosphorylated) (Addition of phosphoryl Lysosomal enzymes, Transcription factors group to protein) 4. Glycosylation (>50% of all proteins are glycosylated) (Addition of sugar Membrane, Secretory proteins group to protein) 5. Lipidation (Co-translation, addition of lipid to protein) Lipids are membrane anchoring molecules, lipoproteins 6. Acetylation (80-90% of all proteins are acetylated) histones are proteins that the DNA is Histone proteins (Post-translation, wrapped around, so adding an acytel group to these proteins will activate them. They are related to gene expression,) 10/3/2022 5 Proteins reach different sites in a eukaryotic cell PM retained Cytosol rER retained Nucleus Mitochondria Peroxisome Post-translational modifications Category-1 Lysosome Co-translational modifications Category-2 How proteins reach their target site? (Modification occurs after translation) (Modification occurs during translation) - Proteins synthesized on free ribosomes will remain inside the cell and will go to the nucleus, mitochondria, peroxisome, or cytosol. - Proteins INITIALLY synthesized on free ribosomes (then bind to —> If proteins don’t reach their target sites —> Disease RER) will go to RER membrane, Golgi apparatus membrane, plasma membrane, lysosome, or leave the cell (secretory) 10/3/2022 How proteins reach their target site? 1. Targeting sequences embedded in the structure of certain proteins serve as addresses (Part of the protein) 2. Specific modifications in some proteins which are important in achieving proper-folding, stability and activity help these proteins reach their targets (The protein is activated or becomes functional only after modification) 7 10/3/2022 Proteins are placed into two categories Based on the location of their synthesis and their targets Category-1 Category-2 Free ribosome ←Ribosomes attached to rER ← rER • Synthesis starts and completes on the free ribosomes • Post-translational modifications • Synthesis starts on the free ribosomes, but completes in the rough ER • Co-translational modifications Category-1 proteins Category-1 proteins & their targets Free ribosome 1. Mitochondria 2. Nucleus 3. Peroxisomes Proteins 4. Cytosol (Amine/Nitrogen End) (Carboxyl End) • Synthesized on the free ribosomes, and after synthesis, they reach their sites • Have specific ‘Targeting sequence’ as a part of their structure (On N-terminus or C-terminus) • Translocation of these proteins requires chaperones & receptors (TS alone is not enough by itself, it needs chaperones, which protect proteins, and receptors to reach its target site) What is a ‘Targeting sequence (TS)’ & its location in these proteins? 10 10/3/2022 10/3/2022 1. Mitochondrial proteins’ targeting sequence N Targeting sequence (Tells protein to go to mitochondria) C • TS is present as N-terminal sequence in these proteins • For translocation, these proteins bind with chaperone HSP70, and receptors on the mitochondrial membrane • Binding to mitochondria leads to the opening of the membrane channels through which they move into the matrix • After reaching the matrix, the TS is cleaved, protein folds & becomes active (Post-translation) 11 If modifications (cleavage and folding) don’t occur, the protein remains in cytosol inactive —> Disease 2. Nuclear proteins’ targeting sequence N C Targeting sequence (Different than mitochondrial TS) Nucleus • Smaller proteins can diffuse freely through nuclear pores / channels • Larger proteins are imported through the nuclear pores and require energy • TS is N-terminal in these proteins • They require receptors to move through nuclear channels • After reaching the nucleus the protein becomes active, TS remains intact (No cleavage) (Organelle in cell) 3. Peroxisomal proteins’ targeting sequence N S K F C Targeting sequence Peroxisomes • TS is a-three aa residue (-SKF-) C-terminal sequence • These proteins also require binding proteins & receptor for their translocation • Mutations in the ‘TS’ mistarget these proteins to the cytosol rather than to their actual sites and cause peroxisomal diseases S=Serine, K=Lysine, F=Phenylalanine 13 10/3/2 022 4. Cytosolic proteins lack targeting sequence Peroxisomes • Cytoplasmic proteins do not have a targeting sequence • Therefore, proteins without target sequence are retained in the cytoplasm by default Cytosol Nucleus 14 10/3/2022 Category-2 proteins Category-2 proteins & their targets Synthesized on the rER and targeted to the following sites: 1. Membrane 2. Lysosome 3. Secretory (Embed in ER membrane) 4. ER resident (Embed in GA membrane) 5. Golgi resident 16 Co-/post-translational modification 10/3/2022 Category-2 proteins free ribosomes Ribosomes Rough ER • Protein synthesis starts on the free ribosomes • Ribosomes do not have modifying enzymes Proteins will not complete their synthesis on free ribosomes because the ribosomes don’t have modifying enzymes, so the ribosomes will bind to the RER Cis • Modifying enzymes are present in the rER & Golgi Golgi Trans • Therefore, ribosomes must couple to rER What is the ‘signal’ for coupling? ‘Signal peptide’ is a coupling signal SP→ SP→ rough ER Only those ribosomes which are engaged in the synthesis of proteins having an N-terminal signal peptide (SP) are attached to the rER (Rough because of these ribosomes) What is a signal peptide? 18 10/3/2022 Characteristics of the ‘signal peptide (SP)’ (Will be cleaved later) Human growth hormone- Human proinsulinBovine proalbuminMouse antibody H chain- Chicken lysozymeBee promellitinDrosophila glue proteinZea maize protein 19- Yeast invertase- • SP is present at N-terminus • The size & composition of SP vary between proteins • SP is not consensus • The middle of SP sequence is hydrophobic • SP is important & sufficient for the coupling of ribosomes & rER Human influenza virus- What is the mechanism of coupling? Highlight: Hydrophobic - This property allows passage into the ER membrane, as the membrane is also hydrophobic 10/3/2022 Mechanism of ribosome coupling to rER • SP binds to a ‘signal recognition particle’ (SRP) • SRP is made of small RNA (300 nt) & 6 different proteins • SRP-SP complex inhibits the translation and binds to SRP receptor on the rER • SRP then hydrolyzes GTP, & dissociates from the receptor • In some cases, the ‘SP’ is cleaved in the ER by signal peptidase • Therefore, ‘SP’ is not present in many mature proteins CAP SRP is removed and recycled Translocating channel (The inhibition occurs temporarily to prevent any possible misfolding. The SRP brings down the SP to the ER membrane) SP cleaved Proteins are modified inside the lumen (If the SP is not cleaved, it will still reach the site, but the mutation itself may cause a problem) (This results in a conformational change, and translation is reinitiated at the ribosome receptor) 10/3/2022 Integral membrane proteins require additional (Also to know which part of the protein has to pass signal sequences the membrane and where the protein has to reside) Membrane proteins Lysosomal proteins rER Secretory proteins Golgi ER • These proteins are synthesized as part of will detach from ribosome the rER membrane (They and fuse to membrane) • However, they require additional signals such as ‘topogenic’ & ‘anchor sequences’ for crossing & anchoring into the lipid bilayer • Other proteins are synthesized and modified in the ER lumen • Only the intracellular epitope in these proteins are glycosylated or modified ( ) Only the part inside the ER lumen will be modified (as the cytosol does not have modifying enzymes) 10/3/2022 (To make the protein functional) Types of modifications in the rER 1. 2. 3. 4. 5. 22 Formation of –S-S- bonds Oligomerization N-linked glycosylation Folding of proteins Quality control S Chain 1 S Chain 2 Disulfide bond • Disulfide bond (-S-S-) is important for protein-folding • It may be formed intra-chain or interchain (Within the same chain) • Interchain –S-S- holds two polypeptides together → Oligomerization • -S-S bond formation is catalyzed by glutathione oxidase 10/3/2022 3. Protein glycosylation (Not simple/monosaccharides) • Complex carbohydrate moieties are selectively added to certain aa in a polypeptide • Glycosylation is of two types: (Based on type of aa and where it happens) 1. N-Linked glycosylation (Initiated in RER, completed in Golgi apparatus) Occurs at -NH2 group in Asn (N) 2. O-linked glycosylation (Only occurs in Golgi apparatus) Occurs at -OH group in Ser & Thr 23 10/3/2022 3. N-linked glycosylation • It occurs only in the ER because the enzymes are present only in the ER • It does not occur in the cytoplasm (No modifying enzymes in cytosol) Asn • It starts in the rER and completes in Golgi • The part of glycosylation that completes in the Golgi → ‘terminal glycosylation’ • Carbohydrate moiety is attached to the -NH2 group of a selected Asn in the Asn-x-Ser/Ther in a primary protein Asn (Not every Asn aa will undergo N-linked glycosylation; only those following this sequence: Asn, then any random aa, then SER or Ther) 24 10/3/2022 4. Protein folding Polypeptide BIP ER lumen • Because of a very high protein density in the rER, proteins are susceptible to form aggregations due to wrong proteinprotein interactions • To avoid this, rER has abundance of (Help proteins fold properly in chaperones such as heat shock proteins high density ER) (hsp) hsp40, 60, 70, 90 & 100, binding protein (bip), calnexin & careticulin • Chaperones prevent incorrect folding, denaturation & aggregation • Chaperones recognize the exposed (An exposed hydrophobic aa is an of wrong folding, so hydrophobic aa in a protein indication chaperones recognize and bind to it) • Chaperones have ATPase activity (Use ATP to power their reactions) 25 10/3/2022 5. Quality control Cis Correctly folded X Mis-folded • In this process only the correctly folded proteins are allowed to move from rER to the Golgi for Golgi sacs further modifications • Mis-folded proteins can not move to Golgi, and are degraded in the Trans (30% of proteins are mis-folded AND pass the quality control, so that are proteasomes don’t degraded/recycled by these organelles) • The movement of proteins (protein trafficking) occurs via membrane bound globular ‘vesicles’ What is the sensor that recognizes an incorrectly folded protein? (From RER to Golgi Apparatus) 10/3/2022 Mechanism of quality control Contains correctly folded proteins coming from RER (The glucose molecule acts as a marker for the “chaperone police” to recognize and correct mis-folding) ER lumen Chaperone A correctly folded protein moves to Golgi After correct folding, the protein is activated Proteasome 3.Protein folds correctly ER membrane 3 4 4. Protein does not fold correctly. Such proteins are degraded in the proteasomes (Sensor protein) •Glucosyl transferase (GT) recognizes a misfolded protein •GT binds with the exposed hydrophobic aa & adds a glucose (G) unit to a protein •Calnexin captures and allows it to fold (Sensor protein) •Glucosidase II (G-II) recognizes a correctly folded protein with ‘G’ & removes ‘G’ to allow protein moves to Golgi 10/3/20 22 Mechanism of quality control Glucosyltransferase recognizes incorrect folding 1. An Incorrectly folded protein is tagged with a Glucose (G) moiety by glucosyl transferase 2. G-tagged protein is then captured by calnexin which then allows it to fold 3. If the protein folds correctly in the calnexin, Glucosidase II removes the glucose tag from the protein and allows it to exit & join the Golgi 4. If a protein fails to fold correctly, it is sent to proteasome for hydrolysis in the cytoplasm 28 10/3/2022 Modifications in Golgi Types of protein modifications in Golgi 1. Terminal glycosylation (N-linked glycosylation completes) 2. O-linked glycosylation (occurs at the –OH group of Ser/Thr) 3. Sorting/packaging (from trans Golgi net work) 30 moves proteins to different targets Trans Cis (receive proteins from ER) 10/3/2022 O-linked glycosylation • It starts and complete in the Golgi apparatus • It occurs at the -OH group of Ser & Thr in a 1o protein • Only a few selected Ser & Thr aa in a 1o protein are glycosylated (Not every Ser and Thr will modified) Ser Thr 31 10/3/2022 Side Note: Review Physiology Lecture 5 to know about the Clathrin Coat and pathway of lysosomal proteins Lysosomal proteins are sorted by a M6P-receptor PM → Extracellular • These proteins carry mannose-6P (M6P) as a marker (Inside Trans Golgi) • M6P receptor (MPR) sorts these Clathrin coated vesicle→ M6P on these proteins (M6PR binds then sorts) • Sorted proteins exit via clathrin coated vesicles which fuse with 2. M6PR Coat removed before fusion Trans Golgi → (minor pathway) the endosomes • In the endosome due to acidic pH, the enzyme & MPR fall apart ---M6PR • MPR is recycled to the Golgi in a Lysosomal pH 4-5 ← early major pathway (Majority of MPR recycled to Golgi) + protein endosome • MPR is also recycled to the PM in (Mature to late endosomes a minor pathway which retrieves 1. M6PR then bind to lysosomes to release the proteins) mannose-6-P the escaped lysosomal proteins (major pathway) (Some proteins can wrongly leak or escape and leave the cell, so this mechanism is needed to bring them back) Lysosomes are important to degrade and recycle materials including wastes or poisons. If these modifications fail to occur or the proteins don’t reach the lysosome, accumulation of wastes will occur resulting in lysosomal disease 32 10/3/2022 Retention and retrieval of ER resident proteins (4 amino acids that prevent RER proteins from escaping) ER retrieval escaping ←KDEL-receptor N- -KDEL NGolgi -KDEL • rER proteins have KDEL sequence at their C-terminal end • Any protein having KDEL sequence is retained by the rER • Some rER resident proteins (Leak) escape to Golgi from where they are retrieved into rER (In a pH-dependent manner) Retrieval of ER proteins from Golgi retrieval K=Lys D=Asp E=Glu L=Leu vesicle ER (Due to acidic environment) High affinity KDEL receptor KDEL KDEL ER resident proteins KDEL (pH=5.5) vesicle • They bind to a high affinity KDEL- receptor in the Golgi (acidic pH) and exocytosed • In the rER due to higher pH the receptor releases the proteins into the ER lumen Golgi Higher pH (less acidic) escape Lower pH (more acidic) 10/3/2022 34 Sorting & targeting of membrane proteins Vesicle Target membrane Golgi Fusion Glycosylated parts Intracellular Extracellular • No special modification for the integral membrane proteins • They are synthesized as part of the ER membrane (Then move to Golgi…) • Membrane proteins are glycosylated • Glycosylated epitope faces the lumen ER, Golgi & vesicle • Upon targeting the glycosylated part becomes extracellular Membrane proteins glycosylated/modified part always face anteriorly (lumen of ER, Golgi, and vesicle). They only face exteriorly when the vesicles fuse to the PLASMA membrane 35 Protein secretion 1. Secretory proteins do not have any signal or marker 2. They are secreted outside by a default mechanism Outside ER → → Golgi → Any proteins with no marker or signal to bring them to a target site are exocytosed by default PM 36 10/3/2022 Summary: Category-2 proteins Golgi Secretory ER Lysosome PM 37 10/3/2022 Clinical significance (Due to genetic mutations) Defects in modifications → mistargeting proteins / enzymes → Diseases Mucolipidosis II (I-cell disease) 38 10/3/2022 I-cell disease • Lysosomal enzymes are tagged with mannose-6-P or MP by N-acetylglucosamine phosphate transferase • In I-cell disease this enzyme is mutated, enzymes do not carry M6P tag, do not reach lysosomes & instead they reach blood (Escape/leak to extracellular environment —> Blood) • Lysosomes accumulate mucopolysaccharidoses & (Long chains of sugar molecules form inclusion bodies (Aggregates found normally in mucus) of proteins) • Size of lysosomes grows which compromise other cell organelle functions (No degradation due to lack of enzymes) • Inclusion bodies can be seen in the Fibroblasts of such patients Inclusion bodies in fibroblast 39 10/3/2022