Protein+structure+and+Post-translational+Protein+Modification.pdf

Systems, Physiology and Anatomy CLS4011U Protein structure And Post-translational modification Dr Sriharsha Kantamneni  Learning Outcomes Understand protein structure and how structure relates to function. Protein conformat...

Systems, Physiology and Anatomy CLS4011U Protein structure And Post-translational modification Dr Sriharsha Kantamneni  Learning Outcomes Understand protein structure and how structure relates to function. Protein conformation and various types of bonds involved in protein folding Understand the different types of protein post-translational modifications and the enzymes involved. Understand the biological significance of protein post- translational modifications Appreciate there can be complex multi-modifications, that interplay together to influence a common protein function often with complex downstream effects Protein post-translational modification enzymes present important therapeutic opportunities and are key drug targets  Proteins Fundamental cellular component vital for all cellular functions Polymeric (=chain-like molecule made up of monomers) Macromolecules (=very large molecule) Thousands of different proteins exist with different functions The human body can generate ~2 million different types of proteins from ~20,000 genes Haemoglobin 3 3 October 2023  Proteins Fundamental cellular component vital for all cellular functions Polymeric (=chain-like molecule made up of monomers) Macromolecules (=very large molecule) Thousands of different proteins exist with different functions The human body can generate ~2 million different types of proteins from ~20,000 genes Haemoglobin STRUCTURE FUNCTION 4 3 October 2023  Protein Functions - Overview Protein Type Function Examples Structural Support Collagen Storage Storage Casein Transport O2 Transport Haemoglobin Hormonal Metabolism Insulin Receptor Cellular response b-Adrenergic receptor Contractile Movement Actin, myosin Defensive Protection Antibodies Enzymatic Catalysis Digestive enzymes 3 October 2023 5  Proteins Polypeptide = Amino acid monomers linked together by peptide bonds Polypeptides >40 amino acids can fold into a defined shape = Protein 6 3 October 2023  Proteins Polypeptide = Amino acid monomers linked together by peptide bonds Polypeptides >40 amino acids can fold into a defined shape = Protein Sequence of amino acids determines the shape and function of a protein STRUCTURE FUNCTION 7 3 October 2023  Proteins Polypeptide = Amino acid monomers linked together by peptide bonds Polypeptides >40 amino acids can fold into a defined shape = Protein Sequence of amino acids determines the shape and function of a protein STRUCTURE FUNCTION 8 3 October 2023  Protein Structure Newly synthesised proteins attain final 3D conformation, as the lowest and most stable shape, in seconds Small regions of relatively stable secondary structure form first, then development of tertiary structure Tertiary folding results in: Fibrous or Globular protein  Levels of protein structure Primary Structure – Amino acid sequence Secondary Structure – Interactions between adjacent amino acids – Examples: α helices, b sheets, loops/random coils 10 3 October 2023  Levels of protein structure Tertiary Structure – 3D folding of a single polypeptide chain Quaternary Structure – Assembly of multiple proteins into a complex 11 3 October 2023  Primary structure Amino acid sequence from N-terminus to C terminus (displayed left to right) Determined by the DNA sequence of the gene for each protein 12 3 October 2023  Primary structure Sickle RBC Healthy RBC Dictates final protein structure because sequential arrangement of R groups influences subsequent 2°, 3° and 4° structures Genetic mutation can lead to 1° structure changes that can alter structure and function Example: Sickle cell disease Caused by a single mutation in HbA haemoglobin gene 13 3 October 2023  Sickle Cell Disease Normal b-globin (HbA) Gene sequence Mutant b-globin (HbS) Gene sequence Single mutation in b-globin gene (T to A) changes 10 sequence (Glu to Val) Val Glu 14 3 October 2023  Tertiary Structure Overall 3-D shape of the entire polypeptide Held together by:- – Hydrogen bonds Between R Groups – Ionic bonds (=electrostatic attraction) Between CO2- and NH3+ of R Groups – Disulphide bridges (=covalent crosslinks) Between cysteine –SH groups (Cys-S—S-Cys) – Hydrophobic interactions Hydrophobic R Groups cluster inside proteins to shield themselves from water 15 3 October 2023  Tertiary Structure =Van der Waals interaction Ser Asp Lys Asp 16 3 October 2023  Pro-forms of proteins, e.g. production of insulin Proproteins are inactive peptides or proteins that need post-translational modifications to activate them  INS gene product Ribosomes feed the growing amino acid chain (preproinsulin) directly into the ER where the signal peptide (red) is immediately cleaved off by a signal peptidase to yield proinsulin. This is later processed further to mature and active insulin. Mature & active  Production of insulin Post-translation modification events include: 1, Cleavage and removal of signal peptide by signal peptidase in ER. 2. Oxidation of -SH groups to -S-S- (disulphide bridges) in ER. This cross-links specific regions via the -S-S- covalent bond. 3. Cleavage and removal of the C chain in ER. Post-translational modifications can therefore involve: 1.Processing (proteolytic cleavage to an active form) And/Or…..... 2.Covalent modification, which is the chemical modification of a protein after its translation. It is one of the late stages in protein biosynthesis for many proteins.  Biological significance of posttranslational protein modifications - During translation a polypeptide chain containing up to 20 genetically encoded amino acids is synthesized - The posttranslational covalent modifications allow to significantly extend the structural repertoire of proteins - The changes in chemical structure of a protein leads to the change in its spatial structure and biological activity - Some PTMs of proteins are reversible (e.g. acetylation phosphorylation, & methylation). This allows rapid dynamic regulation of a protein activity by controlling the balance of reversible PTMs.  Biological significance of post-translational protein modifications cont.. - The control of PTMs of proteins allow the control of their activity. This principle is widely used in nature to regulate numerous biological processes involving proteins, including metabolism, cellular signaling, gene transcription etc. - Indeed, PTMs and de-modifications of proteins are catalyzed by enzymes, that are involved in the regulation of their target protein activity.  Post-translational modifications are key mechanisms to increase proteomic diversity Post-translational modifications are key mechanisms to increase proteomic diversity. While the genome comprises 20,000 to 25,000 genes, the proteome is estimated to encompass over 1 million proteins. Changes at the transcriptional and mRNA levels increase the size of the transcriptome relative to the genome, and the myriad of different post-translational modifications exponentially increases the complexity of the proteome relative to both the transcriptome and genome  Classification of post-translational protein modifications PTMs involving structural changes in the proteins: 1. Proteolytic cleavage, or cleavage of a protein at a peptide bond. One or several amino acids could be removed from N-terminus of a protein, or protein peptide bond could be cleaved in the internal part of the protein  H2 N COOH Protease H2 N COOH Protease Not reversible! 2. Proline isomerisation – the change in proline residue spatial conformation (transition between cis- and trans- conformations of peptide bonds involving proline). Can seriously affect protein structure adopted. 3. PTMs involving addition of small functional groups - Phosphorylation - Acetylation - Methylation - Hydroxylation - more then 100 modifications of this type are known.  Protein phosphorylation Protein phosphorylation is the process in which phosphate (phosphoryl) group, donated by ATP, is transferred to an acceptor protein. The reaction is catalyzed by a protein kinase. Protein phosphorylation is reversible Kinase, ATP, Mg2+ OH OPO32- Phosphatase Reaction of a protein de-phosphorylation is catalyzed by a protein phosphatase Reversible protein phosphorylation regulates numerous biological processes Pyruvate dehydrogenase is regulated by phosphorylation/dephosphorylation by a protein kinase activated by high [NADH]:[NAD+] and [acetylCoA]:[CoA], but inhibited by pyruvate The cell cycle is controlled cyclins and their cyclin dependent kinases CDKs Cyclin-dependent Kinases and Cyclins CdKs phosphorylate serine and threonine residues, to promote progression of the cycle from one phase to the next CdKs only work when attached to a cyclin The key things to remember are: The type of cyclin influences the behaviour a CdK Cyclin concentration changes drive the cell cycle  Protein phosphorylation Estimated that ~ 30% of proteins are phosphorylated (often multiply) Serine is most commonly phosphorylated amino acid, followed by threonine and is often associated Tyrosine phosphorylation leads to binding of specific proteins that promote protein:protein interactions as part of the signaling networks  Detect phosphorylated proteins by: Phospho-specific antibodies 2-Dimension Phosphopeptide 32 mapping with P  Protein acetylation Protein acetylation is the process in which acetyl group, donated by acetyl Coenzyme A, is transferred to an acceptor amino acid, lysine, in protein. The reaction is catalyzed by a Protein AcetylTransferase (PAT). Process of a protein deacetylation is catalyzed by a Protein DeACetylase (PDAC). PAT PDAC The most characterized targets of protein acetylation are histones. The histone PATs and PDACs are called histone acetyltransferases (HATs) and histone deacetylases (HDACs). But they often have non-histone substrates too. The reversible histone acetylation is important in control of gene transcription. The biological role of non-histone protein acetylation is less understood and is an area of very active research.  Protein methylation - Protein methylation is the process in which methyl group, donated by S-adenosylmethionine, is transferred to an acceptor protein. - The reaction is catalyzed by a protein methyltransferase. Process of a protein demethylation is catalyzed by a protein demethylase. - The 2 major amino acids methylated are Arginine and Lysine -Not all protein methylation modifications are reversible - The best studied example of protein methylation is N- methylation of lysine and arginine side chains of histones involved in gene regulation. Methylation of Arginine and Lysine Illustration of PMTs associated with nucleosome core particles Representation showing post-translational modifications associated with histone particles. Nucleosomes are represented by red spheres wrapped by DNA (shown in gray). Also depicted are the positions of PTMs located on the histone proteins H2A (and H2A.X), H2B, H3, and H4. These PTMs impact gene expression by altering chromatin structure and recruiting histone modifiers. PTM events mediate diverse biological functions such as transcriptional activation and inactivation, chromosome packaging, and DNA damage and repair processes  Histone code hypothesis ‘…multiple histone modifications, acting in a combinatorial or sequential manner on one or mulitple histone N-terminal tails specify unique downstream functions’ 4. PTMs involving changes in chemical nature of amino acids -citrullination, or deimination of arginine converting it to citrulline. - Immune system attacks citrullinated proteins, and is implicated as a cause in auto-immune and arithritis diseases Peptidylarginine deiminases New amino acid! 5. PTMs involving additional of large functional groups and macromolecules -glycosylation (addition of mono- and oligo- saccharides). -addition of other peptides or proteins (mono- and poly ubiquitination, SUMOylation, etc.). -addition of fatty acid and lipid residues.  Protein glycosylation Protein glycosylation is a process of adding mono- or poly- saccharides to a protein. > glycoproteins - Glycosylated proteins are called glycoproteins. Protein glycosylation has significant effects on protein folding, conformation, distribution, stability and activity Most cellular proteins are glycosylated and their glycosylation play numerous biological functions, including control of protein stability, trafficking and recognition Carbohydrates in the form of aspargine-linked (N-linked) or serine/threonine-linked (O-linked) oligosaccharides are major structural components of many cell surface and secreted proteins N-linked and O-linked glycosylation The following modifications occur in the ER and Golgi apparatus N-linked glycosylation Polysaccharide is added as a 14 sugar unit to asparagine residue of the newly synthesised polypeptide in the ER. O-linked glycosylation Sugar added one at a time in Golgi, or in cytoplasm. The sugar is added usually to hydroxyl- group of serine or threonine. In some proteins hydroxy- lysine or hydroxyproline are glycosylated Synthesis of N-linked glycoproteins Processing of N-linked oligosaccharides O-linked glycoproteins In contrast to N-linked glycoproteins, O-linked glycoproteins are formed by addition of one sugar at a time, usually consist of a few residues – Golgi – for secreted proteins – Cytoplasm – for cellular proteins  Types of glycosylation Glycopeptide bonds can be categorized into specific groups based on the nature of the sugar–peptide bond and the oligosaccharide attached, including N-, O- and C-linked glycosylation, glypiation and phosphoglycosylation  Protein polyubiquitination - Ubiquitin is a small protein containing 76 a.a. - The last glycine in Ubiquitin is attached to lysine in proteins - Attachment of mono-ubiquitin to a protein plays multiple biological functions by changing the protein structure -Attachement of polyubiquitin chain (polyubiquitination) to a protein marks the protein for degradation in a proteasome -Ubiquitination requires three types of enzyme: ubiquitin- activating enzymes, ubiquitin conjugating and ubiquitin ligase enzymes (E1, E2 and E3 respectively) -Deubiquitinating enzymes (DUBs) removes it Protein ubiquitination pathway Proteasome degradation  Biological functions of the protein polyubiquitination and proteasomal degradation -Removal of damaged and mis-folded proteins -Control the lifespan of different proteins -Control the multiple cellular processes (cell cycle, mitosis, response to DNA damage etc.) by regulating the availability of key regulatory proteins in these processes  Ubiquitination controls neuronal excitability and synaptic transmission AMPARs (top), chronically elevated synaptic activity or direct stimulation with ephrin-A1 activates EphA4 receptors. This leads to recruitment of the Cdh1 component of the multiprotein ubiquitin ligase anaphase-promoting complex (APC) that, in turn, binds to and ubiquitinates the GluR1 subunit of AMPARs. These ubiquitinated AMPARs are targeted for degradation in the proteasome. For L-type calcium channels (bottom), the Cav1.2 pore-forming subunit must assemble with the cytosolic β-subunit for correct surface expression and protein stability. In the absence of Cavβ, Cav1.2 binds to and is ubiquitinated by the ER-associated ubiquitin ligase RFP2 and is targeted for proteasomal degradation by ERAD. Kantamneni et al 2011 Nature Neuroscience Ubiquitination controls synaptic transmission A B Kantamneni et al Unpublished data  Lipidation Lipidation is a method to target proteins to membranes in organelles (ER, Golgi apparatus, mitochondria), vesicles (endosomes, lysosomes) and the plasma membrane. The four types of lipidation are: C-terminal glycosyl phosphatidylinositol (GPI) anchor N-terminal myristoylation S-myristoylation S-prenylation Each type of modification gives proteins distinct membrane affinities, although all types of lipidation increase the hydrophobicity of a protein and thus its affinity for membranes. The different types of lipidation are also not mutually exclusive, in that two or more lipids can be attached to a given protein.  Protein post-translational modifications and medicine - Defects in protein post-traslational modifications and cell signaling are crucial in pathobiology of numerous diseases. - Enzymes controlling PTMs are often used as therapeutic targets - You will learn more about PTMs and their role in biological regulation during your studies GPCR regulation Kristiansen, 2004 Summary of post-translational modifications