Protein Translation and Clinical Significance PDF

Protein Translation and their clinical significance Raj Kumar, PhD  Part I Protein Translation/synthesis and Structure & function  Key Learning Objectives Explain each step involved during protein translation and the role of mRNA, tRNA, and ribosomes in...

Protein Translation and their clinical significance Raj Kumar, PhD  Part I Protein Translation/synthesis and Structure & function  Key Learning Objectives Explain each step involved during protein translation and the role of mRNA, tRNA, and ribosomes in the process. Identify the structural hierarchy in proteins, and importance of protein folding and denaturation. Examine how genetic mutations or other cellular environment conditions disrupt protein structure and functions leading to pathological/disease conditions.  What are Proteins? Proteins are organic compounds that contain the element nitrogen, carbon, hydrogen, and oxygen. Proteins are the most diverse group of biologically important substances. Proteins are considered to be the central compound necessary for life and are necessary for the building and repair of body tissues. Proteins produce enzymes, receptors, hormones, and other substances the body uses. Without proteins the most basic functions of life could not be carried out.  Proteins are made of amino acids File:Peptide bond formation.svg Image from http://api.ning.com/files/xO6ybWgUbfFlk7GUXm9d8dfR--U-fUdPOJEtDzVGgDY_/aminoacidstruc.jpg A General View of Protein Translation Occurs in cytoplasm Requires charged tRNA and ribosome Translation occurs in only one reading frame mRNA reads in 3-nucleotide stretches (codon) to direct polypeptide sequence  Codon and Anti-codon A codon is a DNA or RNA sequence of three nucleotides (a trinucleotide) that forms a unit of genomic information encoding a particular amino acid or signaling the termination of protein synthesis (stop signals). There are 64 different codons: 61 specify amino acids and 3 are used as stop signals. An anticodon is a trinucleotide sequence located at one end of a tRNA, which is complementary to a corresponding codon in a mRNA sequence. Each time an amino acid is added to a growing polypeptide during protein synthesis, a tRNA anticodon pairs with its complementary codon on the mRNA molecule, ensuring that the appropriate amino acid is inserted into the polypeptide.  Codon and Anti-codon Codon wobble is a phenomenon that occurs when a tRNA anticodon can recognize more than one codon, usually due to atypical base pairing in the third position of the codon. The Wobble hypothesis states that the first two bases of a codon pair precisely with the bases of the tRNA anticodon, but the third bases may wobble. The wobble hypothesis allows mRNA to be translated with fewer tRNAs than would be required without wobble. For 61 codons for amino acids, only about 40 tRNAs are needed because of wobble.  The Genetic Code Start Codon Glycine AUG 2nd BASE GGU 3rd BASE Leucine 1st BASE GGC GGA 2nd BASE GGG CUU 3rd BASE 1st BASE CUC CUA Stop Codon CUG UAA UAG  mRNA (Shine-Dalgarno sequence) The transcription and translation occurs simultaneously in prokaryotes and in eukaryotes the RNA is first transcribed in the nucleus and then translated in the cytoplasm. The mRNAs of many bacteria are polycistronic whereas eukaryote mRNAs are monocistronic. Thus, bacteria may contain several sites for initiating and terminating polypeptide synthesis whereas eukaryotes have only one site for initiation of protein synthesis.  Transfer RNA (tRNA) A Acceptor arm - C attachment of amino C acid Anticodon loop - interacts with mRNA A tRNA is an adaptor molecule composed of RNA, typically 76 to 90 nucleotides in length, that serves as the physical link between the mRNA and the amino acid sequence of proteins.  Charging of tRNAs with amino acids Adenylated amino acid bound to 3’ of tRNA tRNA-synthetase required for charging tRNA tRNA-synthetase are specific for an amino acid Recognition determined by sequences in the anticodon loop Structures of Ribosomes  ❖ At the start of translation, the tRNA with the anticodon to AUG will bind to AUG at site P. ❖ A second tRNA with a different AA will bind to the next codon on the mRNA molecule at the A site. ❖ In step 2, the peptide chain in the P site will attach to the amino acid in the A site. ❖ Once this attachment occurs, the ribosome will shift and move one position (1 codon) forward along the mRNA transcript. ❖ Step 5 (Termination), at some point during translation, the ribosome will encounter stop codon for which there is no tRNA with an anticodon that matches stop codons. Meth Meth Meth Phe Phe Phe Leu Leu Meth UAC E A P P E P A AAA CCG UAC 5’ AUG UUU GGC CUU ACG AUC CGG GCC AUC CUA CCC 3’  Instead, a release factor protein binds at the stop codon. When this happens, the ribosome/mRNA complex will become disrupted and break apart. This action will release the polypeptide chain and signal the end of translation.  Process by which a polypeptide chain acquires its three-dimensional (3-D) structure to achieve the biologically active state is called protein folding. Adapted from: http://withfriendship.com/user/neeha/protein-folding.php  Schematic diagram of some of the states accessible to a polypeptide chain following its biosynthesis Auto degraded peptides U I N Soluble amyloid precursor Consequences of amyloid fibril formation  Proposed mechanism for an IAPP aggregation cascade in type II diabetes mellitus. Recent evidence suggests that formation of toxic aggregates of the islet amyloid polypeptide (IAPP) might contribute to β-cell dysfunction and disease. However, the mechanism of protein aggregation and associated toxicity is still unclear. Protein Folding/degradation Adapted from: http://withfriendship.com/user/neeha/protein-folding.php Thermodynamics of Protein Folding Adapted from: http://withfriendship.com/user/neeha/protein-folding.php  Many faces of proteins Protein misfolding is the basis of numerous human diseases How can proteins fold so fast? The question then is how the ensembles of unfolded or partially folded proteins are in such a small population to avoid aggregation in the biological environment. The answer to this possibly lies with the cooperative nature of protein folding process, which has been associated with protein folding into native-like functional species.  OSMOLYTES Osmolytes (chemical chaperones) form a class of naturally occurring small compounds used by many organisms to enhance proper protein folding. It has been calculated that osmolyte concentrations in whole tissues often reach 400 mmols/kg of cell water. Osmolytes induce folding not by affecting the amino acid side chains, but as a result of their solvophobic effects on the peptide backbone. Because the protein backbone comprises the most numerous functional group in proteins, osmolyte-induced conformations result into native folded functional species. CATEGORIES Amino acids, e.g. proline Polyols, e.g. glycerol Sugars, e.g. trehalose Amine oxides, e.g. TMAO Importance of protein folding in nature These dapper animals are known as Phylum Tardigrada (water bears). Trehalose functions as an osmolyte in these very widespread aquatic organisms that live for stance in mosses, lichens, or beach sand. The water content of body can be reduced from 85% to 3% and body becomes barrel- shaped. In this state they are capable of surviving extremely harsh conditions such as very-very low temperature, boiling temperature, and high pressure.  Protein Folding amino acid chain (primary structure) α helix chain, β sheet etc (secondary structure) domain / subunits (tertiary structure) active native protein (final) No general rule yet…  Hemoglobin Protein http://www.randomhouse.com/knopf/authors/watson/images/rna.jpg Image from- DNA: The secret of life Structure of Hemoglobin protein File:1GZX Haemoglobin.png  Many faces of proteins Adapted from: Thermo Scientific Website: http://www.piercenet.com/browse.cfm?fldID=7CE3FCF5-0DA0-4378-A513-2E35E5E3B49B Writers, erasers and readers of PTMs  Major writers, erasers and readers of posttranslational modifications associated with pathophysiology PTMs AAs Writers Erasers Reader proteins Pathophys Protein Y Kinases Phosphatases SH2 PD α-Synuclein Phosphorylation S, T Kinases Phosphatases 14-3-3 AD Tau Ubiquiti-nation K Ubiquitin ligases Deubiquitinases UBD PD α-Synuclein Acetylation K HATs Histone deacetylases Bromo-domains AD Tau Methylation K, R Methyltransferases Demethylases PHD ALS FUS N-Glycosylation N OST PNGases immune receptors CJD PrPC, PrPSc ALS, amyotrophic lateral sclerosis; AD, Alzheimer’s disease; CJD, Creutzfeldt Jakob disease; PD, Parkinson’s disease; OST, Oligo-saccharyl-transferases; UBD, Ubiquitin binding domains; FUS, Fused in Sarcoma Adapted from HOPES website  Therapeutic solutions 3 main therapeutic approaches to Avoid Misfolding/Aggregation Inhibition of protein aggregation Interference with post-translational peptide changes before the misfolding/aggregation step Upregulation of molecular chaperones or aggregate-clearance mechanisms SUMMARY I A protein, during and after its synthesis at the ribosome, can adopt many different conformational states on the way to its native 3D structure. Imbalances in proteostasis often lead to protein aggregation and disease.  Part II Clinical Applications of Proteins ▪ Diagnostic Tools ▪ Therapeutic Tools ▪ Laboratory Uses  Learning Objectives Identify the differences between functional and non-functional plasma enzymes. Explain the utilities of enzymes and iso-enzymes in the diagnosis of organ-specific pathological conditions. Describe the therapeutic utilities of recombinant proteins to treat pathophysiological conditions.  Diagnostic Tools Plasma Functional Proteins Blood plasma contains many enzymes, which are classified into functional and non-functional plasma enzymes. Functional plasma enzymes Non-functional plasma enzymes Concentration Present in plasma in higher Normally, present in plasma in very in plasma concentrations in comparison to low concentrations in comparison to tissues tissues Function Have known functions No known functions Substrates Their substrates are always present Their substrates are absent from the in the blood blood Site of Liver Different organs: liver, heart, brain synthesis and skeletal muscles Effect of Decrease in liver diseases Different enzymes increase in different diseases organ diseases Examples Clotting factors, Lipoprotein lipase ALT, AST, CK, LDH, alkaline- and pseudo- choline esterase phosphatase, and amylase  Sources of non-functional plasma enzymes ❖ Increase in the rate of enzyme synthesis, e.g., bilirubin increases the rate of synthesis of alkaline phosphatase in obstructive liver diseases. ❖ Obstruction of normal pathway, e.g., obstruction of bile ducts increases alkaline phosphatase. ❖ Increased permeability of cell membrane, e.g., in tissue hypoxia. ❖ Cell damage with the release of its content of enzymes into the blood, e.g., myocardial infarction and viral hepatitis.  Increase of Non-Functional plasma enzyme Tissue damage or necrosis resulting from injury or disease is generally accompanied by increases in the levels of several nonfunctional enzyme Medical importance of non- functional plasma enzymes Measurement of non-functional plasma enzymes is important for: Diagnosis of diseases ✓ diseases of different organs cause elevation of different plasma enzymes. Prognosis of the disease ✓ the effect of treatment can be followed up by measuring plasma enzymes before and after treatment.  Examples of medically important non-functional plasma enzymes ❖ Amylase and lipase enzymes increase in diseases of the pancreas as acute pancreatitis. ❖ Creatine kinase (CK) enzyme increases in heart, brain and skeletal muscle diseases. ❖ Acid phosphatase enzyme increases in prostate cancer. ❖ Alkaline phosphatase (ALP) enzyme increases in obstructive liver diseases, bone diseases and hyperparathyroidism. ❖ Lactate dehydrogenase (LDH) enzyme increases in heart, liver and blood diseases. ❖ Alanine transaminase (ALT) enzyme, a.k.a., serum glutamic pyruvic transaminase (SGPT) increases in liver and heart diseases. ❖ Aspartate transaminase (AST) enzyme, a.k.a., serum glutamic oxaloacetic transaminase (SGOT) increases in liver and heart diseases.  Which two enzymes are more important for the diagnosis of LIVER DISEASE? ❖ Alanine transaminase (ALT) enzyme, a.k.a., serum glutamic pyruvic transaminase (SGPT) increases in liver and heart diseases. ❖ Aspartate transaminase (AST) enzyme, a.k.a., serum glutamic oxaloacetic transaminase (SGOT) increases in liver and heart diseases. Which one is more specific for the diagnosis of LIVER DISEASE? ❖ Alanine transaminase (ALT) enzyme, a.k.a., serum glutamic pyruvic transaminase (SGPT) increases in liver and heart diseases. WHY? Because its plasma concentration increases only in liver diseases AST is expressed in a variety of tissues, including the liver, brain, pancreas, heart, kidneys, lungs, and skeletal muscles. If any of these tissues are damaged, AST will be released into the bloodstream. While increased AST levels are signs of a tissue injury, it doesn't always relate to the liver. ALT is mainly expressed in the liver. ▪ Any elevation of the ALT is a sign that there is a liver injury. ▪ Occasional increases may occur with a short-term infection. ▪ Sustained increases mean there's an underlying liver disease.  Importance of AST/ALT Ratio in the Diagnosis of Liver Disease An AST/ALT ratio of less than one (where the ALT is significantly higher than the AST) is suggestive of non-alcoholic fatty liver disease. An AST/ALT ratio equal to one (where the ALT is equal to the AST) is suggestive of acute viral hepatitis or drug-related liver toxicity. An AST/ALT ratio higher than one (where the AST is higher than ALT) is suggestive of cirrhosis. An AST/ALT ratio higher than 2:1 (where the AST is more than twice as high as the ALT) is suggestive of alcoholic liver disease. ❖ ↑ ↑ ↑ Alkaline phosphatase (ALP) enzyme Obstructive liver diseases ↑ AST & ALT ↑ ↑ ↑ ↑ ALP Obstructive Jaundice ↑ ↑ ↑ ↑ AST & ALT ↑ ALP Hepatitis Isoenzymes As Diagnostic Tools Isoenzymes are different forms of an enzyme that catalyze the same reaction in different cells or tissues of the body. have quaternary structures with slight variations in the amino acids in the polypeptide subunits. There are five isoenzymes of lactate dehydrogenase (LDH) that catalyze the conversion between lactate and pyruvate.  LDH ISOENZYMS LDH exists in 5 forms (isoenzymes) LDH Isoenzyme Tissues LDH-1 is found primarily in heart muscle and red blood cells. LDH-2 is concentrated in while blood cells. LDH-3 is highest in the lung LDH-4 is highest in the kidney, placenta, and pancreas LDH-5 is highest in the liver and in skeletal muscle Diagnostic Importance of LDH Isozymes Creatine kinase as Cardiac marker CK-MB (Cardiac Enzyme) CK-MB (Cardiac Enzyme) As Biomarker of Myocardial Infarction Enzymes As Diagnostic Tools Myocardial infarction may be indicated by an increase in the levels of creatine kinase (CK) and lactate dehydrogenase (LDH). The different forms of an enzyme allow a medical diagnosis of damage or disease to a particular organ or tissue. Proteins as Therapeutic Tools Proteins as Therapeutic Tools 56 Proteins as Therapeutic Tools Why Protein against small drug molecules? The diversity of functional groups in protein. Highly specific function less chance of being mimicked by simple chemical compounds. High specificity in action less potential for protein to interfere with normal biological processes. The body naturally produces many of the proteins that are used as therapeutics, & hence are often well tolerated and are less likely to elicit immune responses. Protein therapeutics with enzymatic or regulatory activity Replacing a protein that is deficient or abnormal Augmenting an existing pathway Providing a novel function or activity Protein therapeutics with special targeting activity Interfering with a molecule or organism Delivering other compounds or proteins Protein vaccines Protecting against a deleterious foreign agent Treating an autoimmune disease. Treating cancer Replacing a protein that is deficient or abnormal Insulin Therapy  TYPES OF INSULIN Rapid Acting Onset: 10-15 min Peak: 60-90 min Usually taken right before eating or to lower high blood glucose level Duration: 4-5 h Short Acting Onset: 30-60 min Peak: 2-4 h Often taken right before eating or to lower high blood glucose level Duration: 5-8 h Intermediate Acting Onset: 0-35 h Peak: 60-90 min Usually taken at bedtime or twice a day (morning and evening) Duration: 4-5 h Extended long-acting Onset: 90 min Peak: None Usually taken once or twice a day (morning and evening) Duration: 24 h Premixed A single vial containing a fixed ratio Depends on the combination of rapid or short/intermediate insulin  CASE STUDY: ENGINEERED INSULINS Healthy humans secrete insulin continuously at a low basal level, with rapid but transient increases triggered by elevated blood glucose concentrations. A combination of fast acting and slow acting insulin thus must usually be administered to diabetics to mimic the natural state. Insulin consists of a 21‐amino acid A chain linked to a 30‐amino acid B chain via two interchain disulfide linkages. At physiological concentrations (10-10 M), insulin molecules exist in monomeric form. However, when present at therapeutic concentrations typical of commercial products (∼10−3 M), individual insulin molecules dimerize, with subsequent oligomerization of three dimers to form a hexamer. When administered via injection, individual insulin molecules must first disassociate from one another before leaking from the site of injection into the bloodstream. As a practical consequence, even such fast acting therapeutic insulins must be administered 30–45 min before a planned meal, and the subsequent mealtime must not be altered or the diabetic risks hypoglycemia. Faster acting insulins that could be administered concurrently with a meal would offer far greater flexibility to diabetics, and the development of such products was made possible by protein engineering. 62  An Engineered Fast Acting Insulin The principle amino acids contributing to dimer formation reside in the B chain are at positions B 8, 9, 12, 13, 16, and particularly 23–28. The main engineering strategies centered around introducing AA substitutions that will discourage monomer interaction in a subset of these positions believed to lie outside the insulin receptor binding site (specifically B 9, 12, and 26–28). Novorapid and Novolog are trade names of one such engineered product in which the proline at position B 28 has been replaced with an Aspartate residue, thereby introducing charge repulsion at the monomer‐monomer surface. The product shows greatly decreased propensity for oligomerization and, as a practical consequence, can be administered directly prior to or during a meal. 63 Question 1: What other amino acids could be potentially introduced into engineered insulin to achieve monomer charge repulsion? Answer: Glutamate. Question 2: What other approaches could be used to discourage dimer formation? Answer: Perhaps the introduction of AAs with bulky side chains to promote steric hindrance. Question 3: Can you see any potential therapeutic disadvantages/complications potentially caused by such engineering? Answer: Alteration of the product's biological activity/potency, generation of an immunogenic product. Question 4: If provided in the laboratory with (unlabeled) samples of both native insulin and the engineered fast acting insulin, can you think of any potential experiments you could undertake to figure out which was which? Answer: Direct amino acid sequencing or perhaps a direct “bioassay” in which both were administered subcutaneously to laboratory animals, whereas measuring how quickly each insulin got into the bloodstream. 64  An Engineered Slow Acting Insulin— In addition to fast acting insulins, slow acting insulin must usually be administered to diabetics to mimic ongoing basal insulin secretion characteristic of the normal state. Traditionally, slow acting insulin was produced by formulation of insulin with substances such as protamines, which further retard entry of the insulin molecules into the bloodstream from the site of injection. Protein engineering again has facilitated the development of long-acting insulin analogues by alteration of amino acid (AA) sequence. Lantus is the trade name given to one such commercialized product, which differs from native insulin in that Asparagine residue 21 of the A chain is replaced by a Glycine, and the B chain is elongated at its C‐terminal end by two Arginines. Consequently, the molecule's isoelectric point (pI) has increased from 5.4 to more neutral values. The engineered insulin is formulated at pH 4.0, a pH at which it is fully soluble. Upon administration, it experiences neutral pH values characteristic of tissue, and it precipitates from solution. The engineered molecules resolubilize only very slowly, thereby greatly prolonging their subsequent release into the bloodstream. 65 Question 1: Why do you think the engineered insulin precipitates from solution at the site of injection? Answer: Because the pH it experiences is close to its pI, and proteins are generally least soluble at their pI values. Question 2: What analytical technique might be particularly effective at distinguishing between native insulin and lantus insulin molecules? Answer: Isoelectric focusing, as it separates proteins based on pI. Question 3: What other engineering approaches might you possibly take to generate a slow acting insulin product? Answer: Perhaps introduce one or more AAs toward the end of the B chain that would promote stronger monomer‐monomer interaction, e.g., introduce a hydrophobic amino acid to introduce interchain hydrophobic attraction.  Summary Proteins are one the most utilized drug targets for the development of novel treatment strategies. Enzymes and iso-enzymes have significant potential in the diagnosis of organ-specific pathological conditions. Recombinant proteins can be used to treat various pathophysiological conditions. REFERENCES Pathophysiology of Disease: An Introduction to Clinical Medicine, 8e, Editors: Gary D. Hammer, Stephen J. McPhee. (https://accesspharmacy.mhmedical.com/Book.aspx?bookid=2468) Any Questions?? Email: [email protected] Office hours: by appointment