Proteins Part 2 2024 - PDF

Cellular Biology & Homeostasis AMINO ACIDS & PROTEINS Part 2 VP 2024 Clara Camargo, DVM Learning Objectives 1. Describe protein synthesis (transcription, post-transcriptional modifications, translation and post-translational modifications) 2. List the outcome of a newly synthesized protein 3. Explai...

Cellular Biology & Homeostasis AMINO ACIDS & PROTEINS Part 2 VP 2024 Clara Camargo, DVM Learning Objectives 1. Describe protein synthesis (transcription, post-transcriptional modifications, translation and post-translational modifications) 2. List the outcome of a newly synthesized protein 3. Explain common mutations, understand the causes and effects DNA and RNA structure Each nucleotide is formed: A pentose sugar A nitrogenous base A phosphate group DNA A-T & C-G (adenine - thymine, cytosine - guanine) RNA A-U&C-G (adenine - uracil, cytosine - guanine) Protein & Protein Synthesis Protein & Protein Synthesis Genetic code characteristics Universal: it is conserved from very Redundant: a given amino acid may early stages of evolution, with only slight have more than one triplet coding for differences in the way it is translated. it. Specific/unambiguous: a particular Nonoverlapping & commaless: the codon always codes for the same amino code is read from a fixed starting point acid. as a continuous sequence of bases without any punctuation between codons. The Central Dogma of Molecular Biology DNA Transcription RNA Translation Protein & Protein Synthesis Protein Tr a n s c r i p ti on Post-Transcriptional Modification PTM Primary transcript RNA pre-mRNA to mature RNA mRNA (messenger RNA) Happens before translation Process involves three major steps: 1. Addition of a 5’ cap 2. Splicing o Removal of introns (non-coding sequences) o Joining of exons (coding sequence) 3. Addition of a 3’ poly-adenylation tail Vital for correcting translation of eukaryote genomes https://www.biointeractive.org/classroom-resources/rna-splicing Post-Transcriptional Modifications 1. Addition of a 5’ cap 2. Splicing o Removal of introns (non-coding sequences) o Joining of exons (coding sequence) 3. Addition of a 3’ poly-adenylation tail snRNP “small nuclear ribonucleoprotein particle” 5’Cap and 3’tail facilitate transport of the mRNA to a ribosome and protect it from molecular degradation PTM - Alternative Splicing Protein Synthesis In which part of the cell is this process occurring? From: Biology dictionary  Same mRNA transcript can be spliced in different ways, yielding different proteins from the same gene.  Specific signal sequences in the mRNA are recognized by an enzyme called “small nuclear ribonucleoprotein particle” (snRNP), regulating gene expression. Steps in Protein Biosynthesis 1. Activation: aminoacyl-tRNA synthetases in cytosol will use ATP to “activate” tRNA. Enzyme is specific to the AA and tRNA, and displays “poof reading” activity 2. Initiation: assembly of components of the ribosome complex 3. Elongation: addition of amino acids to the carboxyl end of the growing peptide chain. Ribosome moves from 5’-end to the 3’-end of the mRNA, GTP is required Step 1: Binding of the aminoacyl-tRNA to the A-site of the ribosome Step 2: Generation of the peptide bond in P-site Step 3: Movement of the ribosome along the mRNA translocates “empty“ tRNA to E-site (exit) Protein Synthesis Steps in Protein Biosynthesis 4. Termination: STOP codons (UAA, UAG, UGA) moves into the ribosome A site → at least 4 high-energy phosphate equivalents (from GTP) are needed for each peptide bond to guarantee translation 5. Folding and post-translational modifications (PTM): polypeptides fold into active, 3-dimensional forms Protein Synthesis Protein Translation - activation AA + ATP + E E = aminoacyl-tRNA synthetases Protein Synthesis Protein Translation – Ribosome complex Protein Synthesis Ribosomes 60S Two subunits: Eukaryotes 60S/40=80S Prokaryotes 50/30=70S 40S A site: ARRIVAL  binds an incoming Aminoacyl-tRNA according to the codon occupying the site P site: PEPTIDE BOND (POLYPEPTIDE)  is occupied by Peptidyl-tRNA which carries the chain of amino acids that has already been synthesized E site: EXIT  occupied by the Empty tRNA as it is about to exit the ribosome Research Ribosome https://www.ks.uiuc.edu/Research/ribosome/ From: Alberts. Mol Biol of the Cell Fate of Newly Made Protein Protein Synthesis Cytosolic ribosomes Depend on  Where the protein is synthesized Synthesize cytosolic proteins and those proteins intended for the nucleus, mitochondria or peroxisomes  Ribosomes free in the cytosol  Ribosomes associated to the rER rER Responsible for synthesizing proteins that are to be exported from the cell (in vesicles) or to be placed in cell membranes Ribosomes – protein factory Associated with rER Cytosolic ribosomes Protein Synthesis Protein Synthesis THE RELATIONSHIP BETWEEN THE ER & GA Both are involved in protein maturation ER: Folding, N-glycosylation, proteolytic cleavage, lipidation Golgi: O-glycosylation, folding, formation of transport vesicle Secretory pathway https://www.youtube.com/watch?v=Cy05M6pY1k4&t=3s Protein Synthesis GOLGI APPARATUS Cis: protein phosphorylation (mainly serine, threonine and tyrosine) Medial and Trans: O-Glycosylation Trans: proteins are repacked - transport vesicles are sent to the specific ‘delivery addresses’  lysosomes  delivered to plasma membrane  exported out of the cell FYI Protein Synthesis Post-Translational Modifications of Proteins  Modifications of many eukaryotic proteins to form mature proteins  Modifications can occur on the amino acid side chains, or at the C (carboxyl group) or N (amino group) termini  Promotes folding, stabilizing proteins  Helps regulating enzyme activity Protein Synthesis Post-Translational Modifications of Proteins Phosphorylation: add phosphate group on the side chain of AA May increase or decrease functional activity Hydroxylation: add hydroxyl group (OH) Can affect protein stability and protein-protein interaction. Serine, threonine or tyrosine New Insights into Protein Hydroxylation and Its Important Role in Human Diseases FYI https://www.sciencedirect.com/science/article/abs/pii/S0304419X16300658 Protein Synthesis Post-Translational Modifications of Proteins Glycosylation: add glycan group Lipidation: add lipid, increasing their (carbohydrate) binding affinity to biological membranes May affect cell-cell adhesion and (cell or organelle membranes) communication Protein Lipidation: Occurrence, Mechanisms, Biological Functions, and Enabling Technologies FYI https://pubs.acs.org/doi/10.1021/acs.chemrev.6b00750 Protein Synthesis Post-Translational Modifications of Proteins Methylation: add methyl (CH3) group Trimming: proteolytic cleavage Acetylation: add acetyl group i.e., activation of trypsinogen into trypsin i.e., Histone (DNA structure) increase/decrease transcription (methylation) regulates gene expression (acetylation) Image source: Research Gate Protein Synthesis PROTEIN SORTING The biological mechanism by which proteins are transported to the appropriate destinations inside or outside of cell Proteins can be targeted to the inner space of an organelle, intracellular or plasma membranes, or to the outside of the cell via secretion This delivery process is carried out based on information contained in the protein itself. Correct sorting is crucial for the cell; errors can lead to diseases. Targeting signals contain the information for cellular transport to correctly transport proteins to their destinations Targeting signals often involve a peptide sequence, generally at the amino terminus of newly synthesized protein In eukaryotes, the signal recognition particle (SRP) helps transfer ribosome with polypeptide to the ER for modification, sorting or further transport Protein Synthesis Protein Mutations POINT MUTATIONS (change in a single nucleotide) Silent mutation: The codon containing the changed base may code for the same amino acid UCA  Ser; UCU  Ser Missense mutation: The codon containing the changed base may code for a different amino acid UCA  Ser; CCA  Pro Nonsense mutation: The codon containing the changed base may become a termination codon UCA  Ser; UAA  Stop SHORTENED PROTEIN Protein Synthesis Protein Mutations – sickle cell Sickle cell anemia in humans is caused by a single nucleotide substitution in the gene for beta-globin (E6V) It occurs mainly in African American populations, 1 of 500 newborn babies in the USA The symptoms are characterized by episodes of pain, chronic hemolytic anemia, & infections Protein Synthesis Protein Mutations Hb sickling in deer FYI Esin A, Bergendahl LT, Savolainen V, Marsh JA, Warnecke T. The genetic basis and evolution of red blood cell sickling in deer. Nat Ecol Evol. 2018 Feb;2(2):367-376. doi: 10.1038/s41559-0170420-3. Epub 2017 Dec 18. PMID: 29255300; PMCID: PMC5777626. Odocoileus virginianus Several deer species No obvious pathological effects Suggestive of adaptative changes (resistant to blood parasites) One amino acid of the β-globin protein is switched from glutamic acid to valine (E22V) Protein Synthesis Protein Mutations OTHER MUTATIONS Frame-shift mutations: 1 or 2 nucleotides are either deleted or inserted to the codon causing large-scale changes to proteins. i.e., dwarfism in Fleckvieh cattle Splice site mutations: change in “introns” removal from pre-mRNA leading to aberrant protein production. i.e., golden retriever muscular dystrophy – GRMD Proteins Mutations - causes  Errors during replication of DNA: I.e. during meiosis, when cells Protein & Protein Synthesis  Damage to DNA by mutagens: o Radiation (γ-radiation, UV radiation) divide to produce gametes leading o Carcinogens & other toxins to spontaneous mutations o Viruses, bacteria & other pathogens DNA damage and mutations https://youtu.be/LfPeSnJhVsw