3 - Proteins, Transport, Degradation Flashcards
Describe the structure of proteins/polypeptides. What are the forces Don’t be a fucking idiot now.
Proteins
Don’t be a fucking idiot now.
Primary structure: peptide chain composed of amino acids. One end has N-terminus and one end has C-terminus. Linear.
Secondary Structure: alpha helices and beta pleated sheets. Hydrogen bonds between the protein backbone (NH and CO interactions) stabilize these structures. side groups stick outward in an ⍺-helix and up and down for β-pleated sheets.
Tertiary Structure: 3D structure of a protein molecule. Can contain alpha helices, beta pleated sheets, and is folded driven by non-specific hydrophobic interactions. Stable only when tertiary interactions such as salt bridges, hydrogen bonds, and disulfide bonds form.
Quaternary Structure: Complexes of 2 or more polypetide (protein) molecules. aka multimers
lets. fucking. go.
POG
Describe Sickle cell hemoglobin. What makes it differ from normal hemoglobin? Describe the structure of normal hemoglobin.
Proteins
A hemoglobin molecule is composed of four protein globin chains, each centered around a heme group (containing iron). In most adult hemoglobin, there are two alpha chains and two beta chains.
Sickle hemoglobin has a Valine 6 instead of a Glutamate 6, causing hydrophobic interactions when proximal to other Sickle hemoglobins. This causes sickle hemoglobin to clump and clog blood vessels.
toughy
How can protein diversity be increased besides from transcriptional and mRNA influence?
Proteins
Post-translational modifications. Changes at transcriptional/mRNA levels increase size of transcriptome. Post-translational modifications exponentially increase the size of the proteome from there.
Expand on the concept of phosphorylation. How is it done? Where is it seen most? Why is it important?
Proteins
Kinases phosphorylate proteins. Phosphatases removes phosphates. Phosphorylation is meant to be a transient process to regulate biological processes. Only Serine, Threonine, and Tyrosine are targets for phosphorylation because of their hydroxyl group. Phosphorylation of Tyrosine, although less prevalent than S or T, regulates the action of RTK’s which are crucial in cell survival.
Signal transduction cascades amplify the signal output. External and internal stimuli induce a wide range of cellular responses through a series of second messengers and enzymes. These signal cascades amplify the initial stimulus for large-scale and/or global cellular responses.
What is gamma carboxylation? What is its clinical significance?
Proteins
gamma carboxylation of the GLUTAMIC ACID residues at the ER form gamma-carboxyglutamyl. This process requires reduced vitamin K, O2, and CO2. Factor 10, thrombin, Factor 7, and Factor 9 of the coagulation cascade are gamma carboxylated. Warfarin, a vitamin k antagonist, is widely known as a blood thinner.
Why is understanding protein synthesis important for understanding disease processes? Describe the process of protein synthesis. Where can it go wrong? How can we take advantage of the importance of protein synthesis clinically?
Proteins
ASCorrect protein synthesis is crucial as mutated proteins can cause disease. Defects in protein translation can also cause disease. However, inhibition of protein synthesis in pathogens can also be used to treat infection.
Mutations in the transcript can disrupt protein synthesis.
Silent mutations: One nucleotide exchanged for another, but the triplet still codes for the same amino acid.
Missense Mutations: One nucleotide exchanged for another, resulting in a different amino acid.
Nonsense mutation: One nucleotide exchanged for another, resulting in a stop codon (UAA, UAG, UGA).
Splice-site mutation: Nucleotide exchange at iron-exon border causes splice variant.
Indel mutation: Insertion/deletion of a nucleotide results in a frameshift possibly causing an early stop codon or random AA sequence.
Translation Inhibition: Antibiotics utilized for the treatment of bacterial infections/toxins inhibit translation. Inhibition can be affected at all stages of translation from initiation to elongation to termination.
How is protein translated by ribosomes? Explain the wobble hypothesis and its significance during your explanation.
Proteins
In eukaryotes, 80S ribosomes are composed of a 40S and 60S subunits. They are found embedded in the ER or floating in the cytosol.
1. Translation initiation complex is formed with the 40S subunit, an initiator tRNA specific for the start codon (methionyl-tRNA), eukaryotic initiation factor 3, and the 60S subunit.
2. Met-tRNA starts in the P site. Aminoacyl-tRNA enter the A site and the anticodon binds to the codon.
3. Petidyl transferase (60S subunit) forms a peptide bond between Met and the amino acid on the tRNA in the A site.
4. Ribosome moves on codon to the right and the free tRNA (Met-tRNA here) is ejected.
5. Repeat until stop codon. A release factor catalylzes the hydrolysis of the polypeptide and the tRNA it is attached to and the ribsomal subunits separate.
The wobble hypothesis provides an explanation for the degeneracy of the genetic code. Degeneracy: multiple codons can code for the same amino acid. The precise pairing between the bases of the codon and the anticodon of tRNA occurs only for the first two bases of the codon. However, the pairing between the third base of the codon and the anticodon can exhibit some flexibility, aka “wobble” or just non standard base pairing. These wobble base pairs exhibit thermodynamic stability comparable to that of Watson-Crick base pairs.
Amino acids are activated by aminoacyl-tRNA synthetases. Each tRNA and aminoacyl-tRNA synthetase is specific to the amino acid it carries the anti-codon for. The 60S subunit contains 3 sites, the (A)cceptance site, the (P)olymerization or (P)eptide Bond site, and the (E)xit site. Eukaryotic release factor 1 (erf1) has 3 domains that are structurally similar the tRNA anticodon loop, the aminoacyl stem, and the T stem of tRNA.
Is protein structure inherent to a specific amino-acid sequence? How do proteins fold? What happens when protein folding goes wrong? Clinical significance?
Proteins
Disulfide bonds can be denatured by adding chemicals or by heat making proteins lose their tertiary structure. Denaturation can be reversed by removing the chemicals or heat. An amino-acid sequence determines the shape of a protein. This reinforces the idea that hydrophobic forces help proteins fold: form a hydrophilic outer shell and a hydrophobic core region. Folding occurs in the cytoplasm and the lumen of the ER. Certain Enzymes and Chaperone proteins ensure correct folding of newly synthesized proteins which is importatn for correct transportation and function of that protein. Protein folding prevents aggregation of unfolded proteins (entropy driven hydrophobic effect process).
Chaperone proteins ensure that proteins do not clump together prior to protein folding. They also protect a protein’s tertiary structure against stress (ex. heat).
In some cases, Chaperonins further protect proteins so that it can fold undisturbed. Chaperonincs can also reverse misfolded structures.
Protein misfolding causes aggregation and disease: tandem triplet repeats in mRNA cause huntington disease and fragile x syndrome.
A suggested therapy for excessive protein aggregation due to protein misfolding in neurodegenerative disease is inducing expression of chaperons and chaperonins.
How are proteins degraded?
Proteins
Proteins are degraded in either the cytosolic ATP dependent pathways or the lysosomal pathway.
The cytosolic ATP dependent pathway is also called the Ubiquitin-Proteasome System where a misfolded protein undergoes polyubitquitination, enters a proteasome, and is degraded into small peptides. Ubiquitin molecules are recycled. The Proteasome contains mainy multicatalytic proteases with protease activity on the inside of the “barrel”. Ubiquitin is activated by ubiquitin activating enzyme, E1, then transferred to ubiquitin conjugating enzyme, E2. Then the ubiquitin is transferred to a lysine residue on the target protein which is catalyzed by ubiquitin ligase, E3. Ubiquitin is frequently copolymerized on target proteins. The more highly ubiquitinated the protein, the more subsceptible it is to proteasomal degradation.
The Lysosomal Pathway depends on trafficking through the Golgi for transport to Lysosomes. After co-translational translocation to the golgi, proteins containing mannose-6-phosphate tags encounter and bind mannose-6-phosphate receptors in clathrin coated regions of the trans golgi (far golgi). Then clathrin-coated sections of the trans golgi bud off enclosing the proteins bound to the m6p receptors inside. The clathrin coat is lost and the transport vesicle fuses with an endolysosome where a decrease in pH releases the protein from the receptor and enzymes can degrade both.
What is glycosylation? When does it occur? Why is it important?
Proteins.
Glycosylation involves covalent addition of glycans (sugars) to specific amino acids. 1/2 of all proteins synthesized by cells are glycosylated. Some glycosylation occurs in the ER and ensures that properly folded proteins are trafficked to the Golgi. Inside the Golgi, further glycosylation ensures delivery of the protein to its correct destination. Large, bulky sugars can affect protein-protein interactions, reducing protein aggregation and regulating protein folding. Glycosylation requires multiple enzymatic steps where enzymes that transfer sugars from sugar nucleotides to proteins are called Glycosyltransferases and enzymes that remove sugars from proteins are Glycosidases. Glycosylation has 2 important types: N-linked and O-linked, but proteins are often glycosylated at multiple sites with different glycosidic linkages. N-linked glycosylation: glycan binds to the amino group of Asparagine in the ER (common). O-linked glycosylation: monosaccharides bind to the hydroxyl group of Serine or Threonine in the ER, Golgi, cytosol, and nucleus. Most N-glycosylated proteins are bound for the membrane oor are secreted.
N-Glycosylation can be broken down into separate events: 1. precursor glycan assembly, 2. attachment, 3. trimming, 4. maturation. N-Glycosylation often occurs co-translationally in the ER.
O-linked Glycosylation: attachment of sugars via GalNAc (N-Acetylgalactosamine). Other sugars can be used including Fructose, Mannose, Glucose, etc. Most common O-glycosylated proteins are mucins. O-linked glycans are critical for the formation of proteoglycan core proteins used to make ECM products. Antibodies are often heavily O-glycosylated.
What is the difference between N-linked and O-linked glycosylation?
Proteins
Why are lysosomes important? Clinical significance?
Proteins
Cells will begin to accumulate relevant substrates if a certain lysosomal enzyme is missing/defective. Lysosomal Storage Disorders are classified by the accumulated substrate. The distribution of accumulated material determines what organs are affected. There are more than 40 diseases classified as lysosomal storage disorders, each resulting from a specific genetic defect that gives rise to an enzymatic deficiency, and the subsequent accumulation of undegraded substrates within lysosomes. A notable example of this:
Gaucher Disease: results from lack of the lysosomal enzyme glucocerebrosidase and accumulation of glucocerebroside in mononuclear phagocytic cells. In the most common, type I variant, affected phagocytes become enlarged (Gaucher cells) and accumulate in liver, spleen, and bone marrow, causing hepatosplenomegaly and bone erosion. Types II and III are characterized by variable neuronal involvement. Gaucher disease has a strong association with Parkinson disease.
What is Autophagy? Why is it important?
Proteins
Autophagy is cellular quality control. It is our body’s process of reusing old and damaged cell parts. Autophagosomes are double-membrane vesicles newly formed during autophagy to engulf a wide range of intracellular material and transport this autophagic cargo to lysosomes (or vacuoles in yeasts and plants) for subsequent degradation. This degradation can be selective through direct, receptor-mediated uptake of cytosolic proteins. Autophagy is an important cellular mechanism allowing the cell to meet various demands, and its disruption compromises homeostasis and leads to various diseases, including metabolic disorders, neurodegeneration and cancer.
What are lysosomes? How do they function?
Proteins
Lysosomes contain about 40 types of hydrolytic enzymes. All are acid hydrolases that have optimal activity at pH 5. These proteases belong to aspartic, cysteine, or serine protease families. Each enzyme recongizes a specific type of structure. The products of lysosomal digestion (aa, simple sugars, nucleotides, etc.) are recycled back to cell for reuse.
Lysosomes use an H+ ATPase pump in the membrane to generate an acidic pH. Lysosomal enzymes are non active in the cytosol’s neutral pH and only activated in acid pH of lysosome.
What is the clinical significance of the proteasome?
Proteins
We can inhibit the proteasome to treat multiple myeloma. Patients with recurring multiple myeloma have few treatment options, but a new drug that is an inhibitor of protease activity in the proteasome, bortezomib, has been added to the arsenal.
* Bortezomib appears to work by inhibiting the degradation of
proteins involved in programmed cell death (apoptosis) of
cancer cells, thereby enhancing the self-destruct signal in
myeloma cells.
How are proteins destined for the plasma membrane or extracellular secretion transported to their destination?
Proteins
Proteins bound for sectrions form the cell contain a signal sequence of 16-30 hydrophobic amino acids at their N-terminus. Membrane proteins also often contain a signal sequence and are inserted into the ER co-translationally. The signal sequence directs the ribosome to bind to a signal recognition particle (SRP) and then binds to an SRP receptor in the ER membrane (cytosolic side). The protein then enters a translocon channel in the ER membrane and a protease cleaves the N-terminal signal from the nascent polypeptide. Once translation is complete, the ribosome subunits dissociate and the protein ends up in the lumen of the ER. In the ER, the protein undergoes initial processing and glycosylation to increase its solubility. Then the protein is transported in vesicles to the Golgi where it undergoes further sorting and tagging to specify its destination.
Transport from the ER to the Golgi involves the polypeptide finding a region of the ER lacking ribosomes. The ER membrane buds and pinches off from the ER around the polypeptide forming a transport vesicle. The transport vesicle fuses with the cis portions of the Golgi Complex and releases the polypeptide inside. The protein is similarly transported from the cis Golgi to the cis cisterna to the medial cisterna, to the trans cisterna, to the trans golgi by COP1 transport vesicles.
How is N-linked glycosylation performed during transport through the golgi?
Proteins
In the cis cisterna, Golgi mannosidase 1 removes multiple mannose sugars. In the medial cisterna, GlcNAc transferase or other transferases add other sugars and Mannosidase 2 removes further mannose (there is likely mannose still remaning. In the trans cisterna, multiple transferases such as Galactose transferase or NANA transferase add different sugars to generate the highly variable complex oligosaccharides. Some glycans do no undergo further glycosylation and are called high mannose oligosaccharides.
How is secretion of peptides from the golgi to the plasma membrane regulated to maintain proper concentrations in the extracellular space?
Proteins
Consitutive secretion: continuous process
Regulated secretion: New proteins reach the end of the trans golgi network and are concentrated there. The secretory vesicle or granule remains within the cytoplasm until the approporiate signal is received for its release. Once the signal is received the vesicle will fuse with the plasma membrane and release its contents to the outside of the cell.
