Thesis Questions Flashcards
Site-directed mutagenesis
- Technique used to introduce precisely targeted changes in a DNA sequence.
- Allows investigation into the functional consequences of specific mutations, such as examining specific amino acids in proteins, and examining their impact on protein structure, function, and interactions.
- I will do site-directed mutagenesis, introducing mutations into the DNA sequence. I will design primers for SDM and they will be designed with a point mutation where only one nucleotide will be replaced.** Performing PCR will allow the DNA polymerase to incorporate the mutant base into the growing DNA strand. **
The PCR mixture is treated with a restriction enzyme, DpnI, which digests the original/parental material by cleaving its methylated DNA. The new mutated PCR product is resistant to this enzyme because it doesn’t have the same methylation pattern. The new mutated DNA is then transformed into E.coli, where it can be replicated.
- This means the transformation primarily introduces the mutated DNA into the bacterial host.
To ensure mutagenesis was successful, I will sequence the modified DNA to confirm the presence of the intended mutations.
Selected residues
- UniProt to generate Erv14 and cargo protein sequences. Copied into AlphaFold to generate a visual representation of the structure and Erv14’s interaction with cargo clients.
- PyMol and RING which allowed me to highlight residues and measure the distance between interactions.
- Put into Excel to sort through all interactions and select residues key for Erv14’s function by the highest frequent interactions.
- Design specific primers using Snapgene and Agilent to ensure precise targeted mutagenesis.
The chemical nature of residues influences their interactions within a protein structure.
- Hydrophobic residues: contribute to the stability of protein cores through hydrophobic interactions. Mutating to hydrophilic/charged residues may disrupt these interactions.
- Hydrophilic residues: found on the protein surface and interact with water/polar molecules.
What happens if Evr14 becomes dysregulated?
- disrupt protein transport, leading to abnormalities in protein trafficking and secretion.
- impact the proper functioning of various cellular processes and organelles.
- allow misfolded proteins to localize rather than be trapped in the ER which can trigger the unfolded protein response (UPR).
- The UPR is a cellular stress response that aims to restore protein-folding homeostasis.
- prolonged UPR activation can lead to cellular dysfunction and contribute to the development of diseases such as neurodegenerative disorders.
- The accumulation of misfolded proteins in the ER, can induce ER stress.
- Prolonged ER stress can lead to apoptosis (programmed cell death) if the stress is too severe or persistent.
- abnormal protein aggregates can form, leading to neuronal dysfunction and cell death.
- Misfolded proteins can interfere with the autophagy process, which is essential for clearing damaged or aggregated proteins. Impaired autophagy can lead to the accumulation of toxic protein aggregates and contribute to cell death.
- Altered vesicular transport and misfolded protein accumulation may impact the regulation of cell growth, differentiation, and survival, promoting tumorigenesis.
What would if i had longer in the lab or i had an additional year?
- Conduct further functional assays to understand the functional characterisation of identified residues in Erv14’s interaction with cargo proteins.
- Explore additional techniques, such as co-immunoprecipitation or yeast two-hybrid assays, to validate and quantify the interactions between Erv14 and its cargo proteins.
- Investigate the three-dimensional structure of Erv14 and its mutants’ using techniques like X-ray crystallography or cryo-electron microscopy to gain insights into the molecular basis of cargo recognition.
- Based on what we observed in the microscopy erv15/erv14 might serve separately different roles. We are currently using Erv14/Erv15 double knockouts to make sure there is no redundancy there,
- Looking at selected mutants under erv14 single knockout background to see if there is any difference in the presence of erv15 so we can dissect which effect we observed. We can see which effects are erv14 or erv15 specific. - The mutants we see a phenotype with we can leverage those mutants to try to screen for any new cargo proteins that are associated with erv14. Expect those residues are important for the erv14 function, so essentially, we may be able to find cargos that are specific to those residues.
In future studies, analogous sites in the cornichon protein homolog of Erv14 could be mutated to assess their impact on neuron development, thereby providing potential links between these proteins and human diseases.
Yeast (conserved) similar in homology to humans and how can help solve diseases in humans?
- Yeast and human cells share conserved vesicular transport pathways, including the endoplasmic reticulum (ER)-Golgi transport mediated by cargo receptors like Erv14. Studying Erv14 in yeast provides insights into these fundamental processes, which are relevant to intracellular trafficking in higher organisms.
- Elucidating the role of Erv14 in yeast helps unravel the general principles of cargo receptor function. Understanding how Erv14 contributes to proper protein localization and trafficking can inform research on similar processes in human cells, aiding in the identification of potential therapeutic targets.
- Given that vesicular transport dysfunction is implicated in neurodegenerative diseases, studying Erv14 in yeast may provide insights into these conditions. Aberrant protein trafficking is a common feature in diseases like Alzheimer’s and Parkinson’s, and understanding Erv14’s role contributes to our understanding of these processes.
- Help find potential drug candidates applicable to human diseases.
- Genetic manipulations in yeast, such as studying Erv14 mutants, provide a platform to model and understand the molecular basis of diseases. Insights gained from these studies can be extrapolated to human systems, offering a simpler and more tractable system for initial investigations.
What do terms such as cornichon, homology, and analogous mean?
Homology: refers to the similarity between organisms due to common ancestry
Cornichon: a family of proteins that regulate the trafficking and function of AMPA receptors, which are important for synaptic transmission and plasticity in the nervous system
Analogous: similar functions in different species but do not share a common evolutionary origin.
Paralog: the result of gene duplication within a single species. Paralogs are related through duplication events within a genome, while analogs are similar in function but not in evolutionary origin.
Why use yeast to study Erv14’s mechanism?
- Early studies revealed that fundamental cellular processes, such as DNA replication, transcription, translation, and basic metabolism, are remarkably conserved between yeast and humans. These shared processes suggested a common ancestry and functional conservation at the molecular level.
Identification of homologous genes
- Advances in DNA sequencing and genetic analysis allowed researchers to compare the genomes of different organisms. The identification of homologous genes—genes with similar sequences and functions—in yeast and humans provided concrete evidence of evolutionary relationships.
- Yeast, particularly Saccharomyces cerevisiae, was established as a model organism for genetic and molecular studies due to its simplicity, rapid growth, and ease of genetic manipulation.
How can AlphaFold determine a protein’s structure?
Highly accurate protein structure prediction with AlphaFold - John Jumper 2021
AlphaFold2 can determine the structure of a protein from its sequence by leveraging the information in multiple sequence alignments (MSAs) of related proteins as raw input features for end-to-end training.
This approach enables AlphaFold2 to predict the 3D atomic coordinates of folded protein structures with high accuracy.
The method has demonstrated the ability to predict a single structure per sequence, utilizing deep-learning models to recognize correlations between protein sequence and structure.
Additionally, AlphaFold2 generates multiple sequence alignments (MSA) of evolutionarily related sequences, identifying residues that coevolve to facilitate structure prediction.
Mutation to an alanine?
- Changing a valine residue to an alanine residue can work as a mutation due to the impact of these amino acid substitutions on protein structure and function.
- The valine-to-alanine mutation is a conservative substitution, meaning that both amino acids have similar properties. Valine and alanine are both nonpolar, aliphatic amino acids, and the substitution between them is less likely to cause significant structural or functional changes in the protein.
- However, subtle alterations in the protein’s properties, such as stability, interactions with other molecules, or subcellular localization, can still occur due to the differences in the side chain size between valine and alanine.
Glycosylation of Mid2 in the golgi appartus but not Yor1 and Gap1?
The glycosylation of Mid2 in the Golgi apparatus is supported by a study conducted by Proszynski and colleagues in 2004. This study found that when cells lacked the PMT4 enzyme, which is crucial for the initial step of O-glycosylation, a protein called Fus1p was not glycosylated. Instead, it accumulated in late Golgi structures.
Additionally, another study, led by Roth in 1984, provided further evidence for Golgi-specific glycosylation processes. This study showed that core O-glycosylation occurs in the cis Golgi cisternae, emphasizing that this process doesn’t take place in the rough endoplasmic reticulum. These findings collectively support the idea that Mid2 glycosylation specifically happens in the Golgi apparatus.
Yor1 and Gap1
- The glycosylation of Yor1 and Gap1 in the Golgi apparatus is not confirmed by a study led by Danhelovska and colleagues in 2021. In their research, they found that depleting the Golgi structure in ACBD3-KO cells didn’t change the glycosylation pattern of the LAMP2 glycoprotein. This suggests that disrupting the Golgi structure doesn’t necessarily impact glycosylation patterns.
- Moreover, another study by Bekier and team in 2017 discovered that knocking out Golgi stacking proteins GRASP55 and GRASP65 led to Golgi stack dispersion, accelerated protein trafficking, and impaired accurate glycosylation of proteins and lipids. This indicates that disrupting the Golgi structure doesn’t always affect glycosylation.
Immunoblotting
Immunoblotting is a method that can help detect glycosylation in cells. A study by Miyata and colleagues in 2013 found that blocking O-glycosylation is a strong signal for the Golgi apparatus, causing Golgi stress. This stress can be identified using immunoblotting techniques.
Additionally, research by Elhammer and Kornfeld in 1984 showed that the first step in O-linked glycosylation happens after the protein is made, either in the smooth endoplasmic reticulum or the Golgi apparatus. This suggests that immunoblotting can be used to find out if glycosylation is happening in these parts of the cell.
Mutation from Alanine (A) to Aspartic acid (D)?
The substitution of an alanine residue to an aspartic acid residue can work as a mutation due to the distinct chemical properties and functional implications of these amino acids.
Alanine: a nonpolar, hydrophobic amino acid.
Aspartic acid: is a negatively charged, polar amino acid.
This substitution introduces a significant change in the chemical nature of the amino acid at the specific position in the protein sequence, potentially altering the protein’s structure, stability, and function.
- The introduction of a charged residue like aspartic acid can affect the local charge distribution and potential interactions within the protein, leading to functional changes.
- Additionally, aspartic acid residues are often involved in phosphorylation events and can serve as important regulatory sites in proteins.
Key papers
- “Sec24 Is a Coincidence Detector that Simultaneously Binds Two Signals to Drive ER Export” 2015 - This study provides insights into the function of Erv14 as a cargo receptor that couples membrane proteins to the COPII coat, emphasizing the importance of Erv14 in driving ER export.
- “A systematic approach to pair secretory cargo receptors with their cargo suggests a mechanism for cargo selection by Erv14” 2012 - “PAIRS approach” which combines genetic manipulations in yeast with microscopy screening to discover receptors used for speciifc cargo. Found deletion of Evr14 had broadest impact on cargo selection, (membrane spanning proteins with long TMDs).
- Erv14 required for ER exit of 32% of membrane proteins (18 of 57). Helps me select long TMD erv14 cargo clients.
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Can you explain western blotting, chemiluminescence, and primary and secondary antibodies?
- used to detect specific proteins in a sample
- separates proteins based on their size through gel electrophoresis, transferring them to a membrane, and then using antibodies to detect the protein.
- Chemiluminescence is used to visualise the protein bands on the membrane. Substrate produces light upon reaction with an enzyme linked to an antibody which allows the visualisation of the protein bands.
- Primary antibodies are the first antibodies used to detect the protein of interest in the sample. They bind specifically to the target protein.
- Secondary antibodies are then used to detect the primary antibodies. They are linked to an enzyme that produces a signal, such as chemiluminescence when it reacts with a substrate. This allows for the visualization of the protein bands on the membrane.
Does perinuclear also mean trapped in the endoplasmic reticulum?
The term “perinuclear” does not specifically mean “trapped in the endoplasmic reticulum.”
- generally refers to the region surrounding the nucleus within a cell. The perinuclear region can encompass various cellular structures, including the endoplasmic reticulum, Golgi apparatus, and other organelles.
