General Flashcards
Phospholamban
- gene and protein
- function
- encoded by PLN gene
- NB in lusitropic effect in heart
- substrate of cAMP-dependent protein kinase (PKA)
- unphosphorylated state inhibits SERCA2, phosphorylation by PKA disinhibits SERCA - allows faster Ca uptake into SER
What are scaffold proteins
Proteins NB in signalling pathways in cells - binds to members of signalling pathways and tethers them to complexes
Function of caveolin
Caveolins are scaffolding proteins/integral membrane proteins NB in caveolae membranes and function of receptor-independent endocytosis
- NB in compartmentalising and concentrating signalling molecules
Role of ALPK3
- codes for alpha protein kinase 3
- believed to act as transcriptional regulator through activation of cardiac transcription factors
Role of junctophilin 2
-encoded by JPH2
- part of junctional complexes NB in mediating cross talk between plasma membrane and ER/SER ion channels
What is nonsense-mediated decay
- which proteins involved
NMD is a translation-coupled mechanism that eliminates mRNAs containing premature termination codons (PTCs)
- UPF proteins (UPF1, 2 and 3)
- exonucleases
What is cosegregation analysis
Technique to establish inheritance pattern of specific variant within a family - constructs pedigree and establishes whether variant co-occurs with phenotype
Mendel’s 3 laws
1- Law of segregation: each individual has 2 alleles, one from each parent, that segregates during gamete formation
2 - Law of Independent Assortment: genes from different chromosomes assort independently during gamete formation
3- Law of Dominance: in heterozygous individuals, 1 allele (dominant) will determine phenotype and recessive allele has no noticeable effect on phenotype
Definition of linkage analysis
Statistical method of mapping genes for heritable traits to their chromosomal locations
- genome-wide markers (microsatellites or SNPs) tested in pedigrees segregating a trait
- using statistical method (of linkage analysis) combines data to determine chromosomal regions likely to harbour genes for trait
- Parametric linkage analysis used for Mendelian traits (vs model-free LA for complex traits)
- LOD and recombination fractions used to test gene locations
LOD score in linkage analysis
Logarithm of the odds score
- evaluates if 2 loci (location of genetic markers) are genetically linked, ie inherited more often than expected by chance
Examples of chromatin annotations
- Histone modifications (H3K4me3 (associated with active promoters), H3K27ac (associated with active enhancers), and H3K9me3 (associated with heterochromatin)
- DNA methylation (at CpG dinucleotides - gene silencing)
- Open chromatin (eg ATAC-seq - identifies regions of open chromatin - assoc with active gene expression)
- Promotor and enhancer regions
- Transcription binding sites
- Topologically associating domains (TAD)
Initiative (projects) for chromatin mapping
- ENCODE (encyclopedia of DNA elements)
- Roadmap Epigenomics
- BLUEPRINT project
What are costameres
- 2 complexes it consists of
Structural-functional component that connects sarcomere to sarcolemma
- DGC (dystrophin-glycoprotein complex)
- Integrin-vinculin-talin complex
What are the protein structures in the DGC (dystrophin-glycoprotein complex)
- Dystroglycans
- Sarcoglycans
- Dystrophin
- Sarcospan
- Syntrophin
Function of costamere
- Keeps sarcolemma in line with sarcomere during contraction
- lateral (perpendicular) transmission of force generated by sarcomere to sarcolemma and ECM
What is dysferlin
-Dystrophin associated fer-like protein
- DYSF gene
- sarcolemma repair and calcium signalling
- dysferlinopathies, muscular dystrophies (incl limb-girdle MD)
Gelsolin
- Severs F-actin
- Caps barbed ends of actin filaments preventing monomer exchange
- Promotes nucleation step of actin polymerization
What are nuclear localisation signals (NLS) and how do they work
- Short sequences of amino acids or motifs on a protein that serves as targeting signal for transportation of protein into nucleus
- Importin-α receptor recognises the sequence and binds protein, then couples onto Importin-β and facilitates transport into nucleus
2 primary types of nuclear localisation signals (NLS)
- classical NLS (cNLS): single, continuous stretch of amino acids, typically lysine rich (PKKKRKV) - K=lysine
- bipartite NLS (bNLS): 2 clusters of amino acid sequences separated by short spacer region
What are filamin proteins
- which NB in heart/muscles
Filamin are class of proteins that holds 2 actin filaments at large angles
- Filamin C NB in cardiac and skeletal muscles, contribute to Z-disc proteins
What is vinculin
-membrane cytoskeletal protein in focal plaques involved in the linkage of integrin adhesion molecules to actin cytoskeleton
-anchors F-actin to membrane
- binds alternatively to a-actinin or talin
What are focal adhesions (FA)
- Large macromolecular assemblies through which mechanical force is transmitted between the ECM and interacting cell.
- Subcellular that mediates signalling events from outside (in response to ECM adhesions/substrate)
What are chimeric proteins
- AKA fusion proteins or hybrid proteins, created by combining genetic sequences of 2 or more different genes or proteins
- Can be natural (eg from chromosomal translocations) or engineered
Examples of engineered chimeric proteins
- Reporter proteins (reporter gene or fluorescent protein): allows tracking of expression and localisation
- Therapeutic proteins: eg antibody-drug conjugates (ADC) in cancer
- Enzyme fusions: biocatalysis - multiple enzyme reactions in a single protein
- Antibody engineering: combining parts of Ab from different species
2 types of actin
- G-actin: globular actin, monomeric form of actin in cytoplasm that can polymerise to form F-actin. Involved in cellular processes and has binding sites for ATP
- F-actin: filamentous actin. Formed from G-actin polymerisation into linear, helical form. Dynamic structure, constantly undergoing assembly/disassembly in response to cellular signals and needs
Some functions of F-actin
- Cell shape and motility
- Muscle contraction
- Cell adhesion (focal adhesions)
- Endocytosis and exocytosis
- Intracellular transport
- Cell divisions
- Neuronal function (maintains dendritic spine structure, NB for neuronal plasticity)
How does the yeast two-hybrid system work (Y2H)
- Process of studying protein interaction where a transcription factor is split into 2 parts: DNA binding domain (DBD) and Activation domain (AD)
- 2 proteins of interest are fused onto these 2 domains respectively
- If the protein interacts with each other, the reconstituted transcription factor becomes active and initiates transcription of a reporter gene in the yeast host
- Results in phenotypic change: growth of yeast cell
Mechanisms of off-target edits in CRISPR
- sgRNA - recognises a “seed sequence” of 5 bps proximal to PAM that determines specificity (ie longer seed + PAM would’ve decreased chances of off-target binding)
- non-specific PAM binding: recognises NRG (where R is G or A)
- Cas9 protein delivery: better with direct delivery as it degrades after cleaving DNA. Plasmid delivery increases off-target edits
3 ways in which first strand cDNA synthesis can be primed
using:
- Random primers
- oligo(dT)
- gene-specific primers (GSPs)
What is the 3’ bias in cDNA synthesis
When using oligo(dT), 3’ bias can occur due to reverse transcriptase (RT) efficiency or RNA fragmentation.
oligo(dT) = short sequences of deoxythymidine. Binds to poly-A tails of RNA (i.e. the 3’ end), ie “primes” cDNA synthesis from the 3’ end. The RT process may not always reach the 5’ end of RNA, leading to biased representation of cDNA library (ie RT efficiency). RNA fragmentation (due to sample processing or unintentional degradation) will also cause bias (ie RT will work from the 3’ end until the fragment site)
What are the factors about amino acid variations increase the likelihood of their deleteriousness?
- variations at conserved sites more likely to cause functional changes
- variations affecting:
– active sites
– interaction sites
– solubility
– protein stability
What are PolyPhen and SIFT scores
In-silico predictors of SNPs/amino acid changes on protein function.
- PolyPhen = Polymorphism phenotyping (amino acids)
- SIFT = Sort Intolerant From Tolerant (SNPs)
What is nebulin
- actin binding protein localised to thin filament of sarcomeres
- coded by gene NEB
- large protein 600-900kDa, binds 200 actin monomers, “ruler” for thin filament
- causes some nemaline myopathies
In Dark et al (LV protocol), what did each of these markers indicate:
- TNNT2
- MYL2
- HAND1
- NR2F2
- HOXB2
- TNNT2 - troponin T = pan-cardiac marker
- MYL2 - myosin light chain 2 = pan-ventricular marker
- HAND1 - heart and neural crest derivatives expressed 1 = LV marker
- NR2F2 - atrial marker
- HOXB2 - atrial marker downstream of RA signalling
What are the factors mechanistic factors affecting sarcomere power in heart?
- beat rate
- sarcomere length (length-dependent activation)
- intracellular Ca flux
- phosphorylation of titin
- Troponin I
- MYBPC3
- Myosin regulatory chain
