MP2 - PTMs Flashcards

Question

What roles do the Golgi and ER play is glycosylation?

Answer 1

The ER is primarily involved in the initial steps of glycosylation, including the assembly of the precursor oligosaccharide chain on a dolichol phosphate molecule (N-linked glycosylation) and transfer to the protein before modifications. The glycoprotein is transported to the Golgi for further modifications and sorting into different vesicles for secretion to different cellular locations.

Answer 2

Structure: - multiple domains and subunits - flexible domain (key to function) Function: - activates ubiquitin and transfers to E2

Answer 3

A hydrophobic surface on ubiquitin that allows for recognition and binding of ubiquitin by various ubiquitin-binding proteins. The hydrophobic nature of the Ile44 patch allows for specific interactions with complementary hydrophobic pockets, leading to the formation of stable protein complexes that facilitate the transfer of ubiquitin from E2 to the target protein.

Answer 4

The ability to bind multiple copies of the same ligand simultaneously i.e., a multivalent molecule. This is due to the presence of multiple binding sites e.g., multiple PTMs.

Answer 5

The BIR domains on IAPs can be bound by IAP inhibitors, which are activated when cytochrome c is released. e.g., SMAC and XIAP-binding protein. SMAC is released from the mitochondria during apoptosis. XIAP BP competes with caspases for binding to XIAP, thereby promoting caspase activity and cell death. IAP inhibitors have been developed as potential cancer therapies to promote apoptosis of cancer cells.

Answer 6

Unlike apoptosis, necroptosis is a type of cell death that's accompanied by a pro-inflammatory response, which can lead to tissue damage and inflammation. It's a form of traumatic cell death that results from acute cellular injury. Formation of the necrosome results in disruption of the plasma membrane and release of intracellular contents, including DAMPs to activate an inflammatory response.

Answer 7

Acetylation of lysine residues on histones is found in enhanced transcription of target genes because it removes lysine's positive charge. This reduces the interaction with the negatively charged DNA.

Answer 8

- cell-cell communication - ECM interactions - protection from, and susceptibility to, pathogens - modulation of immune responses

Answer 9

HPLC allows the separation, identification and quantification of glycan moieties. HPLC methods used for glycoprotein analysis include: - size exclusion chromatography - reverse phase chromatography - ion exchange chromatography

Answer 10

Writer: kinase Reader: SH2 domains, 14-3-3 proteins Eraser: phosphatases Occurs on polar residues and histidine.

Answer 11

Caspases are initially synthesized in cells as inactive zymogens, which require proteolytic cleavage for activation. Cleavage of the inactive zymogen forms the p10 and p20 fragments which associate together to form the active caspase enzyme. This ensures that caspases are not activated prematurely, which could lead to unwanted cell death.

Answer 12

Acetyl CoA

Answer 13

TRABID: specificity is regulated by accessory domains, such as an Ankyrin repeat. AMSH: metalloprotease DUB that's activated in endocytosis.

Answer 14

The complete set of protein kinases encoded by an organism's genome. The human kinome consists of over 500 protein kinases that are classified into different families based on their properties.

Answer 15

Ubiquitin contains 7 internal lysine residues that can also be ubiquitinated. The formation of different Ub chains gives rise to the ubiquitin code. E.g., Lys48 polyubiquitination = degradation Met1 = innate immune signaling

Answer 16

- De-phosphorylation of the kinase activation loop - Receptor internalization - Scaffold proteins They have to be regulated due to their role in signal amplification, and signals cannot be constant or ever-expanding.

Answer 17

Regulatory PTM due to its short half-life. It's thought to compete with O-phosphate via high levels of cross-talk which makes the regulation highly dynamic.

Answer 18

a platform that activates procaspases in response to intrinsic cell death signals such as release of cytochrome c from the mitochondria

Answer 19

A family of DUBs with high ubiquitin chain specificity/selectivity. e.g., OTULIN.

Answer 20

1. Phosphoprotein phosphatase 2. Metallo-dependent protein phosphatase 3. Protein tyrosine phosphatase 4. Asp-based phosphatase

Answer 21

1. oligomannose - purely mannose 2. complex 3. hybrid - a mix of oligomannose and complex

Answer 22

It's highly specific - the smear indicates other types of sugars being present in the sample that couldn't be cleaved. PNGase isn't specific so won't smear.

Answer 23

N-linked: - Asn-X-Ser/Thr - glycan attachment occurs in the ER - complex and heterogeneous structure - common in all tissues O-linked: - Ser/Thr - glycan attachment occurs in the golgi - simpler than N-linked - occurs mostly in mucosal tissues

Answer 24

Writer: ubiquitin ligase (E1, E2 and E3) Reader: Ubiquitin binding domains Eraser: deubiquitinating enzymes (DUBs)

Answer 25

PNGase cleaves N-linked glycan chains from glycoproteins. It's widely used in glycoprotein analysis to remove N-linked glycans from glycoproteins.

Answer 26

1. adding the kinase to the protein 2. purify from source 3. direct incorporation through the addition of unnatural phosphorylated amino acids

Answer 27

It binds the glycocalyx before being internalized and is taken into the ER where it splits into two chains. The a-chain cleaves a residue within the ribosome, blocking protein synthesis.

Answer 28

Glycotype: describes the overall pattern of glycosylation of a protein in a particular tissue or organism. Glycoform: refers to a specific glycan structure that is attached to a protein or lipid.

Answer 29

They're water-soluble due to their -OH and C=O groups, giving them polarity and hence ability to interact with water. Being water-soluble aids in the formation of a hydrated layer around the cell, protecting it from mechanical stresses, and allows them to interact with other soluble molecules in the extracellular environment. Sialic acid - contains a carboxyl group that allows it to become negatively charged.

Answer 30

The probes were internalized, showing most of the GlcNAc moieties were intracellular.

Answer 31

Mass spec is a highly sensitive technique that allows for the structures of glycoproteins to be studied. It usually requires fractionation first, and so sialic acid is hard to use here because its negative charge interferes with how it flies through the flight tube.

Answer 32

Both activation and repression e.g., H3K4me3 activates whilst H3K27me3 represses.

Answer 33

Caspases are a family of proteases (enzymes that break down proteins) that play a crucial role in programmed cell death, also known as apoptosis. They are a group of cysteine proteases, named for their ability to cleave after an aspartic acid residue in their target proteins.

Answer 34

Initiator: activated in response to specific signals and cleave and activate effector caspases. Effector: cleave and activate other downstream targets, ultimately leading to the death of the cell.

Answer 35

E1, E2, and E3 are the three main classes of enzymes involved in the ubiquitination pathway, which is a process by which ubiquitin, a small protein, is covalently attached to target proteins. E1: activate Ub and transfer them to E2 E2: attach Ub to the target E3: recognize the target and facilitate Ub transfer from E2

Answer 36

The tubulin code refers to a set of post-translational modifications that occur on microtubules. The tubulin code is believed to regulate microtubule stability, dynamics, and function. For example, acetylation of lysine residues on tubulin has been shown to promote microtubule stability. Others aren't completely understood.

Answer 37

IAPs block the activity of caspases. They're characterized by one or more BIR domains, which are responsible for their anti-apoptotic activity. They inhibit through ubiquitination of caspases, targeting them for degradation. e.g., XIAP

Answer 38

- So many glycoforms - Low amounts of each - Common monosaccharides have the same mass so struggle to separate them - O-glycan are labile in mass spec - Many don't have a consensus sequence

Answer 39

- glycosyltransferase expression, localization and organisation - glycosidase expression, localization and organisation - sugar concentrations

Answer 40

Parkin is an E3 HECT ligase that is associated with protein-degradation pathways and mitophagy. Mutations in the parkin gene disrupts its function, leading to impaired mitophagy and accumulation of damaged mitochondria in cells. This has been associated with Parkinson's disease.

Answer 41

A large multi-subunit protein complex that acts as an E3 ubiquitin ligase to target specific proteins for degradation in the cell cycle. It's a member of the cullin RING superfamily.

Answer 42

1. Synthesis of the lipid-linked oligosaccharide in the ER. The lipid is dolichol phosphate. 2. Transfer of the LLO onto the protein Asn by the oligosaccharyltransferase enzyme. 3. Calnexin cycle to aid protein folding. 4. Trafficking to the golgi. 5. Golgi sorts the glycoproteins for secretion, and further modifications occur.

Answer 43

Activation of the NF-kB signaling pathway: Ubiquitination of the NF-kB inhibitor (IkBa) leads to its degradation, releasing NF-kB to translocate to the nucleus and activate pro-inflammatory genes. Writer: LUBAC Eraser: OTULIN (and CYLD)

Answer 44

Add tunicamycin to prevent sugar transfer, or add a glycosidase such as endoH.

Answer 45

A family of E3 ubiquitin ligases that play a key role in the regulation of protein degradation pathways. e.g., APC. The cullin protein serves as a scaffold for the assembly of the complex, while the RING finger protein interacts with E2 ligases.

Answer 46

Ubiquitin-like proteins (UBLs) are a family of proteins that share structural and functional similarities with ubiquitin. - SUMO: involved in the DNA damage response - Nedd8: cullin RING ligase regulator Like ubiquitin, UBLs are conjugated to target proteins through a series of enzymatic reactions involving E1, E2, and E3 enzymes, and can also be removed by deconjugation enzymes.

Answer 47

Structure: - small protein - single catalytic domain - Ub binding site for backside binding to form polyubiquitination chains - conserved cysteine residues to form thioester bonds with Ub from E1 Function: - transfers Ub from E1 to the target protein

Answer 48

Patients with higher SMAC expression, promoting apoptosis, have better remission rates from cancer. This led to the idea of synthesizing SMAC mimetics to promote apoptosis within cancer cells. These have good tolerance with low-off target effects, but alone aren't sufficient to reduce tumor size.

Answer 49

Kinases can be regulated by the addition of ubiquitin molecules, either activating or repressing them. e.g., IKKB is inactive until NEMO recruits ubiquitin to activate the IKK complex.

Answer 50

1. Damaged mitochondria prevents PINK1 transport for degradation, resulting in accumulation of PINK1 on the mitochondrial membrane. 2. PINK1 promotes recruitment of Parkin. 3. Parkin attaches Ub to mitochondrial proteins, marking them for degradation. 4. Autophagosome forms an engulfs the damaged mitochondria.

Answer 51

Writer: histone acetyltransferase (HAT) Reader: bromodomain Eraser: deacetylase (HDACs and Sirtuins)

Answer 52

1. HPLC 2. Mass spec 3. NMR (and combinations)

Answer 53

- reversing the Ub signal - rescuing proteins from ub-mediated degradation - recycling Ub from the proteasome - editing of the Ub code

Answer 54

Can be divided into two main classes based on their structure: - HECT - contain a HECT domain with a catalytic cysteine - RING - contain a RING domain, but no catalytic residue Function: - recognize and bind specific target proteins and facilitate transfer of Ub from E2 to the protein. As RING ligases don't have catalytic residues, they function by positioning Ub-E2 in the right place with the target protein.

MP2 - PTMs Flashcards

(78 cards)