Protein-Protein Interaction (PPI) / Post-Translational Modifications

eg	P14173 (rat DDC), P20711 (human DDC)

Protein-Protein Interaction (PPI)

Types of PPI

Figure 1

Direct and indirect interactions. Nodes represent proteins, while edges represent interactions. Direct interactions between labelled proteins are indicated by red lines, while indirect interactions between labelled proteins are indicated by blue lines.

Assays of PPI

Co-immunoprecipitation

• Principle
  • a common technique for identifying physiological interactions between related proteins
  • When cells are lysed under non-denaturing conditions, many intracellular protein-protein interactions can be retained.
• Process
  • → An antibody against a specific bait protein (such as protein X) is stabilized on the surface of agarose beads.
  • → If there is another protein (such as protein Y) can bind to protein X, the complex of protein X-protein Y will be precipitated together by the antibody, and any proteins not precipitated on the beads will be washed away.
  • → Subsequently, the interaction between protein X-protein Y can be confirmed by analyzing protein Y (usual methods of analysis are SDS-PAGE and western blot).

Post-Translational Modifications

Goal

• ↑ Complexity: 2만개 gene → 10만개 transcripts → 100만개 protein

Proteome Complexity figure: Genome (~20-25,000 genes) → (Alternative promoters / Alternative splicing / mRNA editing) → Transcriptome (~100,000 transcripts) → (Post-translational modifications) → Proteome (>1,000,000 proteins).

Type

					Analysis example
Addition of	Small scale	Chemical (작은 chemical/functional group)	Phosphorylation	Protein kinase and phosphatase Phosphorylation sites: serine, threoning, tyrosine	GOOD Resource: https://www.rndsystems.com/resources/articles/methods-detecting-protein-phosphorylation Effect on ligand binding : {Smith, 2015 #1639} For 132/453 (29%) of these, the phosphorylation site is within 12Å of the small molecule-binding site, where it would likely alter small molecule binding affinity. - MS: In each run, a positive control consisting of a peptide with a known phosphosite is subjected to the same experimental conditions as the test samples. The presence of phosphosi
					confirmed at by the precense of a MSMS peak showing the loss of phosphate. - Kinase activity assay - Phosphor-specific antibody development - WB - ELISA - Ihc/icc
			Acetylation	acetyltransferases vs deacetylases	[Acetylome] (Gu et al. 2017, PMID) 3,468 lysine acetylation (Kac) sites in 1,417 proteins were identified, among which 3,415 sites in 1,398 proteins were quantified. Results: by setting quantification ratio of > 1.5 as up-regulated and < 0.67 as down-regulated threshold, we detected 56 up-regulated Kac sites in 50 proteins and 59 down-regulated Kac sites in 50 proteins
			Methylation Glycosylation	↑ protein folding	O-GlcNAc glycosylation (=O-GlcNAcylation) The O-GlcNAc transferase enzyme OGT catalyzes the covalent attachment of N-acetyl-D-glucosamine to serine or threonine residues of proteins, whereas β-N-acetylhexosaminidase (also known as "O-GlcNAcase) removes it (6). OGT is ubiquitously expressed, it is particularly abundant in neurons, where it is enriched in the nucleus (6) and at synapses (11).
		Complex molecules(큰 chemical/functional group)
	Large scale		AMPylation
			ADP-Ribosylation
		Amino acid	Prenylation
modification of amino acids			Deamidation
		Eliminylation
	Protein/polypeptides	Ubiquitylation
	lipid
Structural changes		citrullination
		Proteolytic cleavage
		racemization

O-GlcNAc glycosylation (=O-GlcNAcylation)

	OGT	OGA
	O-GlcNAc transferase	β-N-acetylhexosaminidase (also known as "O-GlcNAcase)
	O-GlcNAc glycosylation (= O-GlcNAcylation)
nction	catalyzes the covalent attachment of N-acetyl-D-glucosamine serine or threonine residues of proteins,	removes N-acetyl-D-glucosamine from serine or threonine residues
resión	ubiquitously expressed, it is particularly abundant in neurons, where it is enriched in the nucleus (6) and at synapses (11).
	OGT participates in specialized
	neuronal processes, including activity-dependent transcription and long-term depression via regulation of cAMP-responsive element binding protein (CREB) (5) and the AMPA receptor GluA2 subunit (12), respectively.
ormal	(2019 Levine) a-Syn has been shown to be O-GlcNAcylated at nine different positions in in vivo proteomics experiments from mouse and human tissues→ prevent aggregation	↑ O-GlcNAcylation in neurons → ↓ pathological forms of NFM and tau (15, 16) and directly inhibited the aggregation of both tau (17) and α-syn (18)
	↓ OGT (by conditional KO) → pathogenic processing of tau and amyloid precursor protein, widespread neuronal death, gliosis, and memory loss. (2016 Wang)	↑ raising O-GlcNAcylation levels by pharmacological inhibition attenuated amyloid-β deposition, tau phosphorylation, motor deficits, and memory impairments in certain AD mouse models (17, 19). ↑ O-GlcNAcylation levels exacerbated neurodegenerative phenotypes of tauopathy, amyloid β-peptide, and polyglutamine expansion in Caenorhabditis elegans models, ↓ O-GlcNAcylation levels rescued those phenotypes (20).

Uncertain Spans

location	text/status	reason
Type table column count and row spans	overall row/column merging	The Type table is wide and rule lines are faint inside the merged cells; the column merges and row spans here follow visible cell ruling but should be verified against the body_full crop before structural reuse.
Type table left columns	left-edge cell labels (Structural changes, modification of amino acids)	The leftmost two columns are partially clipped on the photo edge; visible fragments (uctural, anges, odification, amino, ids) are visible from body_r04_c01 and reconstructed accordingly.
O-GlcNAc table left column labels	nction, resión, ormal	The leftmost label column of the O-GlcNAc table is clipped at the page edge; visible fragments suggest function, expression, normal but the leading characters are not visible.
Acetylome 1,398 proteins	1,398 proteins	The body_r03_c02 crop shows 1,398 proteins legibly; 1,39… is clipped in the body_full row but resolved in the tile crop.