Reference range (normal range)
Figure 1-1 Schematic representation of the UPS.
(a) Ubiquitin is activated by a ubiquitin-activating enzyme (E1) through an ATP-dependent mechanism and transferred to ubiquitin-conjugating enzymes (E2). It is then ligated to lysine residues of protein substrates in a reaction catalysed by many different ubiquitin protein ligases (E3). (b) The 26S proteasome is composed of two major components, the 19S cap and the 20S core protein complexes. The 20S core complex has a cylinder-shaped structure with a narrow opening at both ends, consisting of two outer α rings and two inner β rings (C) The proteolytic activities occur in the three subunits in the β ring. MG-132 inhibits the proteolytic activity primarily on the chymotrypsin-like site. Adapted from Tsai et al. (2014), McNaught et al. (2006), and Goldberg et al. (2012).
proteins to small peptides by using the hydroxyl group of the N-terminal threonine residues; chymotrypsin-like, a trypsin-like, and a caspase-like site (Goldberg, 2012).
UPS impairment in PD patients
| The proteasomal α subunit expression is reduced in substantia nigra dopaminergic neurons of PD patients (St. P. McNaught et al., 2003), | |
| postmortem | (McNaught, 2001 #1177) hymotrypsin- (39%), trypsin- (42%) and postacidic-like (33%) hydrolysing activities of 20/26S proteasome are impaired in substantia nigra in PD |
| (McNaught, 2003 #1176) -subunits(but not -subunits) of 26/20S proteasomes are lost within dopaminergic neurons and 20S proteasomal enzymatic activities are impaired in the SNc in sporadic PD. In addition, while the levels of the PA700 proteasome activator are reduced in the SNc in PD, PA700 expression is increased in other brain regions such as the frontal cortex and striatum. We also found that levels of the PA28 proteasome activator are very low to almost undetectable in the SNc compared to other brain areas in both normal and PD subjects | |
| Detection of ubiquitin, ubiquitinated α-syn and proteasome components in Lewy bodies also supports the involvement of the UPS in the pathogenesis of PD (McNaught et al., 2002). |
Ubiquitin
Gene Ubiquitin
i. There are four genes in the human genome that produce ubiquitin;
| Codes for | ||
| ☐ | UBB, | polyubiquitin precursor proteins |
| ☐ | UBC, | polyubiquitin precursor proteins |
| ☐ | UBA52 | a single copy of ubiquitin fused to the ribosomal proteins L40 |
| ☐ | RPS27A | a single copy of ubiquitin fused to the ribosomal proteins S27a |
Protein ubiquitin
ii. is a small protein iii. The ubiquitin protein itself consists of 76 amino acids and has a molecular mass of about 8.6 kDa.
Detection methods
| Parkin E3 Ligase TR-FRET Kit | Antibody A | Antibody B | Antibody C | ... | ... | |
|---|---|---|---|---|---|---|
| What to detect? | Conjugated pS65 ubiquitin on Parkin | Conjugated ubiquitin | UnConjugated ubiquitin | Both Conjugated ubiquitin & Conjugated ubiquitin | Monoubi (=monomer) | polyubi |
| 여기서 말하는 conjugated 는 = bound to protein (bound to lysine residues on the protein or to the amino group of the protein's N-terminus) 이고 unconjugated는 = free ubi (그냥 cytoplasm에 떠다니는 ubiquitin) 인가 보다 | ||||||
- TUBE (Tandem Ubiquitin Binding Entities)
- A technique to specifically isolate/captue/purify (and stabilize) polyubiquitylated proteins from cell lysates and tissues
- Thi sis not antibody-based
- Products
- Chain-selective
The TR-FRET E3 assay involves terbium-labeled TUBEs that bind to fluorescein-labelled polyubiquitin chains synthesized by the target E3 ligase. Terbium and fluorescein are a FRET pair, so polyubiquitin chains containing fluorescein-labeled ubiquitin yield a FRET signal when bound by a terbium-TUBE. This signal can be monitored over time in a homogenous, high-throughput format, making it ideal for small-molecule screening.
Ubiquitination sites defined on lysines (on the protein substrate겠지!)
| Which Lysine sites? | function |
|---|---|
| K48 and K29 | degradation with the proteasome |
| K63, K11, K6) and monoubiquitinations | endocytic trafficking, inflammation, translation and DNA repair |
Assessment of UPS
| CSF | In vitro | In vivo | Postmortem | Product | Example | ||
|---|---|---|---|---|---|---|---|
| roteolytic ctivity | {Cleeter, 2013 #533} by 'standard technique (Schapira, A.H., Cleeter, M.W., Muddle, J.R., Workman, J.M., Cooper, J.M., King, R.H., 2006. Proteasomal inhibition causes loss of nigral tyrosine hydroxylase neurons. Ann. Neurol. 60, 253-255.)' PGP-like, Chymotrysin-like, Trypsin-like | {McNaught, 2001 #1177}. Proteasomal function was determined by continuously measuring the ⁻uorescence of 7-amido-4-methylcoumarin (AMC) (excitation 380 nm, emission 460 nm) generated from peptide-AMC linkedsubstrates [4]. Reactions were conducted in a ®nal volume of 2 ml containing 50 mM Tris±HCl buffer (pH 7.5), 1 mMEDTA, nigral tissue homogenate or puri®ed recombinant 20S proteasome, and initiated by the addition of Suc-Leu-Leu-Val-Try-AMC (65 mM), Boc-Leu-Arg-Arg-AMC (71 mM) or Z-Leu-Leu-Glu-AMC (75 mM) to determine the chymotrypsin-, trypsin- and postacidic- hydrolysing activities of proteasome, respectively. Reactions were followed for 60 min at 25°C, enzymatic activities determined from linear reaction rates, and expressed as ⁻uorescence units (FU)/min per mg protein. Speci®city of proteasomal assay was con®rmed by the ability of 10 mM lactacystin to near-totally inhibit ⁻uorescence change. Results, | |||||
| aging |
Source: Mizushima 2020
urine
| cohort | Overlap with CSF proteins | |
|---|---|---|
| [Karayel 2021 #2257₩] | HBS and LCC | -63% of CSF-abundant proteins (1728) overlaps with urine-abundant proteins (2365) -46% of urine-abundant proteins (2365) overlaps with CSF-abundant proteins (1728) -36% (1080) of all identified proteins in both biofluids 이 overlapping protein 들은 pearson r=0.49 -The most abundant proteins present in both included ALB, PTGDS, ORM1, SERPINA1, B2M and several apolipoproteins and immunoglobulins |
| • |
| GBA activator | GBA GT | Parkin GT | cGAS | |
|---|---|---|---|---|
| 01110 | - PE : Feb 2021 - Retinal - * histology - PLR: budget | Cognition, natural Hx study | - MC1: Tsujihata san, fibroblast availability, readout, - Other mito bm: mitochondrial isolation | - Cgamp prototype established. |
| TPP | GBA PET TCP |
Uncertain Spans
- ‘Detection methods’ 표 우측 두 컬럼은 머리행이 ’…’으로 표기되어 있어 항목 이름 미확정. 본 페이지에서는 ’…’ 그대로 옮겼다.
- ‘Assessment of UPS’ 표 첫 컬럼 라벨 ‘roteolytic ctivity’는 사진 좌단에서 ‘p’와 ‘a’가 잘려 나간 표기로 보임 — 실제로는 ‘proteolytic activity’로 추정되나 본 페이지에서는 시각 그대로 보존.
- 페이지 하단 wide planning table은 우하단 일부 row가 사진 모서리 밖에 걸쳐 절단되었다 — TPP / GBA PET / TCP 행 아래 추가 행이 있는지 본 페이지에서는 확정 불가.