| Enzyme | substrate | cell type / tissue | animal vs human notes |
|---|---|---|---|
| Cathepsin C | hardly detectable in CNS | protein levels and activity were restored, and Correction: cerezyme & ambroxol → cathepsin L monomeric aSyn decreased (Ambrosi, 2015 #747) cathepsin D cleaves Sap C from precursor prosaposin pathological accumulation of α-syn, concomitant with disruption of autophagy lysosome markers, has been reported in α-galactosidase A-deficient (Fabry) mouse brains [68]. | |
| Alpha-galactosidase | Gb3 | • | Mutations in GLA were found to be overrepresented in PD patients [52]; numerous Fabry patients have been diagnosed with symptoms of Parkinsonism, suggesting an increased risk of developing PD in individuals with GLA mutations [69-71]. Gb3 levels were not significantly different between temporal cortex of control and PD cases [65]; no prodromal clinical features of parkinsonism have been identified to date in Fabry patients [72]. (2019 Huebecker) we did not observe Gb3 substrate accumulation and Gb3 is only expressed at very low levels in the brain. |
| neuraminidase |
xxii) Plasma vs Blood
| analyte | plasma vs blood | Fresh blood | PBMC | lymphocyte | CSF |
|---|---|---|---|---|---|
| Gba activity | - (2018 Medscape) wbc, whole blood, a standard blood test - serum 도 한 건 있음 (xicoy) | ||||
| BMP | (Guedes et al., 2017, PMID: 28890071) SERUM | ||||
| GlcSph | serum | ||||
| Cathepsin D activity | less than 10% of CD is secreted as Pro-CD into the extracellular milieu and is also detected in the serum. 적어서 별로, not correlated with tissue | ||||
| LIMP2 | Serum OK |
Assessment of lysosome
| category | In vitro | Animal brain | Human brain | Human CSF | Mouse | |
|---|---|---|---|---|---|---|
| (global) | 1. Short lived protein | ab234622 (a proprietary Lysosome-Specific Self-Quenched Substrate) |
Very challenging (Mizushima, 2020 #1168) the use of a global translation inhibitor to halt protein synthesis, (eg. cycloheximide, 단점: 이렇게 하면 proteolytic 에 관여하는 (short-lived protein 들도 안 생산) → followed by measurements of decreasing levels of a protein of interest (a "chase") by SDS-PAGE and immunoblotting or by other means. | |||
| Proteolysis | 2. Long lived proteins |
Mazzulli 2011: GD ips neuron: radioactive (3H-leucine) pulse chase. (suppl2) ↑ long lived protein by GBA KD in neurons a decline in proteolysis of long-lived proteins compared to WT cells, and the addition of lysosomal inhibitors did not further affect proteolysis (Figure 2C). Proteolysis measurement of short-lived proteins revealed no change compared to WT cells (Figure 2C, inset) [mouse primary astrocytes] (Henry, 2015 #1174) Pulse-chase assay, long lived protein 용임: [3H]-valine for 24 h. Protein degradation was measured as the amount of acid-precipitatable radioactivity released into the media and is expressed as the percentage of radioactivity released in the media compared with that remaining in the cell lysate; {Jennings 2022 #2283, DNL201} Protein turnover was measured using I-azidohomoalanine (AHA) labeling to quantify long-lived protein degradation. AHA is used as a surrogate for I-methionine, and the AHA probe is incorporated into proteins during protein synthesis. After a short chase to remove short-lived proteins, the amount of long-lived AHA-containing proteins was visualized through a "click" reaction between the AHA probe and a fluorescently tagged alkyne probe. {Logan, 2020 #1215} Denali: Grn KO cell phenotypes were fully rescued with PTV:PGRN, including lysosomal proteolysis (혹시 Cathepsin activity?) and BMP |
위 Henry 방법에서, chloroquine was used to measure lysosomal degradation. 위 Mazulli 에서: For lysosomal, leupeptin / NH4Cl were used. 위 Jennings 2022 #2283 에서 bafilomycin A (to assess the lysosomal component of long-lived degradation) | (Marwaha, 2017 #1325) (Yadavalli, 2023 #2421) Js: the gap here is a big caveat! | ||
| (general) | xxv) #2 Total protein | DQ-Red BSA: (결국 albumin degradation 보는 거네!) which is a BSA (bovine serum albumin) derived cargo heavily labeled with BODIPY TR-X dye, Degradation of DQ-Red BSA in acidic, hydrolase active endo-lysosomes results in smaller protein fragments that have isolated fluorophores, hence de-quenching the dye that can be visualized as a bright fluorescence in cells. The excitation and emission maxima for this dye are ~590 nm and ~620 nm respectively. Under normal conditions, DQ-Red BSA traffics to lysosomes and is cleaved by lysosomal hydrolases, resulting in bright red fluorescent signal {Frost, 2017 #1326} | ||||
| (general) | xxvi) #2 The stable isotope labelling kinetics | |||||
| Lysosomal pH | xxvii) LK |
LysoTracker: Weakly basic amines selectively accumulate in cellular compartments with low internal pH and can be used to investigate the biosynthesis and pathogenesis of lysosomes.1,2 The LysoTracker® probes are fluorescent acidotropic probes for labeling and tracking acidic organelles in live cells.4,5 Lysosensor Yellow/Blue DND-160 (Invitrogen). researchers studying the dynamic aspects of lysosome biogenesis and function in live cells, we offer LysoSensor probes — fluorescent pH indicators that partition into acidic organelles. The LysoSensor™ dyes are acidotropic probes that appear to accumulate in acidic organelles as the result of protonation. Derek: This is a very challenging assay to optimize and may take several attempts. Degradation and enzymatic assays may be more informative endpoints for lysosomal health. | near-infrared (NIR) boron complex (HCy-BIZ-BF2) with lysosome-targeting and pH-sensing properties, Employing a classic rhodamine-hemicyanine hybrid. (js: it seems that rhodamine is a toxic substance) | |||
| Lysosomal hydrolytic enzyme activity |
-Cathepsin B (magic red) https://www.alzforum.org/protocols/vivo-cathepsin-d-and-cathepsin-b-activity-assay (막장보니 in vitro 용이네), -Cathepsin B and L activity were assayed as described previously (Nakanishi et al., 1994) and Cathepsin D activity was assayed using [125I]methemoglobin as previously described (Dottavio-Martin and Ravel, 1978). |
(Shi, 2019 #1722) mouse body Rocha, 2015 #1253: mouse brain homogenate |
postmortem brain 가능 (kurzawa 2012) (Chu, 2009 #266) |
(Youn, 2018 #643) early PD patients ELISA 어디 분명 있을듯 한데 못 찾겠네 | ||
| (autophagic) | Gold nanoparticle (AuNC) |
Gold nanoparticle (AuNC): Following cellular uptake, most nanomaterials will locate to lysosomes [12, 13] 이건 proteolysis는 아니네 (Mizushima, 2020 #1168): detailed review - p62 is also called sequestosome 1 (SQSTM1), and binds to ubiquitinated proteins and interacts with LC3, promoting autophagic degradation of otherwise proteasomal substrates (Osellame et al., 2013). |
Source: Mizushima 2020
Process
| Molecules or ligands internalized from the plasma membrane (endocytosis) | Early endosome | Late endosome | 2. Fused with lysosome or |
| (vesicles up to 1 µm in diameter with connected tubules of approx. 50 nm diameter) | Multivesicular appearance, mainly spherical, lack tubules, and contain many close-packed intraluminal vesicles. | ||
| Marker | Markers include RAB5A and RAB4, Transferrin and … | Markers include RAB7, RAB9, … |
Uncertain Spans
| location | transcription | uncertainty |
|---|---|---|
Plasma vs Blood xicoy | serum 도 한 건 있음 (xicoy) | xicoy may be a Korean shorthand; reading preserved as visible. |
| Henry pulse-chase | [3H]-valine | The radiolabel reading valine may be lecuine due to OCR; preserved as visible. |
| Cathepsin notes | [125I]methemoglobin | The isotope label is rendered as [CJmethemoglobin in the OCR but the visible text reads [125I]methemoglobin; preserved with the conventional reading. |
| Process row | Markers include RAB5A and RAB4, Transferrin and ... | The trailing markers list is cut off at the bottom of the photo; rendered as ellipsis. |