Discovery of small molecules that normalize the transcriptome and enhance cysteine cathepsin activity in progranulin-deficient microglia

The sensitivity of cultured human and hamster fibroblast cells to killing by the lysosomotropic detergent N-dodecylimidazole (C12-Im) was investigated as a function of cellular levels of general lysosomal hydrolase activity, and specifically of cysteine cathepsin activity. Fibroblasts from patients with mucolipidosis II (I-cell disease) lack mannose-6-phosphate-containing proteins, and therefore possess only 10-15% of the normal level of most lysosomal hydrolases. I-cell fibroblasts are about one-half as sensitive to killing by C12-Im as are normal human fibroblasts. Overall lysosomal enzyme levels of CHO cells were experimentally manipulated in several ways without affecting cell viability: Growth in the presence of 10 mM ammonium chloride resulted in a gradual decrease in lysosomal enzyme content to 10-20% of control values within 3 d. Subsequent removal of ammonium chloride from the growth medium resulted in an increase in lysosomal enzymes, to approximately 125% of control values within 24 h. Treatment with 80 mM sucrose caused extensive vacuolization within 2 h; lysosomal enzyme levels remained at control levels for at least 6 h, but increased 15-fold after 24 h of treatment. Treatment with concanavalin A (50 micrograms/ml) also caused rapid (within 2 h) vacuolation with a sevenfold rise in lysosomal enzyme levels occurring only after 24 h. The sensitivity of these experimentally manipulated cells to killing by C12-Im always paralleled the measured intracellular lysosomal enzyme levels: lower levels were associated with decreased sensitivity while higher levels were associated with increased sensitivity, regardless of the degree of vacuolization of the cells. The cytotoxicity of the cysteine proteases (chiefly cathepsin L in our cells) was tested by inactivating them with the irreversible inhibitor E-64 (100 micrograms/ml). Cell viability, protein levels, and other lysosomal enzymes were unaffected, but cysteine cathepsin activity was reduced to less than 20% of control values. E-64-treated cells were almost completely resistant to C12-Im treatment, although lysosomal disruption appeared normal by fluorescent visualization of Lucifer Yellow CH-loaded cells. It is concluded that cysteine cathepsins are the major or sole cytotoxic agents released from lysosomes by C12-Im. These observations also confirm the previous conclusions that C12-Im kills cells as a consequence of lysosomal disruption.

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After one year of the COVID-19 pandemic and hundreds of suggested drugs, will cathepsin L inhibitors be the solution?

Avicenna ◽

10.5339/avi.2022.2 ◽

2021 ◽

Vol 2022 (1) ◽

Author(s):

Amr Ahmed ◽

Mohammad Nezami ◽

Abdullah Alkattan ◽

Ahmed Mohamed ◽

Omar Alshazly ◽

...

Keyword(s):

Active Sites ◽

Viral Infections ◽

Cathepsin L ◽

Docking Studies ◽

Host Cells ◽

Endosomal Membrane ◽

Cysteine Cathepsins ◽

Cathepsin Activity ◽

Cysteine Cathepsin ◽

One Year

Cysteine cathepsins are defined as lysosomal enzymes that are members of the papain family. Cysteine cathepsins (Cts) prevalently exist in whole organisms, varying from prokaryotes to mammals, and possess greatly conserved cysteine residues in their active sites. Cts are engaged in the digestion of cellular proteins, activation of zymogens, and remodeling of the extracellular matrix (ECM). Host cells are entered by SARS-CoV-2 via endocytosis. Cathepsin L and phosphatidylinositol 3-phosphate 5-kinase are crucial in endocytosis by cleaving the spike protein, which permits viral membrane fusion with the endosomal membrane and succeeds in the release of the viral genome to the host cell. Therefore, inhibition of cathepsin L may be advantageous in terms of decreasing infection caused by SARS-CoV-2. Coordinate inhibition of multiple Cts and lysosomal function by different drugs and biological agents might be of value for some purposes, such as a parasite or viral infections and antineoplastic applications. Zn2+ deficiency or dysregulation leads to exaggerated cysteine cathepsin activity, increasing the autoimmune/inflammatory response. For this purpose, Zn2+ metal can be safely combined with a drug that increases the anti-proteolytic effect of endogenous Zn2+, lowering the excessive activity of some CysCts. Biguanide derivative complexes with Zn2+ have been found to be promising inhibitors of CysCts protease reactions. Molecular docking studies of cathepsin L inhibited by the metformin-Zn+2 complex have been performed, showing two strong key interactions (Cys-25&His-163) and an extra H-bond with Asp-163 compared to cocrystallized Zn+2 (PDB ID 4axl).

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C-terminal α-synuclein truncations are linked to cysteine cathepsin activity in Parkinson's disease

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.008930 ◽

2019 ◽

Vol 294 (25) ◽

pp. 9973-9984 ◽

Cited By ~ 11

Author(s):

Ryan P. McGlinchey ◽

Shannon M. Lacy ◽

Katherine E. Huffer ◽

Nahid Tayebi ◽

Ellen Sidransky ◽

...

Keyword(s):

Parkinson’S Disease ◽

Amino Acids ◽

Parkinson's Disease ◽

Amyloid Fibrils ◽

Lewy Bodies ◽

Amyloid Formation ◽

Pathological Feature ◽

Cysteine Cathepsins ◽

Cathepsin Activity ◽

Cysteine Cathepsin

A pathological feature of Parkinson's disease (PD) is Lewy bodies (LBs) composed of α-synuclein (α-syn) amyloid fibrils. α-Syn is a 140 amino acids–long protein, but truncated α-syn is enriched in LBs. The proteolytic processes that generate these truncations are not well-understood. On the basis of our previous work, we propose that these truncations could originate from lysosomal activity attributable to cysteine cathepsins (Cts). Here, using a transgenic SNCAA53T mouse model, overexpressing the PD-associated α-syn variant A53T, we compared levels of α-syn species in purified brain lysosomes from nonsymptomatic mice with those in age-matched symptomatic mice. In the symptomatic mice, antibody epitope mapping revealed enrichment of C-terminal truncations, resulting from CtsB, CtsL, and asparagine endopeptidase. We did not observe changes in individual cathepsin activities, suggesting that the increased levels of C-terminal α-syn truncations are because of the burden of aggregated α-syn. Using LC-MS and purified α-syn, we identified C-terminal truncations corresponding to amino acids 1–122 and 1–90 from the SNCAA53T lysosomes. Feeding rat dopaminergic N27 cells with exogenous α-syn fibrils confirmed that these fragments originate from incomplete fibril degradation in lysosomes. We mimicked these events in situ by asparagine endopeptidase degradation of α-syn fibrils. Importantly, the resulting C-terminally truncated fibrils acted as superior seeds in stimulating α-syn aggregation compared with that of the full-length fibrils. These results unequivocally show that C-terminal α-syn truncations in LBs are linked to Cts activities, promote amyloid formation, and contribute to PD pathogenesis.

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Non-Invasive Imaging of Cysteine Cathepsin Activity in Solid Tumors Using a 64Cu-Labeled Activity-Based Probe

PLoS ONE ◽

10.1371/journal.pone.0028029 ◽

2011 ◽

Vol 6 (11) ◽

pp. e28029 ◽

Cited By ~ 28

Author(s):

Gang Ren ◽

Galia Blum ◽

Martijn Verdoes ◽

Hongguang Liu ◽

Salahuddin Syed ◽

...

Keyword(s):

Solid Tumors ◽

Non Invasive ◽

Cathepsin Activity ◽

Cysteine Cathepsin

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The effects of Cstb duplication on APP/amyloid-β pathology and cathepsin B activity in a mouse model

PLoS ONE ◽

10.1371/journal.pone.0242236 ◽

2021 ◽

Vol 16 (7) ◽

pp. e0242236

Author(s):

Yixing Wu ◽

Heather T. Whittaker ◽

Suzanna Noy ◽

Karen Cleverley ◽

Veronique Brault ◽

...

Keyword(s):

Mouse Model ◽

Cathepsin B ◽

Single Gene ◽

Amyloid Β ◽

Chromosome 21 ◽

Cathepsin Activity ◽

Cysteine Cathepsin ◽

Increased Risk ◽

Old Mice

People with Down syndrome (DS), caused by trisomy of chromosome 21 have a greatly increased risk of developing Alzheimer’s disease (AD). This is in part because of triplication of a chromosome 21 gene, APP. This gene encodes amyloid precursor protein, which is cleaved to form amyloid-β that accumulates in the brains of people who have AD. Recent experimental results demonstrate that a gene or genes on chromosome 21, other than APP, when triplicated significantly accelerate amyloid-β pathology in a transgenic mouse model of amyloid-β deposition. Multiple lines of evidence indicate that cysteine cathepsin activity influences APP cleavage and amyloid-β accumulation. Located on human chromosome 21 (Hsa21) is an endogenous inhibitor of cathepsin proteases, CYSTATIN B (CSTB) which is proposed to regulate cysteine cathepsin activity in vivo. Here we determined if three copies of the mouse gene Cstb is sufficient to modulate amyloid-β accumulation and cathepsin activity in a transgenic APP mouse model. Duplication of Cstb resulted in an increase in transcriptional and translational levels of Cstb in the mouse cortex but had no effect on the deposition of insoluble amyloid-β plaques or the levels of soluble or insoluble amyloid-β42, amyloid-β40, or amyloid-β38 in 6-month old mice. In addition, the increased CSTB did not alter the activity of cathepsin B enzyme in the cortex of 3-month or 6-month old mice. These results indicate that the single-gene duplication of Cstb is insufficient to elicit a disease-modifying phenotype in the dupCstb x tgAPP mice, underscoring the complexity of the genetic basis of AD-DS and the importance of multiple gene interactions in disease.

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T1859 Cysteine-Cathepsin Activity Reveals Dynamics of Polyp Growth

Gastroenterology ◽

10.1016/s0016-5085(08)62700-6 ◽

2008 ◽

Vol 134 (4) ◽

pp. A-578

Author(s):

Elias Gounaris ◽

Fotini Gounari ◽

Ching-Hsuan Tung ◽

Ralph Weissleder ◽

Khashayarsha Khazaie

Keyword(s):

Cathepsin Activity ◽

Cysteine Cathepsin

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Functional in vivo imaging of cysteine cathepsin activity in murine model of inflammation

Bioorganic & Medicinal Chemistry ◽

10.1016/j.bmc.2010.10.028 ◽

2011 ◽

Vol 19 (3) ◽

pp. 1055-1061 ◽

Cited By ~ 34

Author(s):

Dejan Caglič ◽

Anja Globisch ◽

Maik Kindermann ◽

Ngee-Han Lim ◽

Volker Jeske ◽

...

Keyword(s):

Murine Model ◽

In Vivo Imaging ◽

Cathepsin Activity ◽

Cysteine Cathepsin

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Measuring Cysteine Cathepsin Activity to Detect Lysosomal Membrane Permeabilization

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot087114 ◽

2016 ◽

Vol 2016 (5) ◽

pp. pdb.prot087114 ◽

Cited By ~ 5

Author(s):

Urška Repnik ◽

Maruša Hafner Česen ◽

Boris Turk

Keyword(s):

Membrane Permeabilization ◽

Lysosomal Membrane ◽

Lysosomal Membrane Permeabilization ◽

Cathepsin Activity ◽

Cysteine Cathepsin

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The effects of CSTB duplication on APP/amyloid-β pathology and cathepsin activity in a mouse model

10.1101/2020.10.30.362004 ◽

2020 ◽

Author(s):

Yixing Wu ◽

Heather T. Whittaker ◽

Suzanna Noy ◽

Karen Cleverley ◽

Veronique Brault ◽

...

Keyword(s):

Mouse Model ◽

Single Gene ◽

Amyloid Β ◽

Chromosome 21 ◽

Cathepsin Proteases ◽

Cathepsin Activity ◽

Cysteine Cathepsin ◽

Increased Risk ◽

Old Mice

AbstractPeople with Down syndrome (DS), caused by trisomy of chromosome 21 have a greatly increased risk of developing Alzheimer’s disease (AD). This is in part because of triplication of a chromosome 21 gene, APP. This gene encodes amyloid precursor protein, which is cleaved to form amyloid-β that accumulates in the brains of people who have AD. Recent experimental results demonstrate that a gene or genes on chromosome 21, other than APP, when triplicated significantly accelerate amyloid pathology in a transgenic mouse model of amyloid-β deposition. Multiple lines of evidence indicate that cysteine cathepsin activity influences APP cleavage and amyloid-β accumulation. Located on human chromosome 21 (Hsa21) is an endogenous inhibitor of cathepsin proteases, CYSTATIN B (CSTB) which is proposed to regulate cysteine cathepsin activity in vivo. Here we determined if three copies of the mouse gene Cstb is sufficient to modulate beta amyloid (Aβ) accumulation and cathepsin activity in a transgenic APP mouse model. Duplication of Cstb resulted in an increase in transcriptional and translational levels of Cstb in the mouse cortex but had no effect on the deposition of insoluble Aβ plaques or the levels of soluble or insoluble Aβ42, Aβ40, or Aβ38 in 6-month old mice. In addition, the increased CSTB did not alter the activity of cathepsin B enzyme in the cortex of 3-month old mice. These results indicate that the single-gene duplication of Cstb is insufficient to elicit a disease-modifying phenotype in the dupCstb x tgAPP mice, underscoring the complexity of the genetic basis of AD-DS and the importance of multiple gene interactions in disease.

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