عنوان

ציסטאין פרוטאזות מסוג קטפסין: ממחקר בסיסי להשלכות טיפוליות

Number

TL58221

.Language of Text, Soundtrack etc

انگلیسی

Title Proper

ציסטאין פרוטאזות מסוג קטפסין: ממחקר בסיסי להשלכות טיפוליות

General Material Designation

[Thesis]

First Statement of Responsibility

Weiss-Sadan, Tommy

Subsequent Statement of Responsibility

Blum, Galia

Name of Publisher, Distributor, etc.

The Hebrew University of Jerusalem (Israel)

Date of Publication, Distribution, etc.

2019

Text of Note

115 p.

Dissertation or thesis details and type of degree

Ph.D.

Body granting the degree

The Hebrew University of Jerusalem (Israel)

Text preceding or following the note

2019

Text of Note

Cysteine proteases cathepsins are lysosomal enzymes that regulate numerous lysosomal functions but were also appreciated for their extra-lysosomal activities and their contribution to various pathologic conditions. Given the diversity in cathepsin function, we set out to explore their functions in context. To this end, we employed high-throughput techniques in combination with unique chemical tools to address the roles of cathepsins in cell metabolism and autophagy. In addition, we took advantage of our published theranostic probe and present its application to alleviate atherosclerosis disease burden in mice. (a) Cathepsins and cell metabolism Evolution endowed cells the fundamental ability to repurpose nutrients for different metabolic tasks. These include the synthesis of lipids and nucleotides, in case of proliferating cells. Whereas these metabolic functions are regulated at various levels, protein degradation emerged as a central mechanism. The lysosomes, being a central metabolic hub, host a bunch of hydrolase enzymes that regulate protein degradation. Cathepsins B and L are typical lysosomal proteases with partially overlapping biological functions. However, their role in cell metabolism remained elusive. Our phenotypic screens in mouse embryonic fibroblasts (MEFs) suggest a distinct role for cathepsin L in regulating glucose metabolism. Here we discovered that cathepsin L deficiency is associated with rapid proliferation rates and enhanced glycolytic activity. An in-depth investigation of the underlying mechanistic features uncovered the lactate dehydrogenase A (LDHA) as a critical node in these settings. In particular, we found that the lactate dehydrogenase A (LDHA) is overexpressed in cathepsin L deficient cells compared to wild types or cathepsin B knockouts. Also, loss of LDHA function dramatically reduced cell viability in these cells, suggesting a causal relationship between LDHA expression and function. Using unbiased metabolomics analysis, we discovered that cathepsin L knockout cells reprogram their metabolic network to shift biosynthetic precursors to support DNA replication and proliferation. Our data thus, highlight the involvement of cathepsin L in regulating metabolic processes such as glycolysis. (b) Cathepsins and Autophagy Apart from their metabolic functions, the lysosomes also regulate homeostatic processes such as clearance of damaged organelles. This mechanism is essential in hyperlipidemic conditions such as in macrophage foam cells to alleviate the accumulation of toxic material and inflammatory activity in macrophages that underlies the etiology vascular disorders. Cathepsins, being prominently lysosomal enzymes, can participate in such homeostatic processes and protect macrophages from damage due to hyperlipidemic stress. To test this hypothesis, we established an in vitro model of lipid-laden macrophages to explore the roles of cathepsins B, L, and S. Using our chemical tools, namely activity-based probes, herein we show that oxidized lipids suppress cathepsin activity. Furthermore, we show that cathepsin inhibition remarkably attenuated autophagic degradation in macrophage foam cells. Similarly, this feature was independently confirmed by shotgun proteomics that unveiled a pivotal role of cathepsin L in a putative cathepsin degradation network. At the physiological level, cathepsin inhibition resulted in mitochondrial stress, which translated into impaired oxidative metabolism, excessive production of reactive oxygen species, and the activation of cellular stress response driven by ATF4-CHOP transcription factors. In addition, transcriptomic analysis of these cells uncovered some genetic similarities with the inflammatory macrophage phenotype (a.k.a M1 macrophages) and increased expression of inflammatory cytokines. Our data thus, highlight the importance of cathepsins for mitochondrial quality control and the amelioration of vascular inflammation in the settings metabolic stress. (c) A Theranostic Cathepsin Activity-Based Probe for Noninvasive Intervention in Cardiovascular Diseases In atherosclerosis, extra-lysosomal cathepsin activity is associated with degradation of collagen and elastin, and hence, render plaques prone to rupture. Not so long ago, we demonstrated the clinical application of activity-based probes to discriminate patients showing full-blown symptoms of advanced cardiovascular disease compared to asymptomatic patients, based on cathepsin B and S activities. Since macrophages are the principal source for both cysteine proteases and inflammatory mediators, we sought to take advantage of our published photosensitizing quenched activity-based probe (PSqABP) to provides a non-invasive means to detect vulnerable plaques and reduce their inflammatory activity by treating lesional macrophages. Here we show that our PS-qABP (YBN14) accumulates in atherosclerotic lesions and attenuate the disease burden by reducing the content of inflammatory cells. We also tested the potential clinical applications of this therapeutic modality by showing that YBN14 binds to activated cathepsins in patients derived aortic tissue specimens. This study demonstrates for the first time the application of molecular photodynamic therapy to treat vulnerable atherosclerotic plaques by a non-invasive means.  In summary, our work sheds light on the importance of lysosomal cathepsins to human physiology as they coordinate different homeostatic processes such as cell metabolism and mitochondrial surveillance. We could also differentiate cathepsin involvement in pathologic conditions and potentially exploit this to relief cardiovascular disease burden.

Subject Term

Angiogenesis

Subject Term

Atherosclerosis

Subject Term

Autophagy

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Bioinformatics

Subject Term

Cancer

Subject Term

Cell cycle

Subject Term

Cell division

Subject Term

Cell growth

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Chromatography

Subject Term

Enzymes

Subject Term

Kinases

Subject Term

Lipids

Subject Term

Mass spectrometry

Subject Term

Metabolism

Subject Term

Metabolites

Subject Term

Mitochondria

Subject Term

Pathology

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Pharmaceutical sciences

Subject Term

Pharmacology

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Physiology

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Plasmids

Subject Term

Proteins

Subject Term

Proteomics

Weiss-Sadan, Tommy

Blum, Galia

The Hebrew University of Jerusalem (Israel)

Electronic name

p

[Thesis]

276903

a

Y