Intrinsically Connected: Therapeutically Targeting the Cathepsin Proteases and the Bcl-2 Family of Protein Substrates as Co-regulators of Apoptosis

Taken with the growing importance of cathepsin-mediated substrate proteolysis in tumor biology and progression, the focus and emphasis placed on therapeutic design and development is coming into fruition. Underpinning this approach is the invariable progression from the direction of fully characterizing cathepsin protease members and their substrate targets, towards targeting such an interaction with tangible therapeutics. The two groups of such substrates that have gained much attention over the years are the pro- and anti- apoptotic protein intermediates from the extrinsic and intrinsic signaling arms of the apoptosis pathway. As proteins that are central to determining cellular fate, some of them present themselves as very favorable candidates for therapeutic targeting. However, considering that both anti- and pro- apoptotic signaling intermediates have been reported to be downstream substrates for certain activated cathepsin proteases, therapeutic targeting approaches based on greater selectivity do need to be given greater consideration. Herein, we review the relationships shared by the cathepsin proteases and the Bcl-2 homology domain proteins, in the context of how the topical approach of adopting ‘BH3-mimetics’ can be explored further in modulating the relationship between the anti- and pro- apoptotic signaling intermediates from the intrinsic apoptosis pathway and their upstream cathepsin protease regulators. Based on this, we highlight important future considerations for improved therapeutic design.

Integrative p53, micro-RNA and Cathepsin Protease Co-Regulatory Expression Networks in Cancer

As the direct regulatory role of p53 and some of its isoform proteins are becoming established in modulating gene expression in cancer research, another aspect of this mode of gene regulation that has captured significant interest over the years is the mechanistic interplay between p53 and micro-RNA transcriptional regulation. The input of this into modulating gene expression for some of the cathepsin family members has been viewed as carrying noticeable importance based on their biological effects during normal cellular homeostasis and cancer progression. While this area is still in its infancy in relation to general cathepsin gene regulation, we review the current p53-regulated micro-RNAs that are generating significant interest through their regulation of cathepsin proteases, thereby strengthening the link between activated p53 forms and cathepsin gene regulation. Additionally, we extend our understanding of this developing relationship to how such micro-RNAs are being utilized as diagnostic or prognostic tools and highlight their future uses in conjunction with cathepsin gene expression as potential biomarkers within a clinical setting.

Acquisition of Host Cytosolic Protein by Toxoplasma gondii Bradyzoites

ABSTRACTToxoplasma gondii is a protozoan parasite that persists in the central nervous system as intracellular chronic stage bradyzoites that are encapsulated by a thick cyst wall. While the cyst wall separates bradyzoites from the host cytosol, it has been posited that small solutes can traverse the cyst wall to sustain bradyzoites. Recently it was found that host cytosolic macromolecules can cross the parasitophorous vacuole and are ingested and digested by actively replicating acute stage tachyzoites. However, the extent to which bradyzoites have an active ingestion pathway remained unknown. To interrogate this, we modified previously published protocols that look at tachyzoite acquisition and digestion of host proteins by measuring parasite accumulation of a host-expressed reporter protein after impairment of an endolysosomal protease (Cathepsin Protease L, CPL). Using two cystogenic parasite strains (ME49 and Pru), we demonstrate that T. gondii bradyzoites can ingest host-derived cytosolic mCherry. Bradyzoites acquire host mCherry within 4 hours of invasion and post-cyst wall formation. This study provides direct evidence that host macromolecules can be internalized by T. gondii bradyzoites across the cyst wall in infected cells.

Toxoplasma Cathepsin Protease B and Aspartyl Protease 1 Are Dispensable for Endolysosomal Protein Digestion

ABSTRACT The lysosome-like vacuolar compartment (VAC) is a major site of proteolysis in the intracellular parasite Toxoplasma gondii. Previous studies have shown that genetic ablation of a VAC-residing cysteine protease, cathepsin protease L (CPL), resulted in the accumulation of undigested protein in the VAC and loss of parasite viability during the chronic stage of infection. However, since the maturation of another VAC localizing protease, cathepsin protease B (CPB), is dependent on CPL, it remained unknown whether these defects result directly from ablation of CPL or indirectly from a lack of CPB maturation. Likewise, although a previously described cathepsin D-like aspartyl protease 1 (ASP1) could also play a role in proteolysis, its definitive residence and function in the Toxoplasma endolysosomal system were not well defined. Here, we demonstrate that CPB is not necessary for protein turnover in the VAC and that CPB-deficient parasites have normal growth and viability in both the acute and chronic stages of infection. We also show that ASP1 depends on CPL for correct maturation, and it resides in the T. gondii VAC, where, similar to CPB, it plays a dispensable role in protein digestion. Taken together with previous work, our findings suggest that CPL is the dominant protease in a hierarchy of proteolytic enzymes within the VAC. This unusual lack of redundancy for CPL in T. gondii makes it a single exploitable target for disrupting chronic toxoplasmosis. IMPORTANCE Roughly one-third of the human population is chronically infected with the intracellular single-celled parasite Toxoplasma gondii, but little is known about how this organism persists inside people. Previous research suggested that a parasite proteolytic enzyme, termed cathepsin protease L, is important for Toxoplasma persistence; however, it remained possible that other associated proteolytic enzymes could also be involved in the long-term survival of the parasite during infection. Here, we show that two proteolytic enzymes associated with cathepsin protease L play dispensable roles and are dependent on cathepsin L to reach maturity, which differs from the corresponding enzymes in humans. These findings establish a divergent hierarchy of proteases and help focus attention principally on cathepsin protease L as a potential target for interrupting Toxoplasma chronic infection.

Toxoplasma Cathepsin Protease B and Aspartyl Protease 1 play recessive roles in endolysosomal protein digestion during infection

ABSTRACTThe lysosome-like vacuolar compartment (VAC) is a major site of proteolysis in the intracellular parasite Toxoplasma gondii. Previous studies have shown that genetic ablation of a VAC-residing cysteine protease, cathepsin protease L (CPL), resulted in accumulation of undigested protein in the VAC and loss of parasite viability during the chronic stage of infection. However, since the maturation of another VAC localizing protease, cathepsin protease B (CPB), is dependent on CPL, it remained unknown whether these defects result directly from ablation of CPL or indirectly from a lack of CPB maturation. Likewise, although a previously described cathepsin D-like aspartyl protease 1 (ASP1) could also play a role in proteolysis, its definitive residence and function in the Toxoplasma endolysosomal system was not well defined. Here we demonstrate that CPB is not necessary for protein turnover in the VAC and that CPB deficient parasites have normal growth and viability in both the acute and chronic stages of infection. We also show that ASP1 depends on CPL for correct maturation and it resides in the T. gondii VAC where, similar to CPB, it plays a dispensable role in protein digestion. Taken together with previous work, our findings suggest that CPL is the dominant protease in a hierarchy of proteolytic enzymes within the VAC. This unusual lack of redundancy for CPL in T. gondii makes it a single exploitable target for disrupting chronic toxoplasmosis.

Cysteine Cathepsin Protease Inhibition: An update on its Diagnostic, Prognostic and Therapeutic Potential in Cancer

In keeping with recent developments in basic research; the importance of the Cathepsins as targets in cancer therapy have taken on increasing importance and given rise to a number of key areas of interest in the clinical setting. In keeping with driving basic research in this area in a translational direction; recent findings have given rise to a number of exciting developments in the areas of cancer diagnosis; prognosis and therapeutic development. As a fast-moving area of research; the focus of this review brings together the latest findings and highlights the translational significance of these developments.

Regulation of the Erythroid Iron Deprivation Response By a TfR2-Scribble-Epor Integrated Module Coupling Iron Sensing with Receptor Presentation

Iron deficiency in humans causes desensitization of erythroid progenitors to erythropoietin, the principal cytokine for erythroid survival, proliferation, and differentiation. This nutrient deprivation response acts in a lineage-selective, non-apoptotic manner to restrain expansion of the tissue responsible for the majority of iron consumption, thereby triaging iron utilization under conditions of deficiency. The molecular basis for this response is incompletely understood but involves transferrin receptor 2 (TfR2) and aconitase enzymatic activity as extra- and intracellular iron sensors. Here, we identify a multi-component, integrated module connecting these two elements of erythroid iron sensing with endo-lysosomal trafficking, receptor surface delivery, and configuration of EpoR signaling. Specifically, iron and aconitase activity modulate the rate of TfR2 catabolism by cathepsin protease activity. Scribble, a conserved regulator of receptor transport and signaling, binds TfR2 and serves as a downstream effector through to its co-catabolism. Scribble levels in turn influence surface delivery of the erythropoietin receptor (EpoR), which physically interacts with both Scribble and TfR2. The TfR2-dependent downregulation of Scribble associated with iron deprivation diminishes surface EpoR density and skews its signaling from JAK-STAT to Akt pathways. Mice with surface-trapped, endocytosis-defective EpoR fail to develop anemia in response to iron deficiency. These findings establish a tissue-specific nutrient sensing pathway relevant to the pathogenesis of human anemias and their resistance to erythropoietin therapy. Disclosures No relevant conflicts of interest to declare.