Variations in MHC Class II Antigen Processing and Presentation in Health and Disease.

MHC class II (MHC-II) molecules are critical in the control of many immune responses. They are also involved in most autoimmune diseases and other pathologies. Here, we describe the biology of MHC-II and MHC-II variations that affect immune responses. We discuss the classic cell biology of MHC-II and various perturbations. Proteolysis is a major process in the biology of MHC-II, and we describe the various components forming and controlling this endosomal proteolytic machinery. This process ultimately determines the MHC-II-presented peptidome, including cryptic peptides, modified peptides, and other peptides that are relevant in autoimmune responses. MHC-II also variable in expression, glycosylation, and turnover. We illustrate that MHC-II is variable not only in amino acids (polymorphic) but also in its biology, with consequences for both health and disease.

Targeting Cancer Lysosomes with Good Old Cationic Amphiphilic Drugs.

Being originally discovered as cellular recycling bins, lysosomes are today recognized as versatile signaling organelles that control a wide range of cellular functions that are essential not only for the well-being of normal cells but also for malignant transformation and cancer progression. In addition to their core functions in waste disposal and recycling of macromolecules and energy, lysosomes serve as an indispensable support system for malignant phenotype by promoting cell growth, cytoprotective autophagy, drug resistance, pH homeostasis, invasion, metastasis, and genomic integrity. On the other hand, malignant transformation reduces the stability of lysosomal membranes rendering cancer cells sensitive to lysosome-dependent cell death. Notably, many clinically approved cationic amphiphilic drugs widely used for the treatment of other diseases accumulate in lysosomes, interfere with their cancer-promoting and cancer-supporting functions and destabilize their membranes thereby opening intriguing possibilities for cancer therapy. Here, we review the emerging evidence that supports the supplementation of current cancer therapies with lysosome-targeting cationic amphiphilic drugs.

Cellular depletion of major cathepsin proteases reveals their concerted activities for lysosomal proteolysis.

Proteins delivered by endocytosis or autophagy to lysosomes are degraded by exo- and endoproteases. In humans 15 lysosomal cathepsins (CTS) act as important physiological regulators. The cysteine proteases CTSB and CTSL and the aspartic protease CTSD are the most abundant and functional important lysosomal proteinases. Whereas their general functions in proteolysis in the lysosome, their individual substrate, cleavage specificity, and their possible sequential action on substrate proteins have been previously studied, their functional redundancy is still poorly understood. To address a possible common role of highly expressed and functional important CTS proteases, we generated CTSB-, CTSD-, CTSL-, and CTSBDL-triple deficient (KO) human neuroblastoma-derived SH-SY5Y cells and CTSB-, CTSD-, CTSL-, CTSZ and CTSBDLZ-quadruple deficient (KO) HeLa cells. These cells with a combined cathepsin deficiency exhibited enlarged lysosomes and accumulated lipofuscin-like storage material. The lack of the three (SH-SY5Y) or four (HeLa) major CTSs caused an impaired autophagic flux and reduced degradation of endocytosed albumin. Proteome analyses of parental and CTS-depleted cells revealed an enrichment of cleaved peptides, lysosome/autophagy-associated proteins, and potentially endocytosed membrane proteins like the amyloid precursor protein (APP), which can be subject to endocytic degradation. Amino- and carboxyterminal APP fragments accumulated in the multiple CTS-deficient cells, suggesting that multiple CTS-mediated cleavage events regularly process APP. In summary, our analyses support the idea that different lysosomal cathepsins act in concert, have at least partially and functionally redundant substrates, regulate protein degradation in autophagy, and control cellular proteostasis, as exemplified by their involvement in the degradation of APP fragments.

SARS-CoV-2 Spike Protein Induces Time-Dependent CTSL Upregulation in HeLa Cells and Alveolarspheres.

Autophagy and lysosomal pathways are involved in the cell entry of SARS-CoV-2 virus. To infect the host cell, the spike protein of SARS-CoV-2 binds to the cell surface receptor angiotensin-converting enzyme 2 (ACE2). To allow the fusion of the viral envelope with the host cell membrane, the spike protein has to be cleaved. One possible mechanism is the endocytosis of the SARS-CoV-2-ACE2 complex and subsequent cleavage of the spike protein, mainly by the lysosomal protease cathepsin L. However, detailed molecular and dynamic insights into the role of cathepsin L in viral cell entry remain elusive. To address this, HeLa cells and iPSC-derived alveolarspheres were treated with recombinant SARS-CoV-2 spike protein, and the changes in mRNA and protein levels of cathepsins L, B, and D were monitored. Additionally, we studied the effect of cathepsin L deficiency on spike protein internalization and investigated the influence of the spike protein on cathepsin L promoters in vitro. Furthermore, we analyzed variants in the genes coding for cathepsin L, B, D, and ACE2 possibly associated with disease progression using data from Regeneron's COVID Results Browser and our own cohort of 173 patients with COVID-19, exhibiting a variant of ACE2 showing significant association with COVID-19 disease progression. Our in vitro studies revealed a significant increase in cathepsin L mRNA and protein levels following exposure to the SARS-CoV-2 spike protein in HeLa cells, accompanied by elevated mRNA levels of cathepsin B and D in alveolarspheres. Moreover, an increase in cathepsin L promoter activity was detected in vitro upon spike protein treatment. Notably, the knockout of cathepsin L resulted in reduced internalization of the spike protein. The study highlights the importance of cathepsin L and lysosomal proteases in the SARS-CoV-2 spike protein internalization and suggests the potential of lysosomal proteases as possible therapeutic targets against COVID-19 and other viral infections.

Cathepsin X deficiency alters the processing and localisation of cathepsin L and impairs cleavage of a nuclear cathepsin L substrate.

Proteases function within sophisticated networks. Altering the activity of one protease can have sweeping effects on other proteases, leading to changes in their activity, structure, specificity, localisation, stability, and expression. Using a suite of chemical tools, we investigated the impact of cathepsin X, a lysosomal cysteine protease, on the activity and expression of other cysteine proteases and their inhibitors in dendritic cells. Among all proteases examined, cathepsin X gene deletion specifically altered cathepsin L levels; pro-cathepsin L and its single chain accumulated while the two-chain form was unchanged. This effect was recapitulated by chemical inhibition of cathepsin X, suggesting a dependence on its catalytic activity. We demonstrated that accumulation of pro- and single chain cathepsin L was not due to a lack of direct cleavage by cathepsin X or altered glycosylation, secretion, or mRNA expression but may result from changes in lysosomal oxidative stress or pH. In the absence of active cathepsin X, nuclear cathepsin L and cleavage of the known nuclear cathepsin L substrate, Lamin B1, were diminished. Thus, cathepsin X activity selectively regulates cathepsin L, which has the potential to impact the degree of cathepsin L proteolysis, the nature of substrates that it cleaves, and the location of cleavage.

Unravelling the role of cathepsins in cardiovascular diseases.

Lysosomal cathepsins as a regulatory medium have been assessed as potential therapeutic targets for the treatment of various cardiac diseases such as abdominal aortic aneurysm, hypertension, cardiomyopathy, coronary heart disease, atherosclerosis, etc. They are ubiquitous lysosomal proteases with papain-like folded protein structures that are involved in a variety of physiological processes, such as the digestion of proteins, activation of pro-inflammatory molecules, degradation of extracellular matrix components, and maturation of peptide hormones. Cathepsins are classified into three major groups: cysteine cathepsins, aspartic cathepsins, and serine-threonine cathepsins. Each of these groups is further divided into subgroups based on their substrate specificity, structural characteristics, and biochemical properties. Several studies suggest that cathepsins control the degradation of ECM components such as collagen and elastin fibres. These enzymes are highly expressed in macrophages and inflammatory cells, and their upregulation has been demonstrated to be critical in the progression of atherosclerotic lesions. Additionally, increased cathepsin activity has been linked to increased vascular inflammation and oxidative stress, both of which are associated with CVDs. Specifically, the inhibition of cathepsins may reduce the release of pro-apoptotic mediators such as caspase-3 and PARP-1, which are thought to contribute to plaque instability. The potential of cathepsins as biomarkers and therapeutic targets has also been supported by the identification of potential cathepsin inhibitors, which could be used to modulate the activities of cathepsins in a range of diseases. This review shall familiarise the readers with the role of cysteinyl cathepsins and their inhibitors in the pathogenesis of cardiovascular diseases.

The role of multiple vitellogenins in early development of fishes.

Functions of vitellogenins have been in the limelight of fish reproductive physiology research for decades. The Vtg system of acanthomorph teleosts consists of two complete forms of Vtgs (VtgAa and VtgAb) and an incomplete form, VtgC. Insufficient uptake and processing of Vtgs and their yolk proteins lead to inadequate oocyte hydration ensuing failure in acquisition of egg buoyancy and early developmental deficiencies. This review presents a summary of our studies on utilization of multiple Vtgs in species with different egg buoyancy characteristics, as examples. Studies of moronids revealed limited degradation of all three forms of lipovitellin heavy chain derived from their three respective forms of Vtg, by which they contribute to the free amino acid pool driving oocyte hydration during oocyte maturation. In later studies, CRISPR/Cas9 was employed to invalidate zebrafish type I, type II and type III Vtgs, which are orthologs of acanthamorph VtgAa, VtgAb and VtgC, respectively. Results revealed type I Vtg to have essential developmental and nutritional functions in both late embryos and larvae. Genomic disturbance of type II Vtg led to high mortalities during the first 24 h of embryonic development. Despite being a minor form of Vtg in zebrafish and most other species, type III Vtg was also found to contribute essentially to the developmental potential of zebrafish zygotes and early embryos. Apart from severe effects on progeny survival, these studies also disclosed previously unreported regulatory effects of Vtgs on fecundity and fertility, and on embryo hatching. We recently utilized parallel reactions monitoring based liquid chromatography tandem mass spectrometry to assess the processing and utilization of lipovitellins derived from different forms of Vtg in Atlantic halibut and European plaice. Results showed the Lv heavy chain of VtgAa (LvHAa) to be consumed during oocyte maturation and the Lv light chain of VtgAb (LvLAb) to be utilized specifically during late larval stages, while all remaining YPs (LvLAa, LvHAb, LvHC, and LvLC) were utilized during or after hatching up until first feeding in halibut. In plaice, all YPs except LvHAa, which similarly to halibut supports oocyte maturation, are utilized from late embryo to late larval development up until first feeding. The collective findings from these studies affirm substantial disparity in modes of utilization of different types of Vtgs among fish species with various egg buoyancy characteristics, and they reveal previously unknown regulatory functions of Vtgs in maintenance of reproductive assets such as maternal fecundity and fertility, and in embryonic hatching. Despite the progress that has been made over the past two decades by examining multiple Vtgs and their functions, a higher complexity of these systems with much greater diversity between species in modes of Vtg utilization is now evident. Further research is needed to reveal novel ways each species has evolved to utilize these complex multiple Vtg systems and to discover unifying principles for this evolution in fishes of diverse lineages, habitats and life history characteristics.

Association between cathepsins and skin cancers: A bidirectional two-sample Mendelian randomization study.

BACKGROUND: Several cathepsins have been identified as being involved in the development of cancer. Nevertheless, the connection between cathepsins and skin cancers remained highly elusive. METHODS: A bidirectional Mendelian randomization (MR) analysis was performed to investigate the causal association between cathepsins and skin malignancies. The genome-wide association studies (GWAS) data for cathepsins, malignant melanoma (MM), and basal cell carcinoma (BCC) were obtained from European research. The primary method employed was inverse variance weighted. In addition, MR-Egger, weighted median, weighted mode, and simple mode were also executed. Sensitivity analysis was performed using Cochran's Q test, MR-Egger, and MR-PRESSO. RESULTS: From univariable MR (UVMR), cathepsin H, and S were determined to have a causal relationship with BCC. Additionally, cathepsin H was identified as associated with MM. Multivariable MR (MVMR) showed that after correcting for risk factors of skin carcinoma, cathepsin H was detected to be protective against BCC, whereas cathepsin S has been observed as a risk factor for BCC. No substantial pleiotropy and heterogeneity were identified in the sensitivity analysis. CONCLUSION: This study was the first to establish a direct link between cathepsins and skin malignancies. Cathepsin H and S have the potential to serve as new biomarkers for BCC, offering valuable assistance in the prompt identification, treatment, and prevention of the disease. Nevertheless, additional clinical trials are required to validate our findings.

The Intricate Balance between Life and Death: ROS, Cathepsins, and Their Interplay in Cell Death and Autophagy.

Cellular survival hinges on a delicate balance between accumulating damages and repair mechanisms. In this intricate equilibrium, oxidants, currently considered physiological molecules, can compromise vital cellular components, ultimately triggering cell death. On the other hand, cells possess countermeasures, such as autophagy, which degrades and recycles damaged molecules and organelles, restoring homeostasis. Lysosomes and their enzymatic arsenal, including cathepsins, play critical roles in this balance, influencing the cell's fate toward either apoptosis and other mechanisms of regulated cell death or autophagy. However, the interplay between reactive oxygen species (ROS) and cathepsins in these life-or-death pathways transcends a simple cause-and-effect relationship. These elements directly and indirectly influence each other's activities, creating a complex web of interactions. This review delves into the inner workings of regulated cell death and autophagy, highlighting the pivotal role of ROS and cathepsins in these pathways and their intricate interplay.

Application of a crude digestive proteases preparation to improve the ripening of marinated fillets from low-technological value Baltic herring (Clupea harengus membras L.).

BACKGROUND: As a result of climate change (reduced the oxygen content and food available in the waters) and overfishing, ever larger batches of the herring catch are classified as low-value fish and used for feedstuff or canned food production. Fast and complete ripening of marinated fillets, especially from low-value Baltic herring, poses a problem because of the low muscle protease activity and changes in muscle tissue proteins. RESULTS: For the first time, a crude digestive proteases preparation (CDPP) was obtained from herring viscera using a two-stage method consisting of ethanol extraction and then salt precipitation. CDPP had a reduced hemoglobin content, with optimum activity at pH 7.5-8.8 or 60-120 g kg-1 NaCl. At pH 4-5, it still exhibited 24-68% of proteolytic activity. CDPP was used for 4-24 h of brining of fresh and frozen-thawed fillets or injection of fresh fillets before marinating. CDPP-brining increased especially cathepsin D and carboxypeptidase A activities, whereas it decreased cathepsin B and L activities in the marinades. CDPP-brining mitigated the negative effect of freezing-thawing on mass-yield, protease activity, protein hydrolysis, texture profile, colour and sensory quality of the marinated fillets. CDPP-injection was found to be the best method because it increased mass-yield and ripeness of the marinated fillets to a greater extent than CDPP-brining did. The marinades from the CDPP-treated fillets had no bitter taste as a result of the presence of hemoglobin or chymotrypsin, and there were no results indicating lipid oxidation. CONCLUSION: The application of CDPP in marinating technology is a viable approach to enable the use of low-value herring in food production, shorten the marinating time, and improve the ripeness and sensory quality of meat. © 2024 Society of Chemical Industry.

Enzyme-Activatable Near-Infrared Hemicyanines as Modular Scaffolds for in vivo Photodynamic Therapy.

Photodynamic therapy is an anti-cancer treatment that requires illumination of photosensitizers to induce local cell death. Current near-infrared organic photosensitizers are built from large and non-modular structures that cannot be tuned to improve safety and minimize off-target toxicity. This work describes a novel chemical platform to generate enzyme-activatable near-infrared photosensitizers. We optimized the Se-bridged hemicyanine scaffold to include caging groups and biocompatible moieties, and generated cathepsin-triggered photosensitizers for effective ablation of human glioblastoma cells. Furthermore, we demonstrated that enzyme-activatable Se-bridged hemicyanines are effective photosensitizers for the safe ablation of microtumors in vivo, creating new avenues in the chemical design of targeted anti-cancer photodynamic therapy agents.

Insight into crustacean cathepsins: Structure-evolutionary relationships and functional roles in physiological processes.

Cathepsins belong to a group of proteins that are present in both prokaryotic and eukaryotic organisms and have an extremely high degree of evolutionary conservation. These proteins are functionally active in extracellular environments as soluble enzymatic proteins or attached to plasma membrane receptors. In addition, they occur in cellular secretory vesicles, mitochondria, the cytosol, and within the nuclei of eukaryotic cells. Cathepsins are classified into various groups based on their sequence variations, leading to their structural and functional diversification. The molecular understanding of the physiology of crustaceans has shown that proteases, including cathepsins, are expressed ubiquitously. They also contain one of the central regulatory systems for crustacean reproduction, growth, and immune responses. This review focuses on various aspects of the crustaceans cathepsins and emphasizes their biological roles in different physiological processes such as reproduction, growth, development, and immune responses. We also describe the bioactivity of crustaceans cathepsins. Because of the vital biological roles that cathepsins play as cellular proteases in physiological processes, they have been proposed as potential novel targets for the development of management strategies for the aquaculture industries.

4-Cyanamido-substituted benzenesulfonamides act as dual carbonic anhydrase and cathepsin inhibitors.

A set of novel N-cyano-N-substituted 4-aminobenzenesulfonamide derivatives were synthesized and investigated for their inhibitory activity against four cytosolic carbonic anhydrase (CA, EC 4.2.1.1) isoforms (hCA I, II, VII and XIII) and two cathepsins (S and B). N-alkyl/benzyl-substituted derivatives were revealed to be very potent inhibitors against brain-associated hCA VII, but inactive against both cathepsins. On the other hand, N-acyl-substituted derivatives displayed significant inhibitory activities against cathepsin S, but only moderate to poor inhibitory potency against hCA VII. Both hCA VII and cathepsin S have recently been validated as therapeutic targets in neuropathic pain. This study provided an excellent starting point for further structural optimization of this class of bifunctional compounds to enhance their inhibitory activity and selectivity against hCA VII and cathepsin S and to achieve new compounds with an attractive dual mechanism of action as anti-neuropathic agents.

Expression, Intracellular Localization, and Maturation of Cysteine Cathepsins in Renal Embryonic and Cancer Cell Lines.

Cysteine cathepsins play an important role in tumor development and metastasis. The expression of these enzymes is often increased in many types of tumor cells. Cysteine cathepsins contribute to carcinogenesis through a number of mechanisms, including proteolysis of extracellular matrix and signaling molecules on the cell surface, as well as degradation of transcription factors and disruption of signaling cascades in the cell nucleus. Distinct oncogenic functions have been reported for several members of the cysteine cathepsin family in various types of cancer, but a comparative study of all eleven cysteine cathepsins in one experimental model is still missing. In this work, we assessed and compared the expression, localization, and maturation of all eleven cysteine cathepsins in embryonic kidney cells HEK293 and kidney cancer cell lines 769-P and A-498. We found that the expression of cathepsins V, B, Z, L, and S was 3- to 9-fold higher in kidney tumor cells than in embryonic cells. We also showed that all cysteine cathepsins were present in varying amounts in the nucleus of both embryonic and tumor cells. Notably, more than half of the cathepsin Z or K and over 88% of cathepsin F were localized in tumor cell nuclei. Moreover, mature forms of cysteine cathepsins were more prevalent in tumor cells than in embryonic cells. These results can be further used to develop novel diagnostic tools and may assist in the investigation of cysteine cathepsins as potential therapeutic targets.

Alveolar cells in the mammary gland: lineage commitment and cell death.

The mammary gland provides a spectacular example of physiological cell death whereby the cells that produce milk during lactation are removed swiftly, efficiently, and without inducing inflammation upon the cessation of lactation. The milk-producing cells arise primarily during pregnancy and comprise the alveolar lineage that is specified by signalling pathways and factors that are activated in response to pregnancy hormones. There are at least two alveolar sub-lineages, one of which is marked by the presence of binucleate cells that are especially susceptible to programmed cell death during involution. This process of post-lactational regression, or involution, is carefully orchestrated and occurs in two phases, the first results in a rapid switch in cell fate with the secretory epithelial cells becoming phagocytes whereupon they destroy dead and dying cells from milk. This reversible phase is followed by the second phase that is marked by an influx of immune cells and a remodelling of the gland to replace the alveolar cells with re-differentiated adipocytes, resulting in a return to the pre-pregnant state in preparation for any subsequent pregnancies. The mouse mammary gland provides an excellent experimental tool with which to investigate lineage commitment and the mechanisms of programmed cell death that occur in a normal physiological process. Importantly, involution has highlighted a role for lysoptosis, a mechanism of cell death that is mediated by lysosomal cathepsins and their endogenous inhibitors, serpins. In this review, I discuss alveolar lineage commitment during pregnancy and the programmed cell death pathways that destroy these cells during involution.

Isolation of Nocuolin A and Synthesis of New Oxadiazine Derivatives. Design, Synthesis, Molecular Docking, Apoptotic Evaluation, and Cathepsin B Inhibition.

Nocuolin A (1), an oxadiazine, was isolated from the cyanobacterium Nostoc sp. Its chemical structure was elucidated using NMR and mass spectroscopic data. From this compound, two new oxadiazines, 3-[(6R)-5,6-dihydro-4,6-dipentyl-2H-1,2,3-oxadiazin-2-yl]-3-oxopropyl acetate (2) and 4-{3-[(6R)-5,6-dihydro-4,6-dipentyl-2H-1,2,3-oxadiazin-2-yl]-3-oxopropoxy}-4-oxobutanoic acid (3), were synthesised. The chemical structures of these two compounds were elucidated by a combination of NMR and MS analysis. Compound 3 showed cytotoxicity against the ACHN (0.73 ± 0.10 µM) and Hepa-1c1c7 (0.91 ± 0.08 µM) tumour cell lines. Similarly, compound 3 significantly decreased cathepsin B activity in ACHN and Hepa-1c1c7 tumour cell lines at concentrations of 1.52 ± 0.13 nM and 1.76 ± 0.24 nM, respectively. In addition, compound 3 showed no in vivo toxicity in a murine model treated with a dose of 4 mg/kg body weight.

Reassessing enzyme kinetics: Considering protease-as-substrate interactions in proteolytic networks.

Enzymes are catalysts in biochemical reactions that, by definition, increase rates of reactions without being altered or destroyed. However, when that enzyme is a protease, a subclass of enzymes that hydrolyze other proteins, and that protease is in a multiprotease system, protease-as-substrate dynamics must be included, challenging assumptions of enzyme inertness, shifting kinetic predictions of that system. Protease-on-protease inactivating hydrolysis can alter predicted protease concentrations used to determine pharmaceutical dosing strategies. Cysteine cathepsins are proteases capable of cathepsin cannibalism, where one cathepsin hydrolyzes another with substrate present, and misunderstanding of these dynamics may cause miscalculations of multiple proteases working in one proteolytic network of interactions occurring in a defined compartment. Once rates for individual protease-on-protease binding and catalysis are determined, proteolytic network dynamics can be explored using computational models of cooperative/competitive degradation by multiple proteases in one system, while simultaneously incorporating substrate cleavage. During parameter optimization, it was revealed that additional distraction reactions, where inactivated proteases become competitive inhibitors to remaining, active proteases, occurred, introducing another network reaction node. Taken together, improved predictions of substrate degradation in a multiple protease network were achieved after including reaction terms of autodigestion, inactivation, cannibalism, and distraction, altering kinetic considerations from other enzymatic systems, since enzyme can be lost to proteolytic degradation. We compiled and encoded these dynamics into an online platform (https://plattlab.shinyapps.io/catKLS/) for individual users to test hypotheses of specific perturbations to multiple cathepsins, substrates, and inhibitors, and predict shifts in proteolytic network reactions and system dynamics.

Lysosomes as a Target of Anticancer Therapy.

Lysosomes are organelles containing acidic hydrolases that are responsible for lysosomal degradation and the maintenance of cellular homeostasis. They play an important role in autophagy, as well as in various cell death pathways, such as lysosomal and apoptotic death. Various agents, including drugs, can induce lysosomal membrane permeability, resulting in the translocation of acidic hydrolases into the cytoplasm, which promotes lysosomal-mediated death. This type of death may be of great importance in anti-cancer therapy, as both cancer cells with disturbed pathways leading to apoptosis and drug-resistant cells can undergo it. Important compounds that damage the lysosomal membrane include lysosomotropic compounds, antihistamines, immunosuppressants, DNA-damaging drugs, chemotherapeutics, photosensitizers and various plant compounds. An interesting approach in the treatment of cancer and the search for ways to overcome the chemoresistance of cancer cells may also be combining lysosomotropic compounds with targeted modulators of autophagy to induce cell death. These compounds may be an alternative in oncological treatment, and lysosomes may become a promising therapeutic target for many diseases, including cancer. Understanding the functional relationships between autophagy and apoptosis and the possibilities of their regulation, both in relation to normal and cancer cells, can be used to develop new and more effective anticancer therapies.

Liposomal Delivery of Saquinavir to Macrophages Overcomes Cathepsin Blockade by Mycobacterium tuberculosis and Helps Control the Phagosomal Replicative Niches.

Mycobacterium tuberculosis is able to establish a chronic colonization of lung macrophages in a controlled replication manner, giving rise to a so-called latent infection. Conversely, when intracellular bacteria undergo actively uncontrolled replication rates, they provide the switch for the active infection called tuberculosis to occur. Our group found that the pathogen is able to manipulate the activity of endolysosomal enzymes, cathepsins, directly at the level of gene expression or indirectly by regulating their natural inhibitors, cystatins. To provide evidence for the crucial role of cathepsin manipulation for the success of tuberculosis bacilli in their intracellular survival, we used liposomal delivery of saquinavir. This protease inhibitor was previously found to be able to increase cathepsin proteolytic activity, overcoming the pathogen induced blockade. In this study, we demonstrate that incorporation in liposomes was able to increase the efficiency of saquinavir internalization in macrophages, reducing cytotoxicity at higher concentrations. Consequently, our results show a significant impact on the intracellular killing not only to reference and clinical strains susceptible to current antibiotic therapy but also to multidrug- and extensively drug-resistant (XDR) Mtb strains. Altogether, this indicates the manipulation of cathepsins as a fine-tuning strategy used by the pathogen to survive and replicate in host cells.

Proteolytic Resistance Determines Albumin Nanoparticle Drug Delivery Properties and Increases Cathepsin B, D, and G Expression.

Proteolytic activity is pivotal in maintaining cell homeostasis and function. In pathological conditions such as cancer, it covers a key role in tumor cell viability, spreading to distant organs, and response to the treatment. Endosomes represent one of the major sites of cellular proteolytic activity and very often represent the final destination of internalized nanoformulations. However, little information about nanoparticle impact on the biology of these organelles is available even though they represent the major location of drug release. In this work, we generated albumin nanoparticles with a different resistance to proteolysis by finely tuning the amount of cross-linker used to stabilize the carriers. After careful characterization of the particles and measurement of their degradation in proteolytic conditions, we determined a relationship between their sensitivity to proteases and their drug delivery properties. These phenomena were characterized by an overall increase in the expression of cathepsin proteases regardless of the different sensitivity of the particles to proteolytic degradation.