Initiation of acute pancreatitis in mice is independent of fusion between lysosomes and zymogen granules.

The co-localization of the lysosomal protease cathepsin B (CTSB) and the digestive zymogen trypsinogen is a prerequisite for the initiation of acute pancreatitis. However, the exact molecular mechanisms of co-localization are not fully understood. In this study, we investigated the role of lysosomes in the onset of acute pancreatitis by using two different experimental approaches. Using an acinar cell-specific genetic deletion of the ras-related protein Rab7, important for intracellular vesicle trafficking and fusion, we analyzed the subcellular distribution of lysosomal enzymes and the severity of pancreatitis in vivo and ex vivo. Lysosomal permeabilization was performed by the lysosomotropic agent Glycyl-L-phenylalanine 2-naphthylamide (GPN). Acinar cell-specific deletion of Rab7 increased endogenous CTSB activity and despite the lack of re-distribution of CTSB from lysosomes to the secretory vesicles, the activation of CTSB localized in the zymogen compartment still took place leading to trypsinogen activation and pancreatic injury. Disease severity was comparable to controls during the early phase but more severe at later time points. Similarly, GPN did not prevent CTSB activation inside the secretory compartment upon caerulein stimulation, while lysosomal CTSB shifted to the cytosol. Intracellular trypsinogen activation was maintained leading to acute pancreatitis similar to controls. Our results indicate that initiation of acute pancreatitis seems to be independent of the presence of lysosomes and that fusion of lysosomes and zymogen granules is dispensable for the disease onset. Intact lysosomes rather appear to have protective effects at later disease stages.

Cathepsin B Gene Knockout Improves Behavioral Deficits and Reduces Pathology in Models of Neurologic Disorders.

Cathepsin B (CTSB) is a powerful lysosomal protease. This review evaluated CTSB gene knockout (KO) outcomes for amelioration of brain dysfunctions in neurologic diseases and aging animal models. Deletion of the CTSB gene resulted in significant improvements in behavioral deficits, neuropathology, and/or biomarkers in traumatic brain injury, ischemia, inflammatory pain, opiate tolerance, epilepsy, aging, transgenic Alzheimer's disease (AD), and periodontitis AD models as shown in 12 studies. One study found beneficial effects for double CTSB and cathepsin S KO mice in a multiple sclerosis model. Transgenic AD models using amyloid precursor protein (APP) mimicking common sporadic AD in three studies showed that CTSB KO improved memory, neuropathology, and biomarkers; two studies used APP representing rare familial AD and found no CTSB KO effect, and two studies used highly engineered APP constructs and reported slight increases in a biomarker. In clinical studies, all reports found that CTSB enzyme was upregulated in diverse neurologic disorders, including AD in which elevated CTSB was positively correlated with cognitive dysfunction. In a wide range of neurologic animal models, CTSB was also upregulated and not downregulated. Further, human genetic mutation data provided precedence for CTSB upregulation causing disease. Thus, the consilience of data is that CTSB gene KO results in improved brain dysfunction and reduced pathology through blockade of CTSB enzyme upregulation that causes human neurologic disease phenotypes. The overall findings provide strong support for CTSB as a rational drug target and for CTSB inhibitors as therapeutic candidates for a wide range of neurologic disorders. SIGNIFICANCE STATEMENT: This review provides a comprehensive compilation of the extensive data on the effects of deleting the cathepsin B (CTSB) gene in neurological and aging mouse models of brain disorders. Mice lacking the CTSB gene display improved neurobehavioral deficits, reduced neuropathology, and amelioration of neuronal cell death and inflammatory biomarkers. The significance of the compelling CTSB evidence is that the data consilience validates CTSB as a drug target for discovery of CTSB inhibitors as potential therapeutics for treating numerous neurological diseases.

A LRRK2/dLRRK-mediated lysosomal pathway that contributes to glial cell death and DA neuron survival.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial and sporadic Parkinson's disease. A plethora of evidence has indicated a role for LRRK2 in endolysosomal trafficking in neurons, while LRRK2 function in glia, although highly expressed, remains largely unknown. Here, we present evidence that LRRK2/dLRRK mediates a lysosomal pathway that contributes to glial cell death and the survival of dopaminergic (DA) neurons. LRRK2/dLRRK knockdown in the immortalized microglia or flies results in enlarged and swelling lysosomes fewer in number. These lysosomes are less mobile, wrongly acidified, exhibit defective membrane permeability and reduced activity of the lysosome hydrolase cathepsin B. In addition, LRRK2/dLRRK depletion causes glial apoptosis, DA neurodegeneration, and locomotor deficits in an age-dependent manner. Taken together, these findings demonstrate a functional role of LRRK2/dLRRK in regulating the glial lysosomal pathway; deficits in lysosomal biogenesis and function linking to glial apoptosis potentially underlie the mechanism of DA neurodegeneration, providing insights on LRRK2/dLRRK function in normal and pathological brains.

Cathepsin B as a key regulator of ferroptosis in microglia following intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a subtype of stroke marked by elevated mortality and disability rates. Recently, mounting evidence suggests a significant role of ferroptosis in the pathogenesis of ICH. Through a combination of bioinformatics analysis and basic experiments, our goal is to identify the primary cell types and key molecules implicated in ferroptosis post-ICH. This aims to propel the advancement of ferroptosis research, offering potential therapeutic targets for ICH treatment. Our study reveals pronounced ferroptosis in microglia and identifies the target gene, cathepsin B (Ctsb), by analyzing differentially expressed genes following ICH. Ctsb, a cysteine protease primarily located in lysosomes, becomes a focal point in our investigation. Utilizing in vitro and in vivo models, we explore the correlation between Ctsb and ferroptosis in microglia post-ICH. Results demonstrate that ICH and hemin-induced ferroptosis in microglia coincide with elevated levels and activity of Ctsb protein. Effective alleviation of ferroptosis in microglia after ICH is achieved through the inhibition of Ctsb protease activity and protein levels using inhibitors and shRNA. Additionally, a notable increase in m6A methylation levels of Ctsb mRNA post-ICH is observed, suggesting a pivotal role of m6A methylation in regulating Ctsb translation. These research insights deepen our comprehension of the molecular pathways involved in ferroptosis after ICH, underscoring the potential of Ctsb as a promising target for mitigating brain damage resulting from ICH.

Lysosomal ceramides regulate cathepsin B-mediated processing of saposin C and glucocerebrosidase activity.

Variants in multiple lysosomal enzymes increase Parkinson's disease (PD) risk, including the genes encoding glucocerebrosidase (GCase), acid sphingomyelinase (ASMase) and galactosylceramidase. Each of these enzymes generates ceramide by hydrolysis of sphingolipids in lysosomes, but the role of this common pathway in PD pathogenesis has not yet been explored. Variations in GBA1, the gene encoding GCase, are the most common genetic risk factor for PD. The lysosomal enzyme cathepsin B has recently been implicated as an important genetic modifier of disease penetrance in individuals harboring GBA1 variants, suggesting a mechanistic link between these enzymes. Here, we found that ceramide activates cathepsin B, and identified a novel role for cathepsin B in mediating prosaposin cleavage to form saposin C, the lysosomal coactivator of GCase. Interestingly, this pathway was disrupted in Parkin-linked PD models, and upon treatment with inhibitor of ASMase which resulted in decreased ceramide production. Conversely, increasing ceramide production by inhibiting acid ceramidase activity was sufficient to upregulate cathepsin B- and saposin C-mediated activation of GCase. These results highlight a mechanistic link between ceramide and cathepsin B in regulating GCase activity and suggest that targeting lysosomal ceramide or cathepsin B represents an important therapeutic strategy for activating GCase in PD and related disorders.

MiR-223 enhances lipophagy by suppressing CTSB in microglia following lysolecithin-induced demyelination in mice.

BACKGROUND: Lipid droplet (LD)-laden microglia is a key pathological hallmark of multiple sclerosis. The recent discovery of this novel microglial subtype, lipid-droplet-accumulating microglia (LDAM), is notable for increased inflammatory factor secretion and diminished phagocytic capability. Lipophagy, the autophagy-mediated selective degradation of LDs, plays a critical role in this context. This study investigated the involvement of microRNAs (miRNAs) in lipophagy during demyelinating diseases, assessed their capacity to modulate LDAM subtypes, and elucidated the potential underlying mechanisms involved. METHODS: C57BL/6 mice were used for in vivo experiments. Two weeks post demyelination induction at cervical level 4 (C4), histological assessments and confocal imaging were performed to examine LD accumulation in microglia within the lesion site. Autophagic changes were observed using transmission electron microscopy. miRNA and mRNA multi-omics analyses identified differentially expressed miRNAs and mRNAs under demyelinating conditions and the related autophagy target genes. The role of miR-223 in lipophagy under these conditions was specifically explored. In vitro studies, including miR-223 upregulation in BV2 cells via lentiviral infection, validated the bioinformatics findings. Immunofluorescence staining was used to measure LD accumulation, autophagy levels, target gene expression, and inflammatory mediator levels to elucidate the mechanisms of action of miR-223 in LDAM. RESULTS: Oil Red O staining and confocal imaging revealed substantial LD accumulation in the demyelinated spinal cord. Transmission electron microscopy revealed increased numbers of autophagic vacuoles at the injury site. Multi-omics analysis revealed miR-223 as a crucial regulatory gene in lipophagy during demyelination. It was identified that cathepsin B (CTSB) targets miR-223 in autophagy to integrate miRNA, mRNA, and autophagy gene databases. In vitro, miR-223 upregulation suppressed CTSB expression in BV2 cells, augmented autophagy, alleviated LD accumulation, and decreased the expression of the inflammatory mediator IL-1ß. CONCLUSION: These findings indicate that miR-223 plays a pivotal role in lipophagy under demyelinating conditions. By inhibiting CTSB, miR-223 promotes selective LD degradation, thereby reducing the lipid burden and inflammatory phenotype in LDAM. This study broadens the understanding of the molecular mechanisms of lipophagy and proposes lipophagy induction as a potential therapeutic approach to mitigate inflammatory responses in demyelinating diseases.

Modelling how increased Cathepsin B/L and decreased TMPRSS2 usage for cell entry by the SARS-CoV-2 Omicron variant may affect the efficacy and synergy of TMPRSS2 and Cathepsin B/L inhibitors.

The SARS-CoV-2 Omicron variant harbours many mutations in its spike protein compared to the original SARS-CoV-2 strain, which may alter its ability to enter cells, cell tropism, and response to interventions blocking virus entry. To elucidate these effects, we developed a mathematical model of SARS-CoV-2 entry into target cells and applied it to analyse recent in vitro data. SARS-CoV-2 can enter cells via two pathways, one using the host proteases Cathepsin B/L and the other using the host protease TMPRSS2. We found enhanced entry efficiency of the Omicron variant in cells where the original strain preferentially used Cathepsin B/L and reduced efficiency where it used TMPRSS2. The Omicron variant thus appears to have evolved to use the Cathepsin B/L pathway better but at the expense of its ability to use the TMPRSS2 pathway compared to the original strain. We estimated >4-fold enhanced efficiency of the Omicron variant in entry via the Cathepsin B/L pathway and >3-fold reduced efficiency via the TMPRSS2 pathway compared to the original or other strains in a cell type-dependent manner. Our model predicted that Cathepsin B/L inhibitors would be more efficacious and TMPRSS2 inhibitors less efficacious in blocking Omicron variant entry into cells than the original strain. Furthermore, model predictions suggested that drugs simultaneously targeting the two pathways would exhibit synergy. The maximum synergy and drug concentrations yielding it would differ for the Omicron variant compared to the original strain. Our findings provide insights into the cell entry mechanisms of the Omicron variant and have implications for intervention targeting these mechanisms.

A cathepsin B/GSH dual-responsive fluorinated peptide for effective siRNA delivery to cancer cells.

Small interfering RNA (siRNA) can be exploited to silence specific genes associated with cancer development, and successful siRNA therapy is highly dependent on the efficiency of the siRNA delivery vector. Herein, a well-designed novel redox- and enzyme-responsive fluorinated polyarginine (PFC-PR) was developed to be used as an anti-cancer siRNA carrier. The multiple guanidine groups could provide positive charges and bind with siRNA efficiently, and further fluorination modification enhanced the interaction with siRNA, resulting in a more stable PFC-PR/siRNA nanocomplex, improving serum tolerance, and promoting cellular uptake and endosome escape. Meanwhile, the PFC-PR was responsive to overexpressed cathepsin B and high levels of glutathione in cancer cells, conferring its ability to enhance siRNA release within cancer cells and making it cancer-targeting. Consequently, PFC-PR showed good biocompatibility and high gene silencing efficiency, which could inhibit cancer cell growth when delivered the siRNA targeting vascular endothelial growth factor, suggesting that it can be potentially used for anti-cancer gene therapy applications.

Association of AXIN1 rs12921862 C/A and rs1805105 G/A and CTSB rs12898 G/A polymorphisms with papillary thyroid carcinoma: A case-control study.

BACKGROUND: Papillary thyroid cancer (PTC) is the most common type of thyroid cancer which its precise etiology remains unknown. However, environmental and genetic factors contribute to the etiology of PTC. Axis inhibition protein 1 (Axin1) is a scaffold protein that exerts its role as a tumor suppressor. In addition, Cathepsin B (Ctsb) is a cysteine protease with higher expression in several types of tumors. Therefore, the aim of this study was to investigate the possible association of AXIN1 rs12921862 C/A and rs1805105 G/A and CTSB rs12898 G/A polymorphisms with PTC susceptibility. MATERIALS & METHODS: In total, 156 PTC patients and 158 sex-, age-, and BMI-matched control subjects were enrolled in the study. AXIN1 rs12921862 C/A and rs1805105 G/A and CTSB rs12898 G/A polymorphisms were genotyped using the PCR-RFLP method. RESULTS: There was a relationship between AXIN1 rs12921862 C/A polymorphism and an increased risk of PTC in all genetic models except the overdominant model. The AXIN1 rs1805105 G/A polymorphism was associated with an increased PTC risk only in codominant and overdominant models. The frequency of AXIN1 Ars12921862 Ars1805105 haplotype was higher in the PTC group and also this haplotype was associated with an increased risk of PTC. Moreover, the AXIN1 rs12921862 C/A polymorphism was not associated with PTC clinical and pathological findings, but AXIN1 rs1805105 G/A polymorphism was associated with almost three folds of larger tumor size (≥1 cm). There was no association between CTSB rs12898 G/A polymorphism and PTC and its findings. CONCLUSION: The AXIN1 rs12921862 C/A and rs1805105 G/A polymorphisms were associated with PTC. AXIN1 rs1805105 G/A polymorphism was associated with higher tumor size.

Cathepsin B Is Not an Intrinsic Factor Related to Asparaginase Resistance of the Acute Lymphoblastic Leukemia REH Cell Line.

L-Asparaginase (ASNase) is a biopharmaceutical used as an essential drug in the treatment of acute lymphoblastic leukemia (ALL). Yet, some cases of ALL are naturally resistant to ASNase treatment, which results in poor prognosis. The REH ALL cell line, used as a model for studying the most common subtype of ALL, is considered resistant to treatment with ASNase. Cathepsin B (CTSB) is one of the proteases involved in the regulation of in vivo ASNase serum half-life and it has also been associated with the progression and resistance to treatment of several solid tumors. Previous works have shown that, in vitro, ASNase is degraded when incubated with REH cell lysate, which is prevented by a specific CTSB inhibitor, suggesting a function of this protease in the ASNase resistance of REH cells. In this work, we utilized a combination of CRISPR/Cas9 gene targeting and enzymatic measurements to investigate the relevance of CTSB on ASNase treatment resistance in the ALL model cell line. We found that deletion of CTSB in REH ALL cells did not confer ASNase treatment sensitivity, thus suggesting that intrinsic expression of CTSB is not a mechanism that drives the resistant nature of these ALL cells to enzymes used as the first-line treatment against leukemia.

Effects of temperature on cathepsin B, cathepsin D and acid phosphatase during embryo development of the hard tick Haemaphysalis longicornis.

The effects of temperature on the expression patterns and enzyme activity of cathepsin B (HlCatB), cathepsin D (HlCatD) and acid phosphatase (HlACP) during the embryo development of Haemaphysalis longicornis (bisexual population) were investigated in this study. Eggs were exposed to 20 °C (low temperature), 26 °C (normal temperature), and 30 °C (high temperature) immediately after laying, and collected on odd days of embryo development to measure HlCatB, HlCatD and HlACP gene expression using quantitative real-time PCR, as well as three enzyme activities using spectrophotometry. Then the associations between mRNA expression levels of three enzymes and their enzyme activities were assessed. Compared with normal temperature, the mRNA expression peaks of HlCatB were higher and appeared later at low and high temperatures and the activity of HlCatB increased on most days of embryonic development at high temperature. As for HlCatD, the expression peak appeared later at low temperature, but earlier at high temperature. The activity peaks of HlCatD were lower and appeared earlier at low and high temperatures. As for HlACP, the expression peak was higher and appeared later at low temperature, whereas it formed no prominent peak at high temperature. The activity peak of HlACP was higher at low temperature, but lower at high temperature. The linear regression analysis showed that activities of three enzymes were associated with their mRNA expression levels (P < 0.05). Three enzymes are involved in the embryo adaptation to temperature stress. Moreover, the mRNA expression level may be another factor affecting its enzyme activity.

Research progress of cathepsin B in brain aging and Alzheimer's diseases.

Cathepsin B (CatB), a cysteine protease derived from lysosomes, was initially thought to non-selectively degrade proteins from phagocytosis and autophagy in lysosomes. However, CatB has been demonstrated to selectively degrade and specifically activate target proteins, thereby regulating the process of physiological and pathological responses. The expression, enzymatic activity, and cellular localization of CatB are significantly altered in brain aging and age-related neurodegenerative diseases. Therefore, the pathological function of CatB has attracted much attention in neuroscience research. In this review, we systematically summarize the molecular functions of CatB in brain aging and Alzheimer's disease and discuss the current problems in neuropathological studies of CatB, which lay a foundation for a comprehensive understanding of the pathogenesis of aging and Alzheimer's disease.

FRET-Based Cathepsin Probes for Simultaneous Detection of Cathepsin B and D Activities.

Fluorescent cathepsin probes were prepared by modification of peptidic substrates for cathepsin B (CTSB) and cathepsin D (CTSD) with FRET pairs. Fluorophores with distinguishable emission characteristics were applied to CTSB and CTSD probes with their appropriate quenchers to simultaneously monitor the activity of CTSB and/or CTSD. Conjugation of both the CTSB and CTSD probes with short single-stranded DNA drastically increased their reactivity to cathepsins over the parent probes possibly by improving their solubility. The activity of CTSB and CTSD were simultaneously detected by using these orthogonal FRET-based cathepsin probes.

Double deficiency of cathepsin B and L in the mouse pancreas alters trypsin activity without affecting acute pancreatitis severity.

BACKGROUND: Premature intracellular trypsinogen activation has long been considered a key initiator of acute pancreatitis (AP). Cathepsin B (CTSB) activates trypsinogen, while cathepsin L (CTSL) inactivates trypsin(ogen), and both proteins play a role in the onset of AP. METHODS: AP was induced by 7 hourly intraperitoneal injections of cerulein (50 µg/kg) in wild-type and pancreas-specific conditional Ctsb knockout (CtsbΔpan), Ctsl knockout (CtslΔpan), and Ctsb;Ctsl double-knockout (CtsbΔpan;CtslΔpan) mice. Pancreatic samples were collected and analyzed by histology, immunohistochemistry, real-time PCR, and immunoblots. Trypsin activity was measured in pancreatic homogenates. Peripheral blood was collected, and serum amylase activity was measured. RESULTS: Double deletion of Ctsb and Cstl did not affect pancreatic development or mouse growth. After 7 times cerulein injections, double Ctsb and Ctsl deficiency in mouse pancreases increased trypsin activity to the same extent as that in Ctsl-deficient mice, while Ctsb deficiency decreased trypsin activity but did not affect the severity of AP. CtsbΔpan;CtslΔpan mice had comparable serum amylase activity and histopathological changes and displayed similar levels of proinflammatory cytokines, apoptosis, and autophagy activity compared with wild-type, CtsbΔpan, and CtslΔpan mice. CONCLUSION: Double deletion of Ctsb and Ctsl in the mouse pancreas altered intrapancreatic trypsin activity but did not affect disease severity and inflammatory response after cerulein-induced AP.

CHO cathepsin B identified as the protease responsible for a target bispecific antibody fragmentation.

In a previous work we demonstrated that CHO protease caused fragmentation of an expressed bispecific antibody (bsAb) and this detrimental host cell protein (HCP) can be effectively removed through an optimized Protein A wash step. In addition, preliminary evidence suggested that the responsible protease belongs to the threonine or cysteine protease family. In the current study, this protease was further identified as cathepsin B. First, we identified several CHO proteases in the further fractionated Protein A wash using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and this allowed us to select four candidate proteases. Next, by examining the cleavage pattern of each individual protease and comparing it with that observed during purification, cathepsin B was identified as the protease responsible for the observed bsAb fragmentation.

Characteristics of cathepsin members and expression responses to poly I:C challenge in Pacific cod (Gadus macrocephalus).

Cathepsins are major lysosomal enzymes that participate in necessary physiological processes, including protein degradation, tissue differentiation, and innate or adaptive immune responses. According to their proteolytic activity, vertebrate cathepsins are classified as cysteine proteases (cathepsins B, C, F, H, K, L, O, S, V, W, and X or Z), aspartic proteases (cathepsin D and E), and serine proteases (cathepsin A and G). Several cathepsins were reported in teleosts, however, no cathepsin gene has been identified from Pacific cod so far. In the present study, a total of 13 cathepsin genes were identified for Pacific cod. The evolutionary path of each cathepsin gene was demonstrated via analysis of phylogenetic trees, multiple alignments, conserved domains, motif compositions, and tertiary structures. Tissue distribution analysis showed that all cathepsin genes were ubiquitously expressed in eight healthy tissues but they exhibited diverse levels of expression. Several cathepsin genes were found to be highly expressed in the kidney, spleen, head kidney and liver, whereas low or modest levels were detected in the gills, skin, intestines, and heart. Temporal-specific expression of cathepsins in early developmental stages of Pacific cod were also conducted. CTSK, S, F, and Z were highly expressed at 1 dph and 5 dph and decreased later, while CTSL, L1, and L.1 transcript levels gradually increased in a time-dependent manner. Additionally, the expression profiles of cathepsin genes in Pacific cod were evaluated in the spleen and liver after poly I:C challenge. The results indicated that all cathepsin genes were significantly upregulated upon poly I:C stimulation, suggesting that they play key roles in antiviral immune responses in Pacific cod. Our findings establish a foundation for future exploration of the molecular mechanisms of cathepsins in modulating antiviral immunity in Pacific cod.

The secreted inhibitor of invasive cell growth CREG1 is negatively regulated by cathepsin proteases.

Previous clinical and experimental evidence strongly supports a breast cancer-promoting function of the lysosomal protease cathepsin B. However, the cathepsin B-dependent molecular pathways are not completely understood. Here, we studied the cathepsin-mediated secretome changes in the context of the MMTV-PyMT breast cancer mouse model. Employing the cell-conditioned media from tumor-macrophage co-cultures, as well as tumor interstitial fluid obtained by a novel strategy from PyMT mice with differential cathepsin B expression, we identified an important proteolytic and lysosomal signature, highlighting the importance of this organelle and these enzymes in the tumor micro-environment. The Cellular Repressor of E1A Stimulated Genes 1 (CREG1), a secreted endolysosomal glycoprotein, displayed reduced abundance upon over-expression of cathepsin B as well as increased abundance upon cathepsin B deletion or inhibition. Moreover, it was cleaved by cathepsin B in vitro. CREG1 reportedly could act as tumor suppressor. We show that treatment of PyMT tumor cells with recombinant CREG1 reduced proliferation, migration, and invasion; whereas, the opposite was observed with reduced CREG1 expression. This was further validated in vivo by orthotopic transplantation. Our study highlights CREG1 as a key player in tumor-stroma interaction and suggests that cathepsin B sustains malignant cell behavior by reducing the levels of the growth suppressor CREG1 in the tumor microenvironment.

Functional selection of protease inhibitory antibodies.

Critical for diverse biological processes, proteases represent one of the largest families of pharmaceutical targets. To inhibit pathogenic proteases with desired selectivity, monoclonal antibodies (mAbs) hold great promise as research tools and therapeutic agents. However, identification of mAbs with inhibitory functions is challenging because current antibody discovery methods rely on binding rather than inhibition. This study developed a highly efficient selection method for protease inhibitory mAbs by coexpressing 3 recombinant proteins in the periplasmic space of Escherichia coli-an antibody clone, a protease of interest, and a ß-lactamase modified by insertion of a protease cleavable peptide sequence. During functional selection, inhibitory antibodies prevent the protease from cleaving the modified ß-lactamase, thereby allowing the cell to survive in the presence of ampicillin. Using this method to select from synthetic human antibody libraries, we isolated panels of mAbs inhibiting 5 targets of 4 main protease classes: matrix metalloproteinases (MMP-14, a predominant target in metastasis; MMP-9, in neuropathic pain), ß-secretase 1 (BACE-1, an aspartic protease in Alzheimer's disease), cathepsin B (a cysteine protease in cancer), and Alp2 (a serine protease in aspergillosis). Notably, 37 of 41 identified binders were inhibitory. Isolated mAb inhibitors exhibited nanomolar potency, exclusive selectivity, excellent proteolytic stability, and desired biological functions. Particularly, anti-Alp2 Fab A4A1 had a binding affinity of 11 nM and inhibition potency of 14 nM, anti-BACE1 IgG B2B2 reduced amyloid beta (Aß40) production by 80% in cellular assays, and IgG L13 inhibited MMP-9 but not MMP-2/-12/-14 and significantly relieved neuropathic pain development in mice.

Role of cathepsin B of Naegleria fowleri during primary amebic meningoencephalitis.

Naegleria fowleri causes primary amoebic meningoencephalitis in humans and experimental animals. It has been suggested that cysteine proteases of parasites play key roles in metabolism, nutrient uptake, host tissue invasion, and immune evasion. The aim of this work was to evaluate the presence, expression, and role of cathepsin B from N. fowleri in vitro and during PAM. Rabbit-specific polyclonal antibodies against cathepsin B were obtained from rabbit immunization with a synthetic peptide obtained by bioinformatic design. In addition, a probe was designed from mRNA for N. fowleri cathepsin B. Both protein and messenger were detected in fixed trophozoites, trophozoites interacted with polymorphonuclear and histological sections of infected mice. The main cathepsin B distribution was observed in cytoplasm or membrane mainly pseudopods and food-cups while messenger was in nucleus and cytoplasm. Surprisingly, both the messenger and enzyme were observed in extracellular medium. To determine cathepsin B release, we used trophozoites supernatant recovered from nasal passages or brain of infected mice. We observed the highest release in supernatant from recovered brain amoebae, and when we analyzed molecular weight of secreted proteins by immunoblot, we found 30 and 37 kDa bands which were highly immunogenic. Finally, role of cathepsin B during N. fowleri infection was determined; we preincubated trophozoites with E-64, pHMB or antibodies with which we obtained 60%, 100%, and 60% of survival, respectively, in infected mice. These results suggest that cathepsin B plays a role during pathogenesis caused by N. fowleri mainly in adhesion and contributes to nervous tissue damage.

MicroRNA-214-3p Ameliorates LPS-Induced Cardiomyocyte Injury by Inhibiting Cathepsin B.

Myocardial injury is a common complication of sepsis. MicroRNA (miRNA) miR-214-3p is protective against myocardial injury caused by sepsis, but its mechanism in lipopolysaccharide (LPS)- induced cardiomyocyte injury is still unclear. An AC16 cell injury model was induced by LPS treatment. Cell Counting Kit-8 and flow cytometry assay showed decreased cell viability and increased apoptosis in LPS-treated AC16 cells. The levels of caspase- 3, Bax, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), myosin 6 (Myh6), myosin 7 (Myh7), reactive oxygen species (ROS), and malondialdehyde (MDA) were increased in LPS-treated AC16 cells, but the levels of Bcl-2 and superoxide dismutase (SOD) were decreased. MiR-214-3p was down-regulated and cathepsin B (CTSB) was upregulated in LPS-treated AC16 cells. At the same time, miR-214-3p could target CTSB and reduce its expression. We also found that a miR-214-3p mimic or CTSB silencing could significantly reduce LPSinduced apoptosis, decrease ROS, MDA, caspase-3, and Bax and increase SOD and Bcl-2. CTSB silencing could significantly reduce ANP, BNP, Myh6, and Myh7 in LPS-treated AC16 cells. The effects of CTSB silencing were reversed by a miR-214-3p inhibitor. In summary, miR-214-3p could inhibit LPSinduced myocardial injury by targeting CTSB, which provides a new idea for myocardial damage caused by sepsis.