Search Results (9)

Alzheimer’s disease (AD) is the most prevalent cause of dementia and can be conceptualized as a tauopathy initiated by the accumulation of amyloid-β (Aβ) in the brain. The clinical introduction of anti-Aβ antibody therapies has marked the beginning of a new era in disease-modifying treatment for dementia. While the deleterious effects of Aβ on postsynaptic spines and axonal microtubules have been increasingly clarified, recent studies have shifted attention beyond extracellular Aβ deposition as senile plaques to the pathogenic significance of intracellular Aβ. In particular, accumulating evidence highlights lysosomes as critical sites of intracellular Aβ toxicity. Interactions between Aβ and gangliosides, v-ATPase-dependent lysosomal acidification, and lysosomal membrane integrity are the key determinants of disease progression. In parallel, additional molecular players, including components of the complement cascade and asparaginyl endopeptidase, have been implicated in linking Aβ pathology to tau dysregulation and neurodegeneration. As therapeutic strategies targeting Aβ enter clinical practice, these emerging pathways represent promising targets for the next generation of AD treatment. Here, we summarize current insights and ongoing therapeutic developments centered on these mechanisms. Full article

Antibody arrays play a pivotal role in the detection and quantification of biomolecules, with their effectiveness largely dependent on efficient protein immobilization. Traditional methods often use heterobifunctional cross-linking reagents for attaching functional residues in proteins to corresponding chemical groups on the substrate surface. However, this method does not control the antibody’s anchoring point and orientation, potentially leading to reduced binding efficiency and overall performance. Another method using anti-antibodies as intermediate molecules to control the orientation can be used but it demonstrates lower efficiency. Here, we demonstrate a site-specific protein immobilization strategy utilizing OaAEP1 (asparaginyl endopeptidase) for building a nanobody array. Moreover, we used a nanobody-targeting enhanced green fluorescent protein (eGFP) as the model system to validate the protein immobilization method for building a nanobody array. Finally, by rapidly enriching eGFP, this method further highlights its potential for rapid diagnostic applications. This approach, characterized by its simplicity, high efficiency, and specificity, offers an advancement in the development of surface-modified protein arrays. It promises to enhance the sensitivity and accuracy of biomolecule detection, paving the way for broader applications in various research and diagnostic fields. Full article

Legumain is a lysosomal cysteine protease that has been implicated in an increasing amount of physiological and pathophysiological processes. However, the upstream mechanisms regulating the expression and function of legumain are not well understood. Here, we provide in vitro and in vivo data showing that vitamin D₃ (VD₃) enhances legumain expression and function. In turn, legumain alters VD₃ bioavailability, possibly through proteolytic cleavage of vitamin D binding protein (VDBP). Active VD₃ (1,25(OH)₂D₃) increased legumain expression, activity, and secretion in osteogenic cultures of human bone marrow stromal cells. Upregulation of legumain was also observed in vivo, evidenced by increased legumain mRNA in the liver and spleen, as well as increased legumain activity in kidneys from wild-type mice treated with 25(OH)D₃ (50 µg/kg, subcutaneously) for 8 days compared to a control. In addition, the serum level of legumain was also increased. We further showed that active legumain cleaved purified VDBP (55 kDa) in vitro, forming a 45 kDa fragment. In vivo, no VDBP cleavage was found in kidneys or liver from legumain-deficient mice (Lgmn^−/−), whereas VDBP was cleaved in wild-type control mice (Lgmn^+/+). Finally, legumain deficiency resulted in increased plasma levels of 25(OH)D₃ and total VD₃ and altered expression of key renal enzymes involved in VD₃ metabolism (CYP24A1 and CYP27B1). In conclusion, a regulatory interplay between VD₃ and legumain is suggested. Full article

Stem-cell-based therapy is very promising for Alzheimer’s disease (AD), yet has not become a reality. A critical challenge is the transplantation microenvironment, which impacts the therapeutic effect of stem cells. In AD brains, amyloid-beta (Aβ) peptides and inflammatory cytokines continuously poison the tissue microenvironment, leading to low survival of grafted cells and restricted efficacy. It is necessary to create a growth-supporting microenvironment for transplanted cells. Recent advances in AD studies suggest that the asparaginyl endopeptidase (AEP) is a potential intervention target for modifying pathological changes. We here chose APP/PS1 mice as an AD model and employed pharmacological inhibition of the AEP for one month to improve the brain microenvironment. Thereafter, we transplanted neural stem cells (NSCs) into the hippocampus and maintained therapy for one more month. We found that inhibition of AEPs resulted in a significant decrease of Aβ, TNF-α, IL-6 and IL-1β in their brains. In AD mice receiving NSC transplantation alone, the survival of NSCs was at a low level, while in combination with AEP inhibition pre-treatment the survival rate of engrafted cells was doubled. Within the 2-month treatment period, implantation of NSCs plus pre-inhibition of the AEP significantly enhanced neural plasticity of the hippocampus and rescued cognitive impairment. Neither NSC transplantation alone nor AEP inhibition alone achieved significant efficacy. In conclusion, pharmacological inhibition of the AEP ameliorated brain microenvironment of AD mice, and thus improved the survival and therapeutic efficacy of transplanted stem cells. Full article

The cysteine protease legumain (also known as asparaginyl endopeptidase or δ-secretase) is the only known mammalian asparaginyl endopeptidase and is primarily localized to the endolysosomal system, although it is also found extracellularly as a secreted protein. Legumain is involved in the regulation of diverse biological processes and tissue homeostasis, and in the pathogenesis of various malignant and nonmalignant diseases. In addition to its proteolytic activity that leads to the degradation or activation of different substrates, legumain has also been shown to have a nonproteolytic ligase function. This review summarizes the current knowledge about legumain functions in health and disease, including kidney homeostasis, hematopoietic homeostasis, bone remodeling, cardiovascular and cerebrovascular diseases, fibrosis, aging and senescence, neurodegenerative diseases and cancer. In addition, this review addresses the effects of some marketed drugs on legumain. Expanding our knowledge on legumain will delineate the importance of this enzyme in regulating physiological processes and disease conditions. Full article

Legumain is a lysosomal cysteine protease with strict specificity for cleaving after asparagine residues. By sequence comparison, legumain belongs to MEROPS clan CD of the cysteine proteases, which indicates its structural and mechanistic relation to caspases. Contrasting caspases, legumain harbors a pH-dependent ligase activity in addition to the protease activity. Although we already have a significant body of knowledge on the catalytic activities of legumain, many mechanistic details are still elusive. In this study, we provide evidence that extended active site residues and substrate conformation are steering legumain activities. Biochemical experiments and bioinformatics analysis showed that the catalytic Cys189 and His148 residues are regulated by sterically close Glu190, Ser215 and Asn42 residues. While Glu190 serves as an activity brake, Ser215 and Asn42 have a favorable effect on legumain protease activity. Mutagenesis studies using caspase-9 as model enzyme additionally showed that a similar Glu190 activity brake is also implemented in the caspases. Furthermore, we show that the substrate’s conformational flexibility determines whether it will be hydrolyzed or ligated by legumain. The functional understanding of the extended active site residues and of substrate prerequisites will allow us to engineer proteases with increased enzymatic activity and better ligase substrates, with relevance for biotechnological applications. Full article

Alzheimer’s disease (AD) is incurable dementia closely associated with aging. Most cases of AD are sporadic, and very few are inherited; the pathogenesis of sporadic AD is complex and remains to be elucidated. The asparaginyl endopeptidase (AEP) or legumain is the only recognized cysteine protease that specifically hydrolyzes peptide bonds after asparagine residues in mammals. The expression level of AEPs in healthy brains is far lower than that of peripheral organs. Recently, growing evidence has indicated that aging may upregulate and overactivate brain AEPs. The overactivation of AEPs drives the onset of AD through cleaving tau and amyloid precursor proteins (APP), and SET, an inhibitor of protein phosphatase 2A (PP2A). The AEP-mediated cleavage of these peptides enhances amyloidosis, promotes tau hyperphosphorylation, and ultimately induces neurodegeneration and cognitive impairment. Upregulated AEPs and related deleterious reactions constitute upstream events of amyloid/tau toxicity in the brain, and represent early pathological changes in AD. Thus, upregulated AEPs are an emerging drug target for disease modification and a potential biomarker for predicting preclinical AD. However, the presence of the blood–brain barrier greatly hinders establishing body-fluid-based methods to measure brain AEPs. Research on AEP-activity-based imaging probes and our recent work suggest that the live brain imaging of AEPs could be used to evaluate its predictive efficacy as an AD biomarker. To advance translational research in this area, AEP imaging probes applicable to human brain and AEP inhibitors with good druggability are urgently needed. Full article

Legumain, a novel asparaginyl endopeptidase, has been observed to be overexpressed in several types of human solid tumors. Elevated levels of legumain are found in human cancers, and this oncoprotein may facilitate tumor invasion and metastasis when overexpressed. These findings suggest that legumain plays a malignant role in cancer biology. However, currently, no publications have identified the role of legumain in the development of canine cancers. The present study first compared the expression patterns of legumain in paraffin-embedded canine tumor tissues, with those of normal tissues, by immunohistochemistry. A total of 100 canine tumor samples, including mast cell tumors, soft tissue sarcoma, hemangiosarcoma, lymphoma, mammary gland carcinoma, hepatoid gland tumor, squamous cell carcinoma, trichoblastoma, and melanoma were evaluated. Compared with the normal tissues, all tumor samples displayed high intensities of legumain expression. Mesenchymal-type tumors displayed immunoreactivity for legumain, with an average expression of 40.07% ± 1.70%, which was significantly lower than those of epithelial tumors and other types of tumors, which had median expressions of 49.12% ± 1.75% and 47.35% ± 2.71%, respectively (p < 0.05). These findings indicate that legumain has a high potential to be a candidate for distinguishing tumors from normal tissues. Although further studies on a larger number of cases are necessary to clarify the clinical application of legumain, the overexpression patterns of legumain in canine tumor tissues are reported, for the first time, in this study. Full article

Asparagine endopeptidase (AEP), also called legumain, is currently the only known cysteine protease that specifically cleaves peptide bonds in asparaginyl residue in the mammalian genome. Since 2003, AEP has been reported to be widely expressed in a variety of carcinomas and is considered a potential therapeutic target. In the following years, researchers intensively investigated the substrates of AEP and the mechanism of AEP in partial tumors. With the identification of substrate proteins such as P53, integrin αvβ3, MMP-2, and MMP-9, the biochemical mechanism of AEP in carcinomas is also more precise. This review will clarify the probable mechanisms of AEP in the progression of breast carcinoma, glioblastoma, gastric carcinoma, and epithelial ovarian carcinoma. This review will also discuss the feasibility of targeted therapy with AEP inhibitor (AEPI) in these carcinomas. Full article