Mannose-6-phosphate pathway: a review of its role in lysosomal function and dysfunction (2023)

Cathepsins can be found in the extracellular space, cytoplasm and nucleus. It was initially suggested that the primary physiological function of cathepsins is to degrade intracellular proteins and that they also play a role in pathological processes including inflammation and apoptosis. However, the many actions of cathepsins outside the cell and their complicated biological actions have attracted much interest. Cathepsins play important roles in a variety of diseases, regulating parenchymal cell proliferation, cell migration, viral invasion, inflammation, and immune responses through extracellular matrix remodeling, signaling disruption, leukocyte recruitment, and cell adhesion. In this review, we outline the physiological role of cathepsins in the extracellular space, the crucial pathological roles that cathepsins play in disease, and recent advances in the detection and therapy of specific inhibitors and fluorescent probes in the associated dysfunction.

CLN2 neuronal ceroid lipofuscinosis is a rare inherited neurodegenerative disease characterized by deleterious sequence variants inTPP1leading to reduced or abolished function of the lysosomal enzyme tripeptidyl peptidase 1 (TPP1). Children with this disorder show progressive neurological decline and vision loss beginning around 2 to 4 years of age. Eye diseases are characterized by progressive retinal degeneration and impairment of retinal function, culminating in complete loss of vision. Similar retinal pathology occurs in a canine model of CLN2 disease with a null variantTPP1. A study using the canine model was conducted to evaluate the effectiveness of ocular gene therapy to provide a continuous, long-term source of human TPP1 (hTPP1) to the retina, inhibit retinal degeneration, and preserve retinal function.TPP1^−/−Dogs received an intravitreal injection of 1 x 10¹²AAV2.CAG viral genomes.hTPP1in one eye and AAV2.CAG.GFPin the contralateral eye at 4 months of age. eye exams,liveOcular imaging and electroretinography were repeated monthly to assess retinal structure and function. Retinal morphology, hTPP1 and GFP expression in the retina, optic nerve, and lateral geniculate nucleus, and vitreous hTPP1 concentrations were evaluated after dogs with terminal neurological disease were euthanized at approximately 10 months of age. Intravitreal administration of AAV2.CAG.hTPP1resulted in stable and widespread expression of hTPP1 throughout the inner retina, prevented disease-related declines in retinal function, and inhibited disease-related cell loss and accumulation of storage bodies in the retina for at least 6 months. Uveitis has occurred in eyes treated with thehTPP1Vector, but this did not prevent therapeutic efficacy. The severity of uveitis was improved by anti-inflammatory treatments. These results demonstrate that a single intravitreal injection of AAV2.CAG.hTPP1is an effective treatment to inhibit ocular disease progression in canine CLN2 disease.

Lysosomal storage diseases (LSD) are a group of rare life-threatening diseases caused by lysosomal dysfunction, usually due to the lack of a single enzyme required for macromolecule metabolism, leading to lysosomal accumulation of certain substrates with serious consequences, leading to morbidity and early death. There is currently no definitive cure for LSD, and despite the approval of certain therapies, their effectiveness is limited. Therefore, a suitable nanocarrier may help to improve the effectiveness of some of these therapies. Liposomes have excellent properties as drug carriers, as they can encapsulate active therapeutic compounds that provide protection, biocompatibility and selectivity. Here we discuss the potential of liposomes for LSD treatment and perform a detailed analysis of promising liposomal formulations still in preclinical development stage from different perspectives including treatment strategy, manufacturing, characterization and future directions for implementing liposomal formulations for LSD. LSD.

The formation of a cell wall is vital for the survival and growth of a fungal cell. Fungi express members of the GH76 family of α-1,6-mannanases, which play important roles in cell wall biogenesis. In this report we characterize theNeurospora crassaDFG-5 α-1,6-mannanase and show that it binds to the α-1,6-mannose backbone of an N-linked galactomannan found in cell wall glycoproteins. We show that DFG-5 has enzymatic activity and demonstrate that it processes the α-1,6-mannose skeleton of N-linked galactomannan. Site-directed mutagenesis and complementation experiments show that D116 and D117 localize to the active site of DFG-5. Also important for enzyme function are D76 and E130, located in a groove on the opposite side of the protein. Cell wall glycoproteins are co-purified with DFG-5, showing a specific association between DFG-5 and cell wall glycoproteins. DFG-5 can discriminate between cell wall and secreted glycoproteins and does not bind N-linked galactomannans present on secreted glycoproteins. DFG-5 plays a key role in directing extracellular glycoproteins to their final destination. By processing galactomannans into cell wall proteins, DFG-5 aims to incorporate them into the cell wall by lichenine transferases. The N-linked galactomannans in secreted proteins are not processed by DFG-5, which targets these proteins for release into the extracellular medium.

The lysosome represents a central degradative compartment of eukaryotic cells, but little is known about the biogenesis and function of this organelle in parasitic protists. While the mannose-6-phosphate (M6P)-dependent system for lysosomal targeting is dominant in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study we examined the phagolysosomal proteome of the human parasiteTrichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. Organelles were purified using Percoll and OptiPrep gradient centrifugation and a new purification protocol based on phagocytosis of lactoferrin-coated magnetic nanoparticles. The analysis revealed a lysosomal proteome composed of 462 proteins, which were classified into 21 classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases and glycosidases. Identification of a large group of proteins involved in vesicular transport (80) and turnover of actin cytoskeletal rearrangement (29) indicate a dynamic phagolysosomal compartment. It has previously been demonstrated that several cysteine ​​proteases, such as TvCP2, are secreted. Our experiments showed that TvCP2 secretion was strongly inhibited by chloroquine, which increases intralysosomal pH, suggesting that TvCP2 secretion occurs through lysosomes and not through the classic secretion pathway. Unexpectedly, however, we identified aberrant homologues of the M6P TvMPR receptor in the phagolysosomal proteomeT. vaginalislack enzymes for the formation of M6P. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine ​​protease CLCP, which has two glycosylation sites. Mutation of one of the sites redirected CLCP to the secretory pathway. Likewise, the introduction of glycosylation sites in secreted β-amylase redirected this protein to lysosomes. Unlike other parasitic protistsT. vaginalisappears to use glycosylation as a recognition marker for lysosomal hydrolases. Our results provide a first insight into the complexity of theT. vaginalisPhagolysosomes, their biogenesis and their role in the unconventional secretion of cysteine ​​peptidases.

Lysosomal diseases are a class of genetic disorders caused primarily by the loss of lysosomal hydrolases, resulting in lysosomal and cellular dysfunction. Enzyme replacement therapy (ERT), in which the recombinant enzyme is administered intravenously, internalized by cells, and transported to the lysosome, has been used to treat various lysosomal disorders. However, current ERT regimens do not correct disease phenotypes in all affected organs because the biodistribution of enzyme uptake does not match that of affected cells that require the enzyme. We present here targeted ERT, an approach that uses antibody-enzyme fusion proteins to target the enzyme to specific cell types. The antibody portion recognizes transmembrane proteins involved in lysosomal transport, which are also preferentially expressed in cells most affected by the disease. Using Pompe's disease (PD) as an example, we show that targeted ERT is superior to ERT in treating the skeletal muscle phenotypes of mice with PD, both as protein replacement therapy and gene therapy.

Brain Research, Vol. 1683, 2018, pp. 12-16

Lysosome-associated membrane glycoprotein 2 (lamp2) plays a crucial role in chaperone-mediated autophagy (CMA) and macroautophagy. Its Lamp2a isoform is reduced in the substantia nigra of Parkinson's disease (PD). Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the best-known common cause of late-onset Parkinson's disease; Although LRRK2 is thought to regulate macroautophagy, the impact of LRRK2 mutations on CNS Lamp2 levels is unknown. To investigate this issue, we compared Lamp2 levels in cerebrospinal fluid (CSF) between patients with sporadic PD (sPD) (n=31), patients with LRRK2-PD (n=20), and healthy controls with or without LRRK2 mutations. - (LRRK2 CTL=30, CTL=27). We also examined Lamp2 correlations with age, oxidative stress, PD progression, and PD duration. Mean Lamp2 concentrations (pg/mL) were LRRK2 PD = 127, sPD = 333, CTL = 436 and LRRK2 CTL = 412. Logarithmically transformed Lamp2 concentrations adjusted for gender effects (and excluding male patients with LRRK2 PD due to low number). , were lower in female LRRK2-PD subjects than in LRRK2-CTL (p = 0.002) and CTL (p = 0.005) subjects (p = 0.06 for bulb2 comparison between female LRRK2-PD subjects and patients with sPD). Lamp2 did not appear to be related to age, PD progression or PD duration; however, three of four Spearman rho values ​​for correlations between Lamp2 and markers of oxidative stress in Parkinson's patients were ≥ 0.30. These results suggest that CSF-Lamp2 levels may be reduced in female patients with LRRK2-PD compared to healthy subjects with or without LRRK2 mutations.

Forensic Science International, Band 335, 2022, Artikel 111284

In forensic investigations, recognizing a suspect's DNA from the available evidence is crucial. In an outdoor crime scene, evidence can be mixed with dirt. However, it is speculated that soil inhibits DNA extraction from forensic samples. In the field of soil microbiology, it is necessary to extract DNA directly from the soil to analyze its microbial composition. In this study, we investigated whether skim milk used in extracting DNA from soil samples can be applied in forensic science to improve the efficiency of extracting human DNA from soil associated with forensic evidence such as blood, cheek cells and skin cells mixed together. . The use of auxiliary reagents, skimmed milk and bovine serum albumin (BSA), are known as blocking reagents. In the experiment to select auxiliary reagents, such as a blood sample, the use of skim milk and BSA increased DNA recovery. Therefore, we monitored the yield of DNA extracted from blood, cheek cells and skin cells when skim milk and BSA were used as additional reagents. The DNA recovery rate was high in all samples when skimmed milk was added. However, STR analysis detected a non-specific peak in DNA extracted in the presence of skimmed milk that was not detected in the presence of BSA, indicating its suitability for forensic analysis. Our study suggests that the addition of BSA can efficiently aid in the extraction of DNA from forensic evidence mixed with soil.

Cell, Vol. 162, Issue 2, 2015, pp. 259-270

Despite being surrounded by different nutrients, mammalian cells preferentially metabolize glucose and free amino acids. Recently, Ras-induced macropinocytosis of extracellular proteins has been shown to reduce a transformed cell's dependence on extracellular glutamine. Here we show that protein macropinocytosis can also serve as a source of essential amino acids. Lysosomal degradation of extracellular proteins can maintain cell survival and induce mTORC1 activation, but does not result in significant cell accumulation. In contrast to its growth-promoting activity under conditions of amino acid excess, we found that activation of mTORC1 suppresses proliferation when cells rely on extracellular proteins as an amino acid source. Inhibition of mTORC1 leads to increased catabolism of endocytosed proteins and increases cell proliferation under nutrient starved conditions in vitro and in vascularly compromised tumors in vivo. Thus, by preventing extracellular proteins from being fed, mTORC1 couples growth to the availability of free amino acids. These findings may have important implications for the use of mTOR inhibitors as therapy.

Journal of Molecular Biology, Band 429, Ausgabe 5, 2017, S. 633-646

Several microtubule stabilizing (MT) agents (MSAs) have shown or predicted potential as anticancer agents, but a detailed structural basis for their mechanism of action is still lacking. We obtained high-resolution (3.9–4.2 Å) cryoelectron microscopy (cryo-EM) reconstructions of MTs stabilized by the taxane site ligands taxol and zampanolide and pelorusid, which targets a specific non-taxoid pocket targeting β -tubulin. We found that each molecule has uniquely different structural effects on the structure of the MT network. Acting primarily on lateral contacts, pelorusid affects the “joining” of heterologous interactions, forcing a conformation more similar to that of homologous contacts (i.e., non-seaming), thus regulating the MT network. In contrast, taxol or zampanolide binding induces MT heterogeneity. In double-bonded MTs, pelorusid replaces the heterogeneity induced by taxol binding. Our structural analysis illustrates different mechanisms of these drugs to stabilize the MT network and is relevant to the potential use of combinations of MSAs to regulate MT activity and increase therapeutic potential.

International Review of Cellular and Molecular Biology, Band 320, 2015, S. 75-101

Considerable research supports a model in which hydrolytic enzymes are sorted by mammalian lysosomes to their fates in a receptor-dependent mechanism. The ligand for mammalian selection receptors is mannose-6-phosphate (M6P). Two M6P receptors have been defined in mammals. Here, we review the basic evidence supporting this mechanism and highlight remaining gaps in our understanding of the mammalian mechanism, including evidence for M6P-independent classification and its relevance to the classification of lysosomal enzymes in metazoans.

Molecular Genetics and Metabolism, Band 111, Ausgabe 3, 2014, S. 369-373