Lysosomal Nucleases Research Articles

Endogenous chondroitin extends lifespan by inhibiting VHA-7-mediated tubular lysosome formation

Chondroitin extends lifespan and healthspan in C. elegans, but the relationship between extracellular chondroitin and intracellular anti-aging mechanisms is unknown. The basement membrane (BM) that contains chondroitin proteoglycans is anchored to cells via hemidesmosomes (HDs), and it accumulates damage with aging. In this study, we found that chondroitin regulates aging through the formation of HDs and inhibition of tubular lysosomes (TLs). Reduction of chondroitin due to a mutation in sqv-5/Chondroitin synthase (ChSy) causes the earlier and excessive formation of TLs and leakage of the lysosomal nuclease in a manner dependent on VHA-7, the a-subunit of V-type ATPase. VHA-7, whose mutation suppresses the short lifespan of the sqv-5 mutant, is initially localized to the basal side of the hypodermal cells and transported to lysosomes with aging. These results demonstrate that endogenous chondroitin suppresses aging by inhibiting the earlier excessive formation of TLs. This is a novel anti-aging mechanism that is controlled by the BM.

Quantification and characterization of the 5′ exonuclease activity of the lysosomal nuclease PLD3 by a novel cell-based assay

Phospholipase D3 (PLD3) and phospholipase D4 (PLD4), the most recently described lysosomal nucleases, are associated with Alzheimer’s disease, spinocerebellar ataxia, and systemic lupus erythematosus. They exhibit 5′ exonuclease activity on single-stranded DNA, hydrolyzing it at the acidic pH associated with the lysosome. However, their full cellular function is inadequately understood. To examine these enzymes, we developed a robust and automatable cell-based assay based on fluorophore- and fluorescence-quencher-coupled oligonucleotides for the quantitative determination of acidic 5′ exonuclease activity. We validated the assay under knockout and PLD-overexpression conditions and then applied it to characterize PLD3 and PLD4 biochemically. Our experiments revealed PLD3 as the principal acid 5′ exonuclease in HeLa cells, where it showed a markedly higher specific activity compared with PLD4. We further used our newly developed assay to determine the substrate specificity and inhibitory profile of PLD3 and found that proteolytic processing of PLD3 is dispensable for its hydrolytic activity. We followed the expression, proteolytic processing, and intracellular distribution of genetic PLD3 variants previously associated with Alzheimer’s disease and investigated each variant's effect on the 5′ nuclease activity of PLD3, finding that some variants lead to reduced activity, but others not. The development of a PLD3/4-specific biochemical assay will be instrumental in understanding better both nucleases and their incompletely understood roles in vitro and in vivo.

THE ACTIVITY OF LYSOSOMAL NUCLEASES IN DIFFERENT AGE GROUPS OF SALMON SALMO SALAR L.

At present time, due to the deterioration of environment, the pollution of rivers by industrial and domestic wastewater, as well as unsustainable fishing resulted in decrease of Atlantic salmon reserves all over the world. In the north-west part of Russia, the one of few rivers where the natural reproduction of this the most valuable commercial species is preserved, is Varzuga River (Murmansk region). To develop strategies for sustainable conservation of salmon populations the monitoring should be conducted in order to maintain stable living conditions in the river ecosystem. It is known that the enzymes involved in the protein, energy and carbohydrate metabolism are quite informative indicators of fish growth, development and adaptation to environmental changes. Along with parameters mentioned above, lysosomal hydrolyses, including acid nucleases involved in compensatory reconstructions of nucleic acid and the closely related protein metabolism are used to assess the status of aquatic ecosystems. The activity of lysosomal nucleases (RNAse, DNAse) in muscles of different age groups (0+, 1+, 2+) of juvenile Atlantic salmon Salmo salar L. from Varzuga River (Sobachiy porog) was investigated in the present study. It was shown that the activity of enzymes studied and the protein content in skeletal muscles increased linearly and were maximal in two-years-old salmon. The absolute values of lysosomal RNAse activity were slightly higher than DNAse activity. The data obtained indicate that the active participation of lysosomal nucleases in adaptive reactions of juvenile salmon on the early stages of post-embryonic development. First of all, it results in the intensification of biosynthetic processes of protein. To conclude the living conditions in Varzuga River are favorable for the growth and development of juvenile Atlantic salmon.

Dnase2a Deficiency Uncovers Lysosomal Clearance of Damaged Nuclear DNA via Autophagy

Deficiencies in DNA-degrading nucleases lead to accumulation of self DNA and induction of autoimmunity in mice and in monogenic and polygenic human diseases. However, the sources of DNA and the mechanisms that trigger immunity remain unclear. We analyzed mice deficient for the lysosomal nuclease Dnase2a and observed elevated levels of undegraded DNA in both phagocytic and nonphagocytic cells. In nonphagocytic cells, the excess DNA originated from damaged DNA in the nucleus based on colocalization studies, live-cell imaging, and exacerbation by DNA-damaging agents. Removal of damaged DNA by Dnase2a required nuclear export and autophagy-mediated delivery of the DNA to lysosomes. Finally, DNA was found to accumulate in Dnase2a(-/-) or autophagy-deficient cells and induce inflammation via the Sting cytosolic DNA-sensing pathway. Our results reveal a cell-autonomous process for removal of damaged nuclear DNA with implications for conditions with elevated DNA damage, such as inflammation, cancer, and chemotherapy.

A cancer cell-activatable aptamer-reporter system for one-step assay of circulating tumor cells.

The current antibody-mediated numeration assays of circulating tumor cells (CTCs) require multiple steps and are time-consuming. To overcome these technical limitations, a cancer cell-activatable aptamer-reporter was formulated by conjugating a biomarker-specific aptamer sequence with paired fluorochrome-quencher molecules. In contrast to the antibody probes, the intact aptamer-reporter was optically silent in the absence of cells of interest. However, when used in an assay, the aptamer selectively targeted cancer cells through interaction with a specific surface biomarker, which triggered internalization of the aptamer-reporter and, subsequently, into cell lysosomes. Rapid lysosomal degradation of the aptamer-reporter resulted in separation of the paired fluorochrome-quencher molecules. The released fluorochrome emitted bright fluorescent signals exclusively within the targeted cancer cells, with no background noise in the assay. Thus, the assays could be completed in a single step within minutes. By using this one-step assay, CTCs in whole blood and marrow aspirate samples of patients with lymphoma tumors were selectively highlighted and rapidly detected with no off-target signals from background blood cells. The development of the cancer cell-activatable aptamer-reporter system allows for the possibility of a simple and robust point-of-care test for CTC detection, which is currently unavailable.

Deoxyribonuclease II is a lysosomal barrier to transfection

DNA delivered in nonviral vectors or as naked DNA must overcome a number of extracellular and intracellular barriers to transfection. Since many vectors deliver DNA into cells by the endocytic route, DNA degradation by lysosomal nucleases has been proposed as a significant barrier to transfection, despite the fact that this has not yet been formally demonstrated to occur. To test this hypothesis, we have investigated the role of deoxyribonuclease II (DNase II), the primary acidic endonuclease active in the lysosome, in transfection. Two genetic systems were engineered in which mammalian cells either overexpressed DNase II or were knocked out for the enzyme. In both models, higher levels of DNase II correlated with decreased transfection efficiency by nonviral DNA delivery vectors. These data provide direct evidence implicating lysosomal DNase II as a barrier to transfection.

Stability of antisense oligonucleotides during incubation with a mixture of isolated lysosomal enzymes

Cellular uptake of antisense oligonucleotides occurs predominantly via the endocytic route but the subsequent intracellular trafficking and metabolic fate is still poorly understood. We have examined the stability of 20-mer phosphodiester (D-oligo) and phosphorothioate (S-oligo) oligonucleotides ([ 32P]-radiolabelled either internally and at the 3′- or 5′- end) during exposure to a mixture of isolated rat liver lysosomal enzymes (Tritosomes). Whereas the oligonucleotides remained stable in aqueous buffer at pH 5.4 for at least 48 h, the D-oligos (at 10 μM) were degraded relatively rapidly ( t 50% = 30–40 min) by Tritosomes and at a rate similar to S-oligos ( t 50% = 50 min). Degradation of both was verified using reverse-phase HPLC and in this case for D- and S-oligos (2 μM) the t 50% was 90 min and 150 min, respectively. Enzymatic digestion by lysosomal nucleases, and not simple acid-catalysed hydrolysis, accounts for the rapid degradation of oligonucleotides.

The Effect of Temperature on the Synthesis of Rhinovirus Type 2 RNA

The reduced yields of rhinovirus type 2 at temperatures above 37 degrees C were shown to result from the degradation of virus-induced RNA, leaving little RNA available for inclusion into mature infectious virions. The degradation occurred about 6 h p.i., and appeared to be selectively effecting the single-stranded species. Lysosomal nucleases do not appear to have a role in this supra-optimal degradation.

Damage to microsomal membrane by lipid peroxidation.

AbstractAlterations of microsomal membrane integrity were examined during lipid peroxidation. Loss of membrane‐bound NADPH cytochromec reductase activity and protein release into the aqueous phase were related to disruption of the lipophilic region of the membrane. Formation of fluorescent products in the lipid phase of the membrane occurred only in the presence of peroxidation products. Changes in the membrane lipid phase during peroxidation included a decrease in the reactive amino groups of the phospholipids and a decrease in detectable phosphatidyl ethanolamine. In an ultrafiltration continuous flow chamber, peroxidized microsomal membranes were hydrolyzed to a lesser degree by solubilized lysosomal cathepsins and to a greater degree by lysosomal nucleases than were nonperoxidized membranes.

Effects of diethyl pyrocarbonate on the isolation and recovery of bound polysomes from a particulate fraction of rat liver

The conventional treatment of rat liver postmitochondrial supernatant with detergents permits the recovery of a roughly equal mixture of undegraded free and bound polysomes [ 1 ] , whereas similar treatment of the pellet containing mitochondria, lysosomes and the bulk of bound polysomes leads to the recovery of largely degraded polysomes [2,3] in spite of the use of naturally-occurring nuclease inhibitor [4]. This presumably is due to detergent-mediated activation or release of nucleases that are refractory to nuclease inhibitor [5]. Intact bound polysomes can be prepared from purified rough membrane fractions; however, the whole procedure requires about 2 days of centrifugation and polysome recovery is low [l]. Recently, diethyl pyrocarbonate (DEP), a potent inhibitor of nucleases, has been employed for the preparation of undegraded polysomes and RNA from higher plants and bacteria [6-81. The inactivation of pancreatic ribonuclease by DEP in vitro is believed to result from the formation of intraand intermolecular peptide-like bonds which cause conformational changes and yield polymeric proteins [9]. It was, therefore, of interest to determine whether mitochondrial and lysosomal nucleases could be inactivated by DEP while still in their latent state, and thereby permit isolation of undegraded bound polysomes from a crude particulate fraction of rat liver. In this report we provide evidence that the nuclease activities of the more rapidly sedimenting fraction of