Péter Löw

The role of ubiquitin-proteasome system in ageing

Maintenance of cellular homeostasis influences ageing and it is determined by several factors, including efficient proteolysis of damaged proteins. The ubiquitin-proteasome system is the major protein degradation pathway in the cell. Specifically, the proteasome is responsible for clearance of abnormal, denatured or in general damaged proteins as well as for the regulated degradation of short-lived proteins. In this review the involvement of the ubiquitin-proteasome pathway in protein degradation at different levels of cellular life is discussed in relation with ageing. Though the exact underlying mechanism is unclear, an age-related decrease in proteasome activity weakens cellular capacity to remove oxidatively modified proteins and favours the development of diseases. Up-regulation of proteasome activity is characteristic of muscle wasting conditions, but may not be rate limiting. Meanwhile, enhanced presence of immunoproteasomes in ageing brain and muscle tissue could reflect a persistent inflammatory defence and anti-stress mechanism. Insulin/IGF-1 signalling regulates ageing in worms, flies and mammals. The insulin/IGF-1 receptor inhibits the forkhead transcription factor, FoxO through activating a cascade of conserved kinases. Longevity increases when FoxO becomes activated in response to reduced insulin/IGF-1 signalling. The ubiquitin-proteasome system plays a major role in signal transduction associated with stress and ageing. The understanding of specific proteolytic targeting paves the way for a new generation of active molecules that may control particular steps of normal and pathological ageing.

Tubulin and microtubule are potential targets for brain hexokinase binding

The metabolite‐modulated association of a fraction of hexokinase to mitochondria in brain is well documented, however, the involvement of other non‐mitochondrial components in the binding of the hexokinase is controversial. Now we present evidence that the hexokinase binds both tubulin and microtubules in brain in vitro systems. The interaction of tubulin with purified bovine brain hexokinase was characterized by displacement enzyme‐linked immunosorbent assay using specific anti‐brain hexokinase serum (IC50=4.0±1.4 μM). This value virtually was not affected by specific ligands such as ATP or glucose 6‐phosphate. Microtubule‐bound hexokinase obtained in reconstituted systems using microtubule and purified hexokinase or brain extract was visualized by transmission and immunoelectron microscopy on the surface of tubules. The association of purified bovine brain hexokinase with either tubulin or microtubules caused about 30% increase in the activity of the enzyme. This activation was also observed in brain, but not in muscle cell‐free extract. The possible physiological relevance of the multiple heteroassociation of brain hexokinase is discussed.

Localisation of 26S proteasomes with different subunit composition in insect muscles undergoing programmed cell death.

The 26S proteasome is a large multisubunit complex involved in degrading both cytoplasmic and nuclear proteins. We have investigated the subcellular distribution of four regulatory ATPase subunits (S6 (TBP7/MS73), S6′ (TBP1), S7 (MSS1), and S10b (SUG2)) together with components of 20S proteasomes in the intersegmental muscles (ISM) of Manduca sexta during developmentally programmed cell death (PCD). Immunogold electron microscopy shows that S6 is located in the heterochromatic part of nuclei of ISM fibres. S6′ is present in degraded material only outside intact fibres. S7 can be detected in nuclei, cytoplasm and also in degraded material. S10b, on the other hand, is initially found in nuclei and subsequently in degraded cytoplasmic locations during PCD. 20S proteasomes are present in all areas where ATPase subunits are detected, consistent with the presence of intact 26S proteasomes. These results are discussed in terms of heterogeneity of 26S proteasomes, 26S proteasome disassembly and the possible role of ATPases in non-proteasome complexes in the process of PCD.

Interaction of a new bis-indol derivative, KAR-2 with tubulin and its antimitotic activity

KAR‐2 (3″‐(β‐chloroethyl)‐2″,4″‐dioxo‐3,5″‐spiro‐oxazolidino‐4‐deacetoxy‐vinblastine), is a bis‐indol derivative; catharantine is coupled with the vindoline moiety which contains a substituted oxazolidino group. Our binding studies showed that KAR‐2 exhibited high affinity for bovine purified brain tubulin (Kd=3 μM) and it inhibited microtubule assembly at a concentration of 10 nM. Anti‐microtubular activity of KAR‐2 was highly dependent on the ultrastructure of microtubules: while the single tubules were sensitive, the tubules cross‐linked by phosphofructokinase (ATP: D‐fructose‐6‐phosphate‐1‐phosphotransferase, EC 2.7.1.11) exhibited significant resistance against KAR‐2. The cytoplasmic microtubules of Chinese hamster ovary mammalian and Sf9 insect cells were damaged by 1 μg ml−1 KAR‐2, as observed by indirect immunofluorescence and transmission electron microscopy. Scanning electron microscopy revealed intensive surface blebbing on both types of cells in the presence of KAR‐2. KAR‐2 was effective in the mouse leukaemia P338 test in vivo without significant toxicity. Studies on a primary cerebro‐cortical culture of rat brain and differentiated PC12 cells indicated that the toxicity of KAR‐2 was significantly lower than that of vinblastine. The additional property of KAR‐2 that distinguishes it from bis‐indol derivatives is the lack of anti‐calmodulin activity.

Production of H₂O₂ in the endoplasmic reticulum promotes in vivo disulfide bond formation.

AIMS Oxidative protein folding in the luminal compartment of endoplasmic reticulum (ER) is thought to be accompanied by the generation of H₂O₂, as side-product of disulfide bond formation. We aimed to examine the role of H₂O₂ produced in the lumen, which on one hand can lead to redox imbalance and hence can contribute to ER stress caused by overproduction of secretory proteins; on the other hand, as an excellent electron acceptor, H₂O₂ might serve as an additional pro-oxidant in physiological oxidative folding. RESULTS Stimulation of H₂O₂ production in the hepatic ER resulted in a decrease in microsomal GSH and protein-thiol contents and in a redox shift of certain luminal oxidoreductases in mice. The oxidative effect, accompanied by moderate signs of ER stress and reversible dilation of ER cisternae, was prevented by concomitant reducing treatment. The imbalance also affected the redox state of pyridine nucleotides in the ER. Antibody producing cells artificially engineered with powerful luminal H₂O₂ eliminating system showed diminished secretion of mature antibody polymers, while incomplete antibody monomers/dimers were accumulated and/or secreted. INNOVATION Evidence are provided by using in vivo models that hydrogen peroxide can promote disulfide bond formation in the ER. CONCLUSION The results indicate that local H₂O₂ production promotes, while quenching of H₂O₂ impairs disulfide formation. The contribution of H₂O₂ to disulfide bond formation previously observed in vitro can be also shown in cellular and in vivo systems.

Immunogold localisation of ubiquitin-protein conjugates in Sf9 insect cells. Implications for the biogenesis of lysosome-related organelles

Immunogold electron microscopy with antibodies which primarily detect ubiquitin‐protein conjugates shows conjugate‐specific gold particles enriched severalfold in acid phosphatase‐positive lysosomes and multivesicular bodies in insect Sf9 cells. The observations demonstrate that ubiquitinated proteins are associated with small acid phosphatase‐containing primary lysosomes (transport vesicles) and indicate a pathway in which primary lysosomes fuse with multivesicular bodies to generate mature lysosome‐related structures.

RELATED ORGANELLES OF THE ENDOSOME-LYSOSOME SYSTEM CONTAIN A DIFFERENT REPERTOIRE OF UBIQUITINATED PROTEINS IN SF9 INSECT CELLS

Two components of the endosomal/lysosomal compartment of Sf9 cells, multivesicular bodies (MVB) and light vacuoles with membrane complexes (LVMC) have been isolated and probed for ubiquitin protein conjugates with a specific antibody. Immunogold electron microscopy indicates that whereas ubiquitin‐protein conjugates are localised to electron dense areas of MVB they are associated with the membranes of LVMC. Five ubiquitinated polypeptides are revealed in MVB by immunoblotting while numerous ubiquitinated species forming a smear following electrophoresis are present in LVMC. We suggest two possible routes for entry of ubiquitin‐protein conjugates into these organelles, via the cell surface and via primary lysosomes.

Intraluminal hydrogen peroxide induces a permeability change of the endoplasmic reticulum membrane

Gulonolactone treatment of mice resulted in the elevation of hepatic ascorbate and hydrogen peroxide levels accompanied by transient liver swelling and reversible dilatation of endoplasmic reticulum cisternae. Although a decrease in glutathione (reduced form)/total glutathione ratio was observed in microsomes, the redox state of luminal foldases remained unchanged and the signs of endoplasmic reticulum stress were absent. Increased permeability of the microsomal membrane to various compounds of low molecular weight was substantiated. It is assumed that Gulonolactone‐dependent luminal hydrogen peroxide formation in the endoplasmic reticulum provokes a temporary increase in non‐selective membrane permeability, which results in the dilation of the organelle and in enhanced transmembrane fluxes of small molecules.

Apocrine secretion in Drosophila salivary glands: subcellular origin, dynamics, and identification of secretory proteins.

In contrast to the well defined mechanism of merocrine exocytosis, the mechanism of apocrine secretion, which was first described over 180 years ago, remains relatively uncharacterized. We identified apocrine secretory activity in the late prepupal salivary glands of Drosophila melanogaster just prior to the execution of programmed cell death (PCD). The excellent genetic tools available in Drosophila provide an opportunity to dissect for the first time the molecular and mechanistic aspects of this process. A prerequisite for such an analysis is to have pivotal immunohistochemical, ultrastructural, biochemical and proteomic data that fully characterize the process. Here we present data showing that the Drosophila salivary glands release all kinds of cellular proteins by an apocrine mechanism including cytoskeletal, cytosolic, mitochondrial, nuclear and nucleolar components. Surprisingly, the apocrine release of these proteins displays a temporal pattern with the sequential release of some proteins (e.g. transcription factor BR-C, tumor suppressor p127, cytoskeletal β-tubulin, non-muscle myosin) earlier than others (e.g. filamentous actin, nuclear lamin, mitochondrial pyruvate dehydrogenase). Although the apocrine release of proteins takes place just prior to the execution of an apoptotic program, the nuclear DNA is never released. Western blotting indicates that the secreted proteins remain undegraded in the lumen. Following apocrine secretion, the salivary gland cells remain quite vital, as they retain highly active transcriptional and protein synthetic activity.

Up- and downregulated genes in muscles that undergo developmentally programmed cell death in the insect Manduca sexta

This study was designed to investigate changes in gene expression associated with stage‐specific programmed cell death (PCD) in intersegmental muscles (ISMs) of the moth, Manduca sexta. The technique of differential display reverse transcription PCR was applied to compare mRNA levels before and after the onset of PCD in ISMs. Expression of E75B transcription factor was repressed while another factor, βFTZ‐F1, stayed at a very low level. However, gene coding for a translation‐initiation factor (eIF1A) was upregulated. Expression of these genes had not been previously reported to be altered in dying ISMs. An ecdysteroid agonist, RH‐5849, that prevented PCD in ISMs also blocked these changes.