Lysosomal multienzyme complex: pros and cons of working together

The ubiquitous distribution of lysosomes and their heterogeneous protein composition reflects the versatility of these organelles in maintaining cell homeostasis and their importance in tissue differentiation and remodeling. In lysosomes, the degradation of complex, macromolecular substrates requires the synergistic action of multiple hydrolases that usually work in a stepwise fashion. This catalytic machinery explains the existence of lysosomal enzyme complexes that can be dynamically assembled and disassembled to efficiently and quickly adapt to the pool of substrates to be processed or degraded, adding extra tiers to the regulation of the individual protein components. An example of such a complex is the one composed of three hydrolases that are ubiquitously but differentially expressed: the serine carboxypeptidase, protective protein/cathepsin A (PPCA), the sialidase, neuraminidase-1 (NEU1), and the glycosidase β-galactosidase (β-GAL). Next to this ‘core’ complex, the existence of sub-complexes, which may contain additional components, and function at the cell surface or extracellularly, suggests as yet unexplored functions of these enzymes. Here we review how studies of basic biological processes in the mouse models of three lysosomal storage disorders, galactosialidosis, sialidosis, and GM1-gangliosidosis, revealed new and unexpected roles for the three respective affected enzymes, Ppca, Neu1, and β-Gal, that go beyond their canonical degradative activities. These findings have broadened our perspective on their functions and may pave the way for the development of new therapies for these lysosomal storage disorders.     

Double deficiency of cathepsins B and L results in massive secretome alterations and suggests a degradative cathepsin-MMP axis

Endolysosomal cysteine cathepsins functionally cooperate. Cathepsin B (Ctsb) and L (Ctsl) double-knockout mice die 4 weeks after birth accompanied by (autophago-) lysosomal accumulations within neurons. Such accumulations are also observed in mouse embryonic fibroblasts (MEFs) deficient for Ctsb and Ctsl. Previous studies showed a strong impact of Ctsl on the MEF secretome. Here we show that Ctsb alone has only a mild influence on extracellular proteome composition. Protease cleavage sites dependent on Ctsb were identified by terminal amine isotopic labeling of substrates (TAILS), revealing a prominent yet mostly indirect impact on the extracellular proteolytic cleavages. To investigate the cooperation of Ctsb and Ctsl, we performed a quantitative secretome comparison of wild-type MEFs and Ctsb ^?/? Ctsl ^?/? MEFs. Deletion of both cathepsins led to drastic alterations in secretome composition, highlighting cooperative functionality. While many protein levels were decreased, immunodetection corroborated increased levels of matrix metalloproteinase (MMP)-2. Re-expression of Ctsl rescues MMP-2 abundance. Ctsl and to a much lesser extent Ctsb are able to degrade MMP-2 at acidic and neutral pH. Addition of active MMP-2 to the MEF secretome degrades proteins whose levels were also decreased by Ctsb and Ctsl double deficiency. These results suggest a degradative Ctsl—MMP-2 axis, resulting in increased MMP-2 levels upon cathepsin deficiency with subsequent degradation of secreted proteins such as collagen α-1 (I).     

Therapeutic strategies to ameliorate lysosomal storage disorders – a focus on Gaucher disease

The lysosomal storage disorders encompass more than 40 distinct diseases, most of which are caused by the deficient activity of a lysosomal hydrolase leading to the progressive, intralysosomal accumulation of substrates such as sphingolipids, mucopolysaccharides, and oligosaccharides. Here, we primarily focus on Gaucher disease, one of the most prevalent lysosomal storage disorders, which is caused by an impaired activity of glucocerebrosidase, resulting in the accumulation of the glycosphingolipid glucosylceramide in the lysosomes. Enzyme replacement and substrate reduction therapies have proven effective for Gaucher disease cases without central nervous system involvement. We discuss the promise of chemical chaperone therapy to complement established therapeutic strategies for Gaucher disease. Chemical chaperones are small molecules that bind to the active site of glucocerebrosidase variants stabilizing their threedimensional structure in the endoplasmic reticulum, likely preventing their endoplasmic reticulum-associated degradation and allowing their proper trafficking to the lysosome where they can degrade accumulated substrate to effectively ameliorate Gaucher disease. Received 22 September 2005; received after revision 15 December 2006; accepted 2 February 2006     

Differential expression of monomeric and proteolytically processed forms of tartrate-resistant acid phosphatase in rat tissues

Purple acid phosphatase (PAP), also known as tartrate-resistant acid phosphatase (TRAP), uteroferrin or type 5 acid phosphatase (Acp5) is synthesized as an N-glycosylated monomeric latent precursor, which can be processed by limited proteolysis to a disulfide-linked two-subunit form with increased enzyme activity. In this study, we disclosed that the proteolytically processed two-subunit form constitutes the major PAP/TRAP variant in monocytic cells in spleen, thymus, liver and colon. In addition significant expression of the monomeric PAP/TRAP, indicating a non-enzymatic function, was detected in epithelial cells of colon, lung and kidney. Interestingly, proteolytic processing alone did not activate the enzyme but rendered the enzyme more susceptible to activation by reductants. Thus, beside limited proteolysis, the subcellular redox state could also be a determinant of enzyme action in vivo. The co-localization of PAP/TRAP and the cysteine protease cathepsin L could suggest a role for cathepsin L in the in vivo proteolytic processing of PAP/TRAP in monocytic cells.Received 10 December 2004; received after revision 19 January 2005; accepted 9 February 2005     

Cathepsin X-mediated β2 integrin activation results in nanotube outgrowth

Membrane nanotubes were recently described as a new principle of cell–cell communication enabling complex and specific messaging to distant cells. Calcium fluxes, vesicles, and cell-surface components can all traffic between cells connected by nanotubes. Here we report for the first time the mechanism of membrane nanotube formation in T cells through LFA-1 (CD11a/CD18; α_Lβ₂) integrin activation by the cysteine protease cathepsin X. Cathepsin X is shown to induce persistent LFA-1 activation. Cathepsin X-upregulated T cells exhibit increased homotypic aggregation and polarized, migration-associated morphology in 2D and 3D models, respectively. In these cells, extended uropods are frequently formed, which subsequently elongate to nanotubes connecting T lymphocytes. Our results demonstrate that LFA-1 activation with subsequent cytoskeletal reorganization induces signal transmission through a physically connected network of T lymphocytes for better coordination of their action at various stages of the immune response. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Received 26 December 2008; received after revision 26 January 2009; accepted 27 January 2009 N. Obermajer, Z. Jevnikar: These authors contributed equally to the present work.     

15-Deoxy-Δ12,14-prostaglandin-J2 reveals a new pVHL-independent, lysosomal-dependent mechanism of HIF-1α degradation

Hypoxia-inducible factor-1α (HIF-1α) protein is degraded under normoxia by its association to von Hippel-Lindau protein (pVHL) and further proteasomal digestion. However, human renal cells HK-2 treated with 15-deoxy-Δ^12,14-prostaglandin-J₂ (15d-PGJ₂) accumulate HIF-1α in normoxic conditions. Thus, we aimed to investigate the mechanism involved in this accumulation. We found that 15d-PGJ₂ induced an over-accumulation of HIF-1α in RCC4 cells, which lack pVHL and in HK-2 cells treated with inhibitors of the pVHL-proteasome pathway. These results indicated that pVHL-proteasome-independent mechanisms are involved, and therefore we aimed to ascertain them. We have identified a new lysosomal-dependent mechanism of HIF-1α degradation as a target for 15d-PGJ₂ based on: (1) HIF-1α colocalized with the specific lysosomal marker Lamp-2a, (2) 15d-PGJ₂ inhibited the activity of cathepsin B, a lysosomal protease, and (3) inhibition of lysosomal activity did not result in over-accumulation of HIF-1α in 15d-PGJ₂-treated cells. Therefore, expression of HIF-1α is also modulated by lysosomal degradation.     

Proteases and proteolysis in the lysosome

Proteins sequestered by a non-selective bulk process within the lysosomes turn over with an apparent half-life of about 8 minutes and this rapid lysosomal proteolysis is initiated by endopeptidases, in particular by the cathepsins D and L. We describe also the cathepsins B and H which show mainly exopeptidase and only low endopeptidase activity. Especially cathepsin H is most probably the only lysosomal aminopeptidase in many cell types. Additionally, the properties of other mammalian lysosomal endo- and exopeptidases are compared. Finally, we discuss some of the conditions for the action of lysosomal proteases as the low intralysosomal pH, the high part of lysosomal thiol groups and the absence of intralysosomal proteinase inhibitors.     

Proteases and proteolysis in the lysosome.

Proteins sequestered by a non-selective bulk process within the lysosomes turn over with an apparent half-life of about 8 minutes and this rapid lysosomal proteolysis is initiated by endopeptidases, in particular by the cathepsins D and L. We describe also the cathepsins B and H which show mainly exopeptidase and only low endopeptidase activity. Especially cathepsin H is most probably the only lysosomal aminopeptidase in many cell types. Additionally, the properties of other mammalian lysosomal endo- and exopeptidases are compared. Finally, we discuss some of the conditions for the action of lysosomal proteases as the low intralysosomal pH, the high part of lysosomal thiol groups and the absence of intralysosomal proteinase inhibitors.     

The inhibition of cathepsin B by plasma haptoglobin biochemistry (enzymes,metabolism)

Summary Purified haptoglobin partially inhibits the activity of a lysosomal thiol proteinase, cathepsin B. This inhibition is reversible in the presence of monospecific antiserum to haptoglobin.This work was supported by an East Anglian Regional Health Authority Grant.     

Interactions of the proteins of neuronal ceroid lipofuscinosis: clues to function

Neuronal ceroid lipofuscinoses (NCL) are caused by mutations in eight different genes, are characterized by lysosomal accumulation of autofluorescent storage material, and result in a disease that causes degeneration of the central nervous system (CNS). Although functions are defined for some of the soluble proteins that are defective in NCL (cathepsin D, PPT1, and TPP1), the primary function of the other proteins defective in NCLs (CLN3, CLN5, CLN6, CLN7, and CLN8) remain poorly defined. Understanding the localization and network of interactions for these proteins can offer clues as to the function of the NCL proteins and also the pathways that will be disrupted in their absence. Here, we present a review of the current understanding of the localization, interactions, and function of the proteins associated with NCL.     

Structural insights into the multiple functions of protein C inhibitor

Protein C inhibitor (PCI) is a widely distributed, multifunctional member of the serpin family of protease inhibitors, and has been implicated in several physiological processes and disease states. Its inhibitory activity and specificity are regulated by binding to cofactors such as heparin, thrombomodulin and phospholipids, and it also appears to have non-inhibitory functions related to hormone and lipid binding. Just how the highly conserved serpin architecture can support the multiple diverse functions of PCI is a riddle best addressed by protein crystallography. Over the last few years we have solved the structure of PCI in its native, cleaved and protein-complexed states. They reveal a conserved serpin fold and general mechanism of protease inhibition, but with some unique features relating to inhibitory specificity/promiscuity, cofactor binding and hydrophobic ligand transport. Received 1 July 2008; received after revision 16 August 2008; accepted 22 August 2008     

Intracellular proteases from the extremely thermophilic archaebacteriumSulfolobus solfataricus

Proteolytic activities from the extremely thermoacidophilic archaebacteriumSulfolobus solfataricus were detected with the aid of synthetic substrates in a cell extract fractionated by gel filtration. Two aminopeptidases (aminopeptidase I and II), three endopeptidases (proteinase I, II and III) and one carboxypeptidase could be identified. Experiments carried out with protease inhibitors led to the identification of the exopeptidases as metalloproteases. Proteinases I and II behaved as chymotrypsin-like serine proteases, and proteinase III as a cysteine protease with a trypsin-like specificity. Molecular weight values assessed with the aid of marker proteins were as follows: aminopeptidase I, >450 kDa; aminopeptidase II, 170 kDa; carboxypeptidase, 160 kDa; proteinase I, 115 kDa; proteinase II, 32 kDa; proteinase III, 27 kDa. On incubation for 15 min they retained most of their activity up to a temperature of 90°C, with the sole exception of proteinase II, which was rapidly inactivated at 60°C. Protease content was also determined in crude extracts from cells grown in a mineral medium both to the stationary and to the exponential phase, with glucose or with yeast extract as carbon sources. No dramatic change was detected depending on the growth phase; however, carboxypeptidase level was three- to four-fold higher when yeast extract was present in the medium instead of glucose; this might suggest an involvement of this enzyme in the digestion of extracellularly available peptides.     

Muscle damage induced by the ionophore A23187 can be prevented by prostaglandin inhibitors and leupeptin

Frog skeletal muscle incubated in vitro with the ionophore A23187 shows extensive morphological alterations. Myofilament disruption, presumably mediated by excess intracellular calcium, can be partially prevented by preincubating the muscle with inhibitors of prostaglandin synthesis and the lysosomal thiol protease inhibitor leupeptin.     

Gaucher disease: Perspectives on a prototype lysosomal disease

Gaucher disease is an autosomal recessive trait and the most common lysosomal storage disease. The pathogenesis evolves from the diminished activity of the lysosomal hydrolase, acid beta-glucosidase and the resultant accumulation of glucosylceramide within lysosomes. The pathogenic mechanisms are poorly understood. During the past 2 decades, progress has been made in understanding the biochemical basis and molecular biology of the disease, but more fundamental knowledge is required to relate these advances to the cell and whole body phenotypes. Despite this lack of understanding, enzyme replacement therapy has proved a successful and effective management for Gaucher disease. However, basic details of this therapeutic efficacy require elucidation. Here, we review the current state of the molecular pathogenesis and provide our perspective of some major issues for continued advances in this prototype lysosomal storage disease.     

Protein digestion in the sea gooseberryPleurobranchia bachei A. Agassiz (Ctenophora: Tentaculata)

Summary Peaks of proteolytic activity of pharyngeal juice occur at pH 5.75 and pH 7.5. The proteases responsible for digestion include a tryptic alkaline protease and a thiol-activated acid protease which is probably cathepsin B. Levels of proteolytic activity parallel those of other carnivorous invertebrates which feed on zooplankton.This study was supported by operating grants from the National Research Council of Canada.We are grateful to Betsy Sweeney for her technical assistance.     

Neuronal ceroid lipofuscinosis protein CLN3 interacts with motor proteins and modifies location of late endosomal compartments

CLN3 is an endosomal/lysosomal transmembrane protein mutated in classical juvenile onset neuronal ceroid lipofuscinosis, a fatal inherited neurodegenerative lysosomal storage disorder. The function of CLN3 in endosomal/lysosomal events has remained elusive due to poor understanding of its interactions in these compartments. It has previously been shown that the localisation of late endosomal/lysosomal compartments is disturbed in cells expressing the most common disease-associated CLN3 mutant, CLN3?ex7-8 (c.462-677del). We report here that a protracted disease causing mutant, CLN3E295K, affects the properties of late endocytic compartments, since over-expression of the CLN3E295K mutant protein in HeLa cells induced relocalisation of Rab7 and a perinuclear clustering of late endosomes/lysosomes. In addition to the previously reported disturbances in the endocytic pathway, we now show that the anterograde transport of late endosomal/lysosomal compartments is affected in CLN3 deficiency. CLN3 interacted with motor components driving both plus and minus end microtubular trafficking: tubulin, dynactin, dynein and kinesin-2. Most importantly, CLN3 was found to interact directly with active, guanosine-5'-triphosphate (GTP)-bound Rab7 and with the Rab7-interacting lysosomal protein (RILP) that anchors the dynein motor. The data presented in this study provide novel insights into the role of CLN3 in late endosomal/lysosomal membrane transport.     

Muscle damage induced by the ionophore A23187 can be prevented by prostaglandin inhibitors and leupeptin

Summary Frog skeletal muscle incubated in vitro with the ionophore A23187 shows extensive morphological alterations. Myofilament disruption, presumably mediated by excess intracellular calcium, can be partially prevented by preincubating the muscle with inhibitors of prostaglandin synthesis and the lysosomal thiol protease inhibitor leupeptin.The author would like to thank Dr R. J. Walter for helpful comments and Ms V. Kriho for valuable technical assistance     

PHAB toxins: a unique family of predatory sea anemone toxins evolving via intra-gene concerted evolution defines a new peptide fold

Sea anemone venoms have long been recognized as a rich source of peptides with interesting pharmacological and structural properties, but they still contain many uncharacterized bioactive compounds. Here we report the discovery, three-dimensional structure, activity, tissue localization, and putative function of a novel sea anemone peptide toxin that constitutes a new, sixth type of voltage-gated potassium channel (K_V) toxin from sea anemones. Comprised of just 17 residues, κ-actitoxin-Ate1a (Ate1a) is the shortest sea anemone toxin reported to date, and it adopts a novel three-dimensional structure that we have named the Proline-Hinged Asymmetric β-hairpin (PHAB) fold. Mass spectrometry imaging and bioassays suggest that Ate1a serves a primarily predatory function by immobilising prey, and we show this is achieved through inhibition of Shaker-type K_V channels. Ate1a is encoded as a multi-domain precursor protein that yields multiple identical mature peptides, which likely evolved by multiple domain duplication events in an actinioidean ancestor. Despite this ancient evolutionary history, the PHAB-encoding gene family exhibits remarkable sequence conservation in the mature peptide domains. We demonstrate that this conservation is likely due to intra-gene concerted evolution, which has to our knowledge not previously been reported for toxin genes. We propose that the concerted evolution of toxin domains provides a hitherto unrecognised way to circumvent the effects of the costly evolutionary arms race considered to drive toxin gene evolution by ensuring efficient secretion of ecologically important predatory toxins.     

Arylsulfatase in coelomocytes of Holothuria polii

Holothuria polii coelomocytes possess arylsulfatase enzymes. Two pH optima were found for arylsulfatase activity in cell lysate preparations, one at pH 5.0 and the other at pH 5.8. Both increased after injection of zymosan particles or formalinized sheep red blood cells (fSR-BC), indicating an active role of the enzymes during phagocytosis of particulate substances. Under a light microscope, the acid hydrolase arylsulfatase were localized in the granules of spherula cells, and therefore considered lysosomal in nature.     

Biogenesis of mitochondrial porin: the import pathway.

We review here the present knowledge about the pathway of import and assembly of porin into mitochondria and compare it to those of other mitochondrial proteins. Porin, like all outer mitochondrial membrane proteins studied so far is made as a precursor without a cleavable 'signal' sequence; thus targeting information must reside in the mature sequence. At least part of this information appears to be located at the amino-terminal end of the molecule. Transport into mitochondria can occur post-translationally. In a first step, the porin precursor is specifically recognized on the mitochondrial surface by a protease sensitive receptor. In a second step, porin precursor inserts partially into the outer membrane. This step is mediated by a component of the import machinery common to the import pathways of precursor proteins destined for other mitochondrial subcompartments. Finally, porin is assembled to produce the functional oligomeric form of an integral membrane protein which is characterized by its extreme protease resistance.