Lysosomal Proteolytic Pathways Research Articles

Essential roles for deubiquitination in Leishmania life cycle progression.

The parasitic protozoan Leishmania requires proteasomal, autophagic and lysosomal proteolytic pathways to enact the extensive cellular remodelling that occurs during its life cycle. The proteasome is essential for parasite proliferation, yet little is known about the requirement for ubiquitination/deubiquitination processes in growth and differentiation. Activity-based protein profiling of L. mexicana C12, C19 and C65 deubiquitinating cysteine peptidases (DUBs) revealed DUB activity remains relatively constant during differentiation of procyclic promastigote to amastigote. However, when life cycle phenotyping (bar-seq) was performed on a pool including 15 barcoded DUB null mutants created in promastigotes using CRISPR-Cas9, significant loss of fitness was observed during differentiation and intracellular infection. DUBs 4, 7, and 13 are required for successful transformation from metacyclic promastigote to amastigote and DUBs 3, 5, 6, 8, 10, 11 and 14 are required for normal amastigote proliferation in mice. DUBs 1, 2, 12 and 16 are essential for promastigote viability and the essential role of DUB2 in establishing infection was demonstrated using DiCre inducible gene deletion in vitro and in vivo. DUB2 is found in the nucleus and interacts with nuclear proteins associated with transcription/chromatin dynamics, mRNA splicing and mRNA capping. DUB2 has broad linkage specificity, cleaving all the di-ubiquitin chains except for Lys27 and Met1. Our study demonstrates the crucial role that DUBs play in differentiation and intracellular survival of Leishmania and that amastigotes are exquisitely sensitive to disruption of ubiquitination homeostasis.

High-throughput proteome analysis reveals targeted TRPM8 degradation in prostate cancer.

The Ca2+-permeable ion channel TRPM8 is a hallmark of the prostate epithelium. We recently discovered that TRPM8 is an ionotropic testosterone receptor. This finding suggested that testosterone-induced TRPM8 activity regulates Ca2+ homeostasis in the prostate epithelium. Since androgens are significantly implicated in prostate cancer development, the role of the novel testosterone receptor TRPM8 in cancer was assessed in our study. Although TRPM8 mRNA levels increase at the early prostate cancer stages, we found that it is not proportionally translated into TRPM8 protein levels. High-throughput proteome analysis revealed that TRPM8 degradation is enhanced in human prostate cancer cells. This degradation is executed via a dual degradation mechanism with the involvement of both lysosomal and proteasomal proteolytic pathways. The evaluation of the TRPM8 expression pattern in prostate cancer patients further confirmed the incidence of TRPM8 removal from the plasma membrane and its internalization pattern coincided with the severity of the tumor. Together, our results indicate that enhanced TRPM8 hydrolysis in prostate cancer could present an adaptation mechanism, sustained via bypassing testosterone-induced rapid Ca2+ uptake through TRPM8, thus, diminishing the rates of apoptosis. In this light, recovery of TRPM8 may pose a novel therapeutic strategy for an anti-tumor defense mechanism.

Autophagy and Alpha-Synuclein: Relevance to Parkinson's Disease and Related Synucleopathies.

Evidence from human postmortem material, transgenic mice, and cellular/animal models of PD link alpha-synuclein accumulation to alterations in the autophagy lysosomal pathway. Conversely, alpha-synuclein mutations related to PD pathogenesis, as well as post-translational modifications of the wild-type protein, result in the generation of aberrant species that may impair further the function of the autophagy lysosomal pathway, thus generating a vicious cycle leading to neuronal death. Moreover, PD-linked mutations in lysosomal-related genes, such as glucocerebrosidase, have been also shown to contribute to alpha-synuclein accumulation and related toxicity, indicating that lysosomal dysfunction may, in part, account for the neurodegeneration observed in synucleinopathies. In the current review, we summarize findings related to the inter-relationship between alpha-synuclein and lysosomal proteolytic pathways, focusing especially on recent experimental strategies based on the manipulation of the autophagy lysosomal pathway to counteract alpha-synuclein-mediated neurotoxicity in vivo. Pinpointing the factors that regulate alpha-synuclein association to the lysosome may represent potential targets for therapeutic interventions in PD and related synucleinopathies.

Chaperone-Mediated Autophagy and Mitochondrial Homeostasis in Parkinson's Disease.

Parkinson's disease (PD), a complex neurodegenerative disorder, is pathologically characterized by the formation of Lewy bodies and loss of dopaminergic neurons in the substantia nigra pars compacta (SNc). Mitochondrial dysfunction is considered to be one of the most important causative mechanisms. In addition, dysfunction of chaperone-mediated autophagy (CMA), one of the lysosomal proteolytic pathways, has been shown to play an important role in the pathogenesis of PD. An exciting and important development is recent finding that CMA and mitochondrial quality control may be linked. This review summarizes the studies revealing the link between autophagy and mitochondrial function. Discussions are focused on the connections between CMA and mitochondrial failure and on the role of MEF2D, a neuronal survival factor, in mediating the regulation of mitochondria in the context of CMA. These new findings highlight the need to further explore the possibility of targeting the MEF2D-mitochondria-CMA network in both understanding the PD pathogenesis and developing novel therapeutic strategies.

Leucine-rich diet supplementation modulates foetal muscle protein metabolism impaired by Walker-256 tumour

BackgroundCancer-cachexia induces a variety of metabolic disorders of protein turnover and is more pronounced when associated with pregnancy. Tumour-bearing pregnant rats have impaired protein balance, which decreases protein synthesis and increases muscle breakdown. Because branched-chain amino acids, especially leucine, stimulate protein synthesis, we investigated the effect of a leucine-rich diet on protein metabolism in the foetal gastrocnemius muscles of tumour-bearing pregnant rats.MethodsFoetuses of pregnant rats with or without Walker 256 tumours were divided into six groups. During the 20 days of the experiment, the pregnant groups were fed with either a control diet (C, control rats; W, tumour-bearing rats; Cp, rats pair-fed the same normoprotein-diet as the W group) or with a leucine-rich diet (L, leucine rats; LW, leucine tumour-bearing rats; and Lp, rats pair-fed the same leucine-rich diet as the LW group). After the mothers were sacrificed, the foetal gastrocnemius muscle samples were resected, and the protein synthesis and degradation and tissue chymotrypsin-like, cathepsin and calpain enzyme activities were assayed. The muscle oxidative enzymes (catalase, glutathione-S-transferase and superoxide dismutase), alkaline phosphatase enzyme activities and lipid peroxidation (malondialdehyde) were also measured.ResultsTumour growth led to a reduction in foetal weight associated with decreased serum protein, albumin and glucose levels and low haematocrit in the foetuses of the W group, whereas in the LW foetuses, these changes were less pronounced. Muscle protein synthesis (measured by L-[3H]-phenylalanine incorporation) was reduced in the W foetuses but was restored in the LW group. Protein breakdown (as assessed by tyrosine release) was enhanced in the L and W groups, but chymotrypsin-like activity increased only in group W and tended toward an increase in the LW foetuses. The activity of cathepsin H was significantly higher in the W group foetuses, but the proteolytic calcium-dependent pathway showed similar enzyme activity. In parallel, an intense oxidative stress process was observed only in the group W foetuses.ConclusionsThese data suggested that the proteasomal and lysosomal proteolytic pathways and oxidative stress are likely to participate in the process of foetal muscle catabolism of Walker’s tumour-bearing pregnant rats. The present work shows that foetal muscle can be protected by supplementation with a leucine-rich diet.

Chaperone-Mediated Autophagy in the Kidney: The Road More Traveled

Chaperone-mediated autophagy (CMA) is a lysosomal proteolytic pathway in which cytosolic substrate proteins contain specific chaperone recognition sequences required for degradation and are translocated directly across the lysosomal membrane for destruction. CMA proteolytic activity has a reciprocal relationship with macroautophagy: CMA is most active in cells in which macroautophagy is least active. Normal renal proximal tubular cells have low levels of macroautophagy, but high basal levels of CMA activity. CMA activity is regulated by starvation, growth factors, oxidative stress, lipids, aging, and retinoic acid signaling. The physiological consequences of changes in CMA activity depend on the substrate proteins present in a given cell type. In the proximal tubule, increased CMA results from protein or calorie starvation and from oxidative stress. Overactivity of CMA can be associated with tubular lysosomal pathology and certain cancers. Reduced CMA activity contributes to protein accumulation in renal tubular hypertrophy, but may contribute to oxidative tissue damage in diabetes and aging. Although there are more questions than answers about the role of high basal CMA activity, this remarkable feature of tubular protein metabolism appears to influence a variety of chronic diseases.

FoxO transcription factors: their roles in the maintenance of skeletal muscle homeostasis.

Forkhead box class O family member proteins (FoxOs) are highly conserved transcription factors with important roles in cellular homeostasis. The four FoxO members in humans, FoxO1, FoxO3, FoxO4, and FoxO6, are all expressed in skeletal muscle, but the first three members are the most studied in muscle. In this review, we detail the multiple modes of FoxO regulation and discuss the central role of these proteins in the control of skeletal muscle plasticity. FoxO1 and FoxO3 are key factors of muscle energy homeostasis through the control of glycolytic and lipolytic flux, and mitochondrial metabolism. They are also key regulators of protein breakdown, as they modulate the activity of several actors in the ubiquitin–proteasome and autophagy–lysosomal proteolytic pathways, including mitochondrial autophagy, also called mitophagy. FoxO proteins have also been implicated in the regulation of the cell cycle, apoptosis, and muscle regeneration. Depending of their activation level, FoxO proteins can exhibit ambivalent functions. For example, a basal level of FoxO factors is necessary for cellular homeostasis and these proteins are required for adaptation to exercise. However, exacerbated activation may occur in the course of several diseases, resulting in metabolic disorders and atrophy. A better understanding of the precise functions of these transcriptions factors should thus lead to the development of new therapeutic approaches to prevent or limit the muscle wasting that prevails in numerous pathological states, such as immobilization, denervated conditions, neuromuscular disease, aging, AIDS, cancer, and diabetes.

Bowman-Birk inhibitors, proteasome peptidase activities and colorectal pre neoplasias induced by 1,2-dimethylhydrazine in Swiss mice

Bowman-Birk inhibitors (BBIs) are protein molecules containing two inhibitory domains for enzymes similar to trypsin and chymotrypsin. Interest in these inhibitors arose from their properties against the cancer chemically induced by 1,2-dimethylhydrazine (DMH). In this study the effect of two BBI preparations (from Glycine max and Macrotyloma axillare) were evaluated for the prevention of colorectal neoplasia induced by intraperitoneal injections of DMH, given at a dose of 30mg/kg, during 12weeks. Mice treated with DMH presented histopathological alterations consistent with tumor development, augmented CD44 expression and increased proteasome peptidase activities. Lysosomal fractions, obtained from the intestines, were chromatographed in a Sepharose-BBI column and increased activity for trypsin and chymotrypsin-like proteases recovered from DMH-treated animals. In parallel, mice treated for eight weeks with BBIs showed a decrease in the chymotrypsin and trypsin-like proteasome activities compared to animals fed on normal diet. For the groups receiving simultaneous treatment with DMH and BBIs, dysplasic lesions were not observed and proteasome peptidase activities were similar to the control group after the 24th week. These results suggest that the mechanism by which BBIs could prevent the appearance of pre neoplastic lesions is associated with inhibition of both the lysosomal and proteasome-dependent proteolytic pathways.

Crosstalk between Hsp70 molecular chaperone, lysosomes and proteasomes in autophagy-mediated proteolysis in human retinal pigment epithelial cells.

The pathogenesis of age-related macular degeneration involves chronic oxidative stress, impaired degradation of membranous discs shed from photoreceptor outer segments and accumulation of lysosomal lipofuscin in retinal pigment epithelial (RPE) cells. It has been estimated that a major part of cellular proteolysis occurs in proteasomes, but the importance of proteasomes and the other proteolytic pathways including autophagy in RPE cells is poorly understood. Prior to proteolysis, heat shock proteins (Hsps), agents that function as molecular chaperones, attempt to refold misfolded proteins and thus prevent the accumulation of cytoplasmic protein aggregates. In the present study, the roles of the Hsp70 molecular chaperone and proteasomal and lysosomal proteolytic pathways were evaluated in human RPE cells (ARPE-19). The Hsp70 and ubiquitin protein levels and localization were analysed by Western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate the morphological changes. Hsp70 levels were modulated using RNA interference and overexpression techniques. Cell viability was measured by colorimetric assay. The proteasome inhibitor MG-132 evoked the accumulation of perinuclear aggregates positive for Hsp70, ubiquitin-protein conjugates and the lysosomal membrane protein LAMP-2. Interestingly, the hsp70 mRNA depletion significantly increased cell death in conjunction with proteasome inhibition. We found that the accumulation of lysosomes was reversible: a cessation of proteasome inhibition led to clearance of the deposits via a mechanism believed to include autophagy. The molecular chaperone Hsp70, proteasomes and autophagy have an important regulatory role in the protein turnover of human RPE cells and may thus open new avenues for understanding degenerative processes in retinal cells.

Crosstalk between Hsp70 molecular chaperone, lysosomes and proteasomes in autophagy‐mediated proteolysis in human retinal pigment epithelial cells

Abstract Purpose The pathogenesis of age‐related macular degeneration involves chronic oxidative stress, impaired degradation of membranous discs shed from photoreceptor outer segments and accumulation of lysosomal lipofuscin in retinal pigment epithelial (RPE) cells. It has been estimated that a major part of cellular proteolysis occurs in proteasomes, however the importance of proteasomes and the other proteolytic pathways including autophagy in RPE cells are poorly understood. In the present study the role of Hsp70 molecular chaperones, proteasomal and lysosomal proteolytic pathways were evaluated in human RPE cells (ARPE‐19). Methods The Hsp70 and ubiquitin protein levels and localization were analyzed by western blotting and immunofluorescense. Confocal and transmission electron microscopy were used to detect cellular organelles and to evaluate morphological changes. Results The proteasome inhibition evoked the accumulation of perinuclear lysosomal vesicles/residual bodies positive for ubiquitin and the lysosomal membrane protein LAMP‐2, and a robust accumulation of Hsp70 protein and ubiquitin‐protein conjugates. We found that the accumulation of lysosomal/residual bodies was reversible, a cessation of proteasome inhibition led to clearance of the deposits via a mechanism that probably includes autophagy. Conclusion We show novel connection of Hsp70 molecular chaperones, proteasomes and autophagy in regulation of protein turnover in human retinal pigment epithelial cells that may thus open new insights to understand degenerative processes in retina cell pathology.

Secondary Bortezomib-Resistance Is Characterized by Upregulation of Proteasomal Subunits and Their Insufficient Inhibition by Bortezomib, Which Can Be Overcome by Alternative Proteasome Inhibitors Combined with Induction of the ER Stress Response.

Resistance towards proteasome inhibition by Bortezomib (Velcade®) represents a challenge for myeloma therapy. Its biology has not yet been characterized in detail. We have demonstrated that Bortezomib-sensitive malignant haematopoetic cells can acquire secondary resistance to Bortezomib in vitro. We here present the first analysis of proteasome biology and activity, alternative proteolytic pathways, ubiquitin-specific proteases (USP) and the ER stress response (unfolded protein response, UPR) upstream of the proteasome, as well as in vitro cytotoxicity of conventional cytotoxic drugs, alternative proteasome inhibitors and agents that target the UPR in Bortezomib-resistant (BR) cells, compared to wild type (WT) controls. BR cells had higher activities of all subunits of the constitutive and the immunoproteasome, as deferred from turnover of fluorogenic substrates as well as affinity-labelling of active proteasome subunits in intact cells. This was mirrored by increased levels of proteasomal β1 and β2, but especially β5 polypeptides, implicating a homeostatic system that senses and corrects low proteasome activity in cells chronically exposed to Bortezomib. While the vinylsulfone-type proteasome inhibitor NLVS abrogated detectable proteasome activity in both BR and WT cells, Bortezomib at therapeutic concentrations eliminated proteasomal β1 and β5-type activity only in WT cells, while BR cells retained residual activity. These changes in proteasome biology appear to be the molecular hallmark of required Bortezomib resistance, since no changes were observed between WT and BR cells in alternative cytosolic or lysosomal proteolytic pathways, UPR activity as well as the gross activity pattern of USP. As expected, this translated into sensitivity against cytotoxic drugs in vitro: BR cells were less sensitive towards alternative proteasome inhibitors. However, while the IC50 for pan-proteasome inhibitors was only roughly doubled in BR cells, it was nearly tenfold elevated for the β5-preferring vinylsulfone inhibitor NLVS. By contrast, sensitivity towards anthracyclines or cytotoxicity induced by ER stressors as well as the synergy between proteasome inhibitors and UPR-activators remained unaffected in BR cells. Based on our data, proteasome inhibitors with activity profiles different from that of Bortezomib, alone or in combination with induction of the UPR, may represent an appropriate concept to overcome secondary Bortezomib resistance.

Suppression of chaperone-mediated autophagy in the renal cortex during acute diabetes mellitus

In the renal hypertrophy that occurs in diabetes mellitus, decreased proteolysis may lead to protein accumulation, but it is unclear which proteins are affected. Because the lysosomal proteolytic pathway of chaperone-mediated autophagy is suppressed by growth factors in cultured cells, we investigated whether the abundance of substrates of this pathway increase in diabetic hypertrophy. Rats with streptozotocin (STZ)-induced diabetes were pair-fed with vehicle-injected control rats. Proteolysis was measured as lysine release in renal cortical suspensions and protein synthesis as phenylalanine incorporation. Target proteins of chaperone-mediated autophagy were measured in cortical lysates and nuclear extracts by immunoblot analysis. Proteins that regulate chaperone-mediated autophagy [the lysosomal-associated membrane protein 2a (LAMP2a) or the heat shock cognate protein of 73 kD (hsc-73)] were measured in lysosomes isolated by density gradient centrifugation. Proteolysis decreased by 41% in diabetic rats; protein synthesis increased at 3 days, but returned to baseline by 7 days. The abundance of proteins containing that chaperone-mediated autophagy KFERQ signal motif increased 38% and individual KFERQ containing proteins [e.g., M2 pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and pax2] were more abundant. LAMP2a and hsc73 decreased by 25% and 81%, respectively, in cortical lysosomes from diabetic vs. control rats. The decline in proteolysis in acute diabetes mellitus is associated with an increase in proteins degraded by chaperone-mediated autophagy and a decrease in proteins which regulate this pathway. This study provides the first evidence that reduced chaperone-mediated autophagy contributes to accumulation of specific proteins in diabetic-induced renal hypertrophy.

Down-regulation of genes in the lysosomal and ubiquitin-proteasome proteolytic pathways in calpain-3-deficient muscle

Calpain-3 deficiency leads to muscular dystrophy in humans and mice and to perturbation of the NFκB/IκB pathway. As this phenotype is mainly atrophic, this study was performed to determine whether protein turnover and/or proteolytic gene expression was altered in muscles following calpain-3 deficiency. In vitro rates of protein turnover and of substrate ubiquitination, cathepsin B and B+L activities, and mRNA levels for several proteolytic genes were measured in skeletal muscles from 4–5 month-old control and calpain-3 knockout mice. Rates of protein synthesis and breakdown, cathepsin activities, and rates of substrate ubiquitination remained stable in muscles from calpain-3 deficient mice. However, and surprisingly, mRNA levels for cathepsin L, the 14-kDa ubiquitin-conjugating enzyme E2, and the C2 subunit of the 20S proteasome decreased by ∼47% ( P<0.005) in the gastrocnemius muscle from calpain-3 deficient mice. In contrast, muscle mRNA levels for ubiquitin and subunit S5a of the 26S proteasome were unaffected by calpain-3 deficiency. Taken together these data demonstrate that the expression of some genes that are involved in distinct proteolytic pathways is selectively and coordinately down-regulated without any effect on proteolysis. This suggests new pathophysiological hypotheses, e.g. a lack of maturation of NFκB precursor and/or a defect in specific substrate targeting.

Heat Shock Proteins and Proteasome Function in Neurodegeneration

In order to survive and maintain cellular homeostasis the different cells of the central nervous system (CNS) must continually adapt to a wide variety of genetic and environmental stimuli. Heat shock proteins (HSPs) are a class of proteins that are rapidly elevated in response to stress, aiding in protein folding, protein unfolding, and protein trafficking. The proteasome is a large intracellular protease that is responsible for the majority of intracellular protein degradation in the CNS. In particular, the proteasome is responsible for the degradation of most oxidized, aggregated, and damaged proteins. Increasing evidence suggests that HSPs may play important roles in the targeting of proteins to the proteasomal and lysosomal proteolytic pathways. Additionally, HSPs may play an important role in protein unfolding necessary for proteasome-mediated proteolytic degradation. Because the proteasome appears to become inhibited in a wide variety of neurodegenerative conditions, and possibly contribute to neurodegeneration in those conditions, studies must be conducted to determine how alterations in HSPs may contribute to proteasome inhibition in neurodegenerative disorders. The focus of this review is to outline what is currently known about HSPs and proteasome function in normal and neurodegenerative settings, and to discuss the possible therapeutic benefit of maintaining a robust HSP-proteasome interplay within the CNS. Keywords: Heat Shock, Proteins, Proteasome, Neurodegeneration, Adenosine Tri-Phosphate, Mitogen, Human DnaJ Homolog

Polyglutamine Expansion, Protein Aggregation, Proteasome Activity, and Neural Survival

Huntington's disease (HD) is one of eight established triplet repeat neurodegenerative disorders, which are collectively caused by the genetic expansion of polyglutamine repeats. While the mechanism(s) by which polyglutamine expansion causes neurodegeneration in each of these disorders is being intensely investigated, the underlying cause of polyglutamine toxicity has not been fully elucidated. A number of studies have focused on the potential role of protein aggregation and disruption of the proteasome proteolytic pathway in polyglutamine-mediated neurodegeneration. However, at present it is not clear whether polyglutamine-mediated protein aggregation is sufficient to induce cell death, nor has it been clearly determined whether proteasome inhibition precedes, coincides, or occurs as the result of the formation of polyglutamine-associated protein aggregation. To address these important components of polyglutamine toxicity, in the present study we utilized neural SH-SY5Y cells stably transfected with polyglutamine-green fluorescent protein constructs to examine the effects of polyglutamine expansion on protein aggregation, proteasome activity, and neural cell survival. Data from the present study demonstrate that polyglutamine expansion does not dramatically impair proteasome activity or elevate protein aggregate formation under basal conditions, but does significantly impair the ability of the proteasome to respond to stress, and increases stress-induced protein aggregation following stress, all in the absence of neural cell death.

Skeletal muscle mRNA levels for cathepsin B, but not components of the ubiquitin‒proteasome pathway, are increased in patients with lung cancer referred for thoracotomy

Muscle wasting is a common and prominent feature of advanced cancer, including lung cancer. Evidence from animal experiments suggests that accelerated proteolysis via the ubiquitin--proteasome pathway is the primary cause of cancer-related cachexia. However, there are few data on the role of this pathway in determining muscle wasting in human cancer. The present study was designed to measure whether skeletal muscle gene expression of components of the ubiquitin-proteasome pathway and/or the lysosomal proteolytic pathway was increased in patients with early lung cancer. A total of 36 patients with lung cancer referred for curative resection and 10 control subjects had biopsies of latissimus dorsi muscle taken at operation. mRNA levels of four components of the ubiquitin-proteasome pathway, i.e. polyubiquitin, C2 alpha proteasome subunit, 14 kDa ubiquitin-carrier protein and ubiquitin-activating protein, and of two lysosomal proteolytic enzymes, i.e. cathepsin B and cathepsin D, were measured using quantitative Northern blotting. mRNA levels for cathepsin B, but not for components of the ubiquitin--proteasome pathway, were higher in patients with cancer compared with controls (P=0.01). Among lung cancer patients, cathepsin B mRNA levels correlated with fat-free mass index (r = -0.57, P=0.003) and tumour stage (r(s)=0.45, P=0.03), and were higher in smokers (P=0.04). Thus gene expression of the lysosomal protease cathepsin B is increased in the skeletal muscle of patients with early lung cancer, and the strong inverse relationship with fat-free mass suggests that cathepsin B may have a role in inducing muscle wasting in the early stages of lung cancer.

Skeletal muscle mRNA levels for cathepsin B, but not components of the ubiquitin–proteasome pathway, are increased in patients with lung cancer referred for thoracotomy

Muscle wasting is a common and prominent feature of advanced cancer, including lung cancer. Evidence from animal experiments suggests that accelerated proteolysis via the ubiquitin-proteasome pathway is the primary cause of cancer-related cachexia. However, there are few data on the role of this pathway in determining muscle wasting in human cancer. The present study was designed to measure whether skeletal muscle gene expression of components of the ubiquitin-proteasome pathway and/or the lysosomal proteolytic pathway was increased in patients with early lung cancer. A total of 36 patients with lung cancer referred for curative resection and 10 control subjects had biopsies of latissimus dorsi muscle taken at operation. mRNA levels of four components of the ubiquitin-proteasome pathway, i.e. polyubiquitin, C2α proteasome subunit, 14kDa ubiquitin-carrier protein and ubiquitin-activating protein, and of two lysosomal proteolytic enzymes, i.e. cathepsin B and cathepsin D, were measured using quantitative Northern blotting. mRNA levels for cathepsin B, but not for components of the ubiquitin-proteasome pathway, were higher in patients with cancer compared with controls (P = 0.01). Among lung cancer patients, cathepsin B mRNA levels correlated with fat-free mass index (r =-0.57, P = 0.003) and tumour stage (rs = 0.45, P = 0.03), and were higher in smokers (P = 0.04). Thus gene expression of the lysosomal protease cathepsin B is increased in the skeletal muscle of patients with early lung cancer, and the strong inverse relationship with fat-free mass suggests that cathepsin B may have a role in inducing muscle wasting in the early stages of lung cancer.

Lysosomal proteases as potential targets for the induction of apoptotic cell death in human neuroblastomas.

Neuroblastoma is the most common type of cancer in infants. In children this tumor is particularly aggressive; despite various new therapeutic approaches, it is associated with poor prognosis. Given the importance of endosomal-lysosomal proteolysis in cellular metabolism, we hypothesized that inhibition of lysosomal protease would impact negatively on neuroblastoma cell survival. Treatment with E-64 or CA074Me (2 specific inhibitors of cathepsin B) or with pepstatin A (a specific inhibitor of cathepsin D) was cytotoxic for 2 neuroblastoma cell lines having different degrees of malignancy. Cell death was associated with condensation and fragmentation of chromatin and externalization of plasma membrane phosphatidylserine, 2 hallmarks of apoptosis. Concomitant inhibition of the caspase cascade protected neuroblastoma cells from cathepsin inhibitor-induced cytotoxicity. These data indicate that prolonged inhibition of the lysosomal proteolytic pathway is incompatible with cell survival, leading to apoptosis of neuroblastoma cells, and that the cathepsin-mediated and caspase-mediated proteolytic systems are connected and cooperate in the regulation of such an event. Since modern antitumor chemotherapy is aimed at restoring the normal rate of apoptosis in neoplastic tissues, the demonstration that endosomal-lysosomal cathepsins are involved in this process may constitute a basis for novel strategies that include cathepsin inhibitors in the therapeutic regimen.

Increased lysosome‐related proteins in the skeletal muscles of distal myopathy with rimmed vacuoles

Investigators have speculated that the degenerative process in distal myopathy with rimmed vacuoles (DMRV) mainly involves the lysosomal system. To investigate possible protein abnormalities related to intracellular lysosomal proteolytic pathways in DMRV-affected muscles, we performed immunohistochemical analyses of certain proteins in muscle biopsy specimens obtained from patients with various neuromuscular diseases, including DMRV, muscular dystrophy, polymyositis, and amyotrophic lateral sclerosis, and in normal human muscles specimens. Immunohistochemically, most muscle fibers in normal control specimens showed little or no reaction for clathrin and α- and γ-subunits of adaptin-constituted adaptin proteins (AP)-1 and AP-2, respectively. Abnormal increases in these proteins were demonstrated mainly in the cytoplasm of atrophic fibers or in necrotic fibers in all diseased specimens. Particularly in DMRV-affected muscles, α- and γ-adaptins were often observed inside or on the rims of vacuoles and in the cytoplasm of vacuolated fibers. Abnormal increases in Golgi-zone protein were also demonstrated in DMRV muscles. The rims of rimmed vacuoles were negative for kinectin, an endoplasmic reticulum–binding protein. Positive staining for both proteins, however, was sometimes seen inside the vacuoles in DMRV-affected fibers. These results suggest increased endocytosis at the plasma membrane as well as secretion involving transport from the trans-Golgi network of the Golgi apparatus in DMRV. Accumulation of various lysosome-related proteins within the rimmed vacuoles indicates at least some of these vacuoles may be autolysosomes. © 2000 John Wiley & Sons, Inc. Muscle Nerve 23: 1686–1693, 2000

Contribution of the ubiquitin-proteasome pathway to overall muscle proteolysis in hypercatabolic patients

The influence of the gene expression of critical components of the cytoplasmic and lysosomal proteolytic pathways on the rate of protein degradation was evaluated in the leg skeletal muscle of 8 severely traumatized patients. Muscle proteolysis was determined as the intramuscular phenylalanine rate of appearance by L-[ring-2H5]phenylalanine infusion and the leg arteriovenous catheterization technique combined with muscle biopsy. Muscle mRNA levels of UbB polyubiquitin and cathepsin B were determined by reverse transcriptase-competitive polymerase chain reaction and expressed as a percent of the mRNA level of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In the patients, individual values for UbB polyubiquitin mRNA levels directly correlated with the rate of muscle proteolysis (r = .76, P < .05), whereas no correlation (r = .10) was found between cathepsin B mRNA levels and proteolysis. Thus, after trauma, the rate of muscle proteolysis appears to be largely regulated by the ubiquitin-proteasome system at the level of gene transcription.