PACSIN2 as a modulator of autophagy and mercaptopurine cytotoxicity: mechanisms in lymphoid and intestinal cells.

PACSIN2 variants are associated with gastrointestinal effects of thiopurines and thiopurine methyltransferase activity through an uncharacterized mechanism that is postulated to involve autophagy. This study aims to clarify the role of PACSIN2 in autophagy and in thiopurine cytotoxicity in leukemic and intestinal models. Higher autophagy and lower PACSIN2 levels were observed in inflamed compared with non-inflamed colon biopsies of inflammatory bowel disease pediatric patients at diagnosis. PACSIN2 was identified as an inhibitor of autophagy, putatively through inhibition of autophagosome formation by a protein-protein interaction with LC3-II, mediated by a LIR motif. Moreover, PACSIN2 resulted a modulator of mercaptopurine-induced cytotoxicity in intestinal cells, suggesting that PACSIN2-regulated autophagy levels might influence thiopurine sensitivity. However, PACSIN2 modulates cellular thiopurine methyltransferase activity via mechanisms distinct from its modulation of autophagy.

Amino acid stress response genes promote L-asparaginase resistance in pediatric acute lymphoblastic leukemia.

Understanding the genomic and epigenetic mechanisms of drug resistance in pediatric acute lymphoblastic leukemia (ALL) is critical for further improvements in treatment outcomes. The role of transcriptomic response in conferring resistance to l-asparaginase (LASP) is poorly understood beyond asparagine synthetase (ASNS). We defined reproducible LASP response genes in LASP-resistant and LASP-sensitive ALL cell lines as well as primary leukemia samples from newly diagnosed patients. Defining target genes of the amino acid stress response-related transcription factor activating transcription factor 4 (ATF4) in ALL cell lines using chromatin immunoprecipitation sequencing (ChIP-seq) revealed 45% of genes that changed expression after LASP treatment were direct targets of the ATF4 transcription factor, and 34% of these genes harbored LASP-responsive ATF4 promoter binding events. SLC7A11 was found to be a response gene in cell lines and patient samples as well as a direct target of ATF4. SLC7A11 was also one of only 2.4% of LASP response genes with basal level gene expression that also correlated with LASP ex vivo resistance in primary leukemia cells. Experiments using chemical inhibition of SLC7A11 with sulfasalazine, gene overexpression, and partial gene knockout recapitulated LASP resistance or sensitivity in ALL cell lines. These findings show the importance of assessing changes in gene expression following treatment with an antileukemic agent for its association with drug resistance and highlight that many response genes may not differ in their basal expression in drug-resistant leukemia cells.

CASPorter: A Novel Inducible Human CASP1/NALP3/ASC Inflammasome Biosensor.

BACKGROUND: Following our 2015 elucidation of the CASP1/NALP3 inflammasome mechanism of glucocorticoid (GC)-resistance in pediatric acute lymphoblastic leukemia (ALL) patients, we engineered a cell-based CASP1/NALP3 reporter system suitable for high-throughput screening (HTS) of small molecule libraries, with the purpose of identifying compounds capable of inhibiting the CASP1/NALP3 inflammasome and synergizing with GC drugs for the treatment of GC-resistant ALL patients and various autoinflammatory diseases. METHODS: A Dox-controlled system was utilized to induce the expression of the ASC transgene in HEK293 cells while simultaneously overexpressing NLRP3 and CASP1. ASC/CASP1/NALP3 inflammasome complex formation was confirmed by co-immunoprecipitation (co-IP) experiments. Next, a LV fluorescence-based biosensor (CASPorter) was transduced in the HEK293-iASC-NLRP3/CASP1 cell line to monitor the real-time activation of CASP1/NALP3 inflammasome in live cells. The applicability and effectiveness of the CASPorter cell line were tested by co-treatment with Dox and four known CASP1/NLRP3 inhibitors (MCC950, Glyburide, VX-765 and VRT-043198). Inflammasome activation and inhibitions were assessed by Western blotting, fluorescence microscopy and flow cytometry (FC) methods. RESULTS: Dox treatment significantly induced ASC expression and increased levels of cleaved and catalytically active CASP1, co-IPs further demonstrated that CASP1 was pulled-down with NLRP3 in HEK293-iASC-NLRP3/CASP1 cells after induction of ASC by Dox treatment. In HEK293-iASC-NLRP3/CASP1-CASPorter cell system, cleavage of the CASP1 consensus site (YVAD) in the CASPorter protein after Dox treatment causing excitation/emission of green fluorescence and the 71% GFP+ cell population increase quantified by FC (78.1% vs 6.90%). Dox-induced activation of the NLRP3 inflammasome was dose-dependently inhibited by Dox co-treatment with four known CASP1/NLRP3 inhibitors. CONCLUSION: We have established a cell-based CASP1/NLRP3 inflammasome model, utilizing a fluorescence biosensor as readout for qualitatively observing and quantitatively determining the activation of caspase 1 and NLRP3 inflammasomes in living cells and easily define the inhibitory effect of inhibitors with high efficacy.

Identification of small molecules that mitigate vincristine-induced neurotoxicity while sensitizing leukemia cells to vincristine.

Vincristine (VCR) is one of the most widely prescribed medications for treating solid tumors and acute lymphoblastic leukemia (ALL) in children and adults. However, its major dose-limiting toxicity is peripheral neuropathy that can disrupt curative therapy. Peripheral neuropathy can also persist into adulthood, compromising quality of life of childhood cancer survivors. Reducing VCR-induced neurotoxicity without compromising its anticancer effects would be ideal. Here, we show that low expression of NHP2L1 is associated with increased sensitivity of primary leukemia cells to VCR, and that concomitant administration of VCR with inhibitors of NHP2L1 increases VCR cytotoxicity in leukemia cells, prolongs survival of ALL xenograft mice, but decreases VCR effects on human-induced pluripotent stem cell-derived neurons and mitigates neurotoxicity in mice. These findings offer a strategy for increasing VCR's antileukemic effects while reducing peripheral neuropathy in patients treated with this widely prescribed medication.

Hypomethylation of NLRP3 gene promoter discriminates glucocorticoid-resistant from glucocorticoid-sensitive idiopathic nephrotic syndrome patients.

To assess whether NLRP3 gene promoter methylation was able to discriminate glucocorticoid (GC)-resistant from GC-sensitive idiopathic nephrotic syndrome (INS), patients with minimal change disease (MCD) or focal segmental glomerulosclerosis (FSGS), we measured the methylation level of NLRP3 promoter in DNA from peripheral blood cells of 10 adult patients with GC-resistant FSGS already in hemodialysis and 18 patients with GC-sensitive INS (13 MCD/5 FSGS) and in 21 pediatric patients with INS with MCD/FSGS before starting any treatment. Association of NLRP3 inflammasome with GC resistance was recapitulated in vitro in monocytic cell lines (THP-1 and U937). In both adults and pediatric patients, NLRP3 promoter methylation was significantly reduced in GC-resistant compared with GC-sensitive patients. Indeed, NLRP3 methylation distinguished GC-resistant and GC-sensitive patients (area under the receiver operating characteristic curve [AUROC] 86.7% in adults, p = 0.00019, and 73.5% in children, p = 0.00097). NLRP3 knock-down augmented sensitivity to GCs in THP-1 cells, whereas NLRP3 inflammasome activation lowered GC receptor concentration, increasing GC resistance in U937 cells. Our results uncovered a new biological mechanism by which patients with INS may acquire GC resistance, that could be used in future as a novel noninvasive diagnostic tool. Study Highlights WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC? ☑ Approximately 80% of patients with idiopathic nephrotic syndrome (INS) respond to glucocorticoids, with the remaining 20% being steroid-resistant. WHAT QUESTION DID THIS STUDY ADDRESS? ☑ Whether NLRP3 gene promoter methylation was able to discriminate glucocorticoid-resistant from glucocorticoid (GC)-sensitive INS. WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE? ☑ In both adults and children, NLRP3 promoter methylation was significantly reduced in leukocytes of patients with GC-resistant compared with GC-sensitive INS. NLRP3 inflammasome activation lowered GC receptor concentration and augmented GC resistance, whereas NLRP3 knockdown increased sensitivity to GCs in cell lines representative of monocytes (U937 and THP1). HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE? ☑ Our findings uncovered a new biological mechanism whereby patients with INS may develop resistance to GCs that could be used in the future as a novel noninvasive diagnostic tool.

Pharmacogenomics of intracellular methotrexate polyglutamates in patients' leukemia cells in vivo.

BACKGROUNDInterpatient differences in the accumulation of methotrexate's active polyglutamylated metabolites (MTXPGs) in leukemia cells influence its antileukemic effects.METHODSTo identify genomic and epigenomic and patient variables determining the intracellular accumulation of MTXPGs, we measured intracellular MTXPG levels in acute lymphoblastic leukemia (ALL) cells from 388 newly diagnosed patients after in vivo high-dose methotrexate (HDMTX) (1 g/m2) treatment, defined ALL subtypes, and assessed genomic and epigenomic variants influencing folate pathway genes (mRNA, miRNA, copy number alterations [CNAs], SNPs, single nucleotide variants [SNVs], CpG methylation).RESULTSWe documented greater than 100-fold differences in MTXPG levels, which influenced its antileukemic effects (P = 4 × 10-5). Three ALL subtypes had lower MTXPG levels (T cell ALL [T-ALL] and B cell ALL [B-ALL] with the TCF3-PBX1 or ETV6-RUNX1 fusions), and 2 subtypes had higher MTXPG levels (hyperdiploid and BCR-ABL like). The folate pathway genes SLC19A1, ABCC1, ABCC4, FPGS, and MTHFD1 significantly influenced intracellular MTXPG levels (P = 2.9 × 10-3 to 3.7 × 10-8). A multivariable model including the ALL subtype (P = 1.1 × 10-14), the SLC19A1/(ABCC1 + ABCC4) transporter ratio (P = 3.6 × 10-4), the MTX infusion time (P = 1.5 × 10-3), FPGS mRNA expression (P = 2.1 × 10-3), and MTX systemic clearance (P = 4.4 × 10-2) explained 42% of the variation in MTXPG accumulation (P = 1.1 × 10-38). Model simulations indicated that a longer infusion time (24 h vs. 4 h) was superior in achieving higher intracellular MTXPG levels across all subtypes if ALL.CONCLUSIONSThese findings provide insights into mechanisms underlying interpatient differences in intracellular accumulation of MTXPG in leukemia cells and its antileukemic effectsFUNDINGTHE National Cancer Institute (NCI) and the Institute of General Medical Sciences of the NIH, the Basque Government Programa Posdoctoral de Perfeccionamiento de Personal Investigador doctor, and the American Lebanese Syrian Associated Charities (ALSAC).

Integrative genomic analyses reveal mechanisms of glucocorticoid resistance in acute lymphoblastic leukemia.

Identification of genomic and epigenomic determinants of drug resistance provides important insights for improving cancer treatment. Using agnostic genome-wide interrogation of mRNA and miRNA expression, DNA methylation, SNPs, CNAs and SNVs/Indels in primary human acute lymphoblastic leukemia cells, we identified 463 genomic features associated with glucocorticoid resistance. Gene-level aggregation identified 118 overlapping genes, 15 of which were confirmed by genome-wide CRISPR screen. Collectively, this identified 30 of 38 (79%) known glucocorticoid-resistance genes/miRNAs and all 38 known resistance pathways, while revealing 14 genes not previously associated with glucocorticoid-resistance. Single cell RNAseq and network-based transcriptomic modelling corroborated the top previously undiscovered gene, CELSR2. Manipulation of CELSR2 recapitulated glucocorticoid resistance in human leukemia cell lines and revealed a synergistic drug combination (prednisolone and venetoclax) that mitigated resistance in mouse xenograft models. These findings illustrate the power of an integrative genomic strategy for elucidating genes and pathways conferring drug resistance in cancer cells.

Inflammasome-mediated glucocorticoid resistance: The receptor rheostat.

In primary acute lymphoblastic leukemia cells exhibiting de novo resistance to glucocorticoids, we recently discovered decreased promoter methylation of caspase 1 (CASP1) and NLR family, pyrin domain containing 3 (NLRP3), which resulted in increased transcription, constitutive NALP3 (NACHT, LRR and PYD domains-containing protein 3) inflammasome activation, and caspase 1-mediated cleavage of the glucocorticoid receptor. This revealed a novel mechanism of glucocorticoid resistance that was recapitulated in model systems.

MicroRNAs Form Triplexes with Double Stranded DNA at Sequence-Specific Binding Sites; a Eukaryotic Mechanism via which microRNAs Could Directly Alter Gene Expression.

MicroRNAs are important regulators of gene expression, acting primarily by binding to sequence-specific locations on already transcribed messenger RNAs (mRNA) and typically down-regulating their stability or translation. Recent studies indicate that microRNAs may also play a role in up-regulating mRNA transcription levels, although a definitive mechanism has not been established. Double-helical DNA is capable of forming triple-helical structures through Hoogsteen and reverse Hoogsteen interactions in the major groove of the duplex, and we show physical evidence (i.e., NMR, FRET, SPR) that purine or pyrimidine-rich microRNAs of appropriate length and sequence form triple-helical structures with purine-rich sequences of duplex DNA, and identify microRNA sequences that favor triplex formation. We developed an algorithm (Trident) to search genome-wide for potential triplex-forming sites and show that several mammalian and non-mammalian genomes are enriched for strong microRNA triplex binding sites. We show that those genes containing sequences favoring microRNA triplex formation are markedly enriched (3.3 fold, p<2.2 × 10(-16)) for genes whose expression is positively correlated with expression of microRNAs targeting triplex binding sequences. This work has thus revealed a new mechanism by which microRNAs could interact with gene promoter regions to modify gene transcription.

NALP3 inflammasome upregulation and CASP1 cleavage of the glucocorticoid receptor cause glucocorticoid resistance in leukemia cells.

Glucocorticoids are universally used in the treatment of acute lymphoblastic leukemia (ALL), and resistance to glucocorticoids in leukemia cells confers poor prognosis. To elucidate mechanisms of glucocorticoid resistance, we determined the prednisolone sensitivity of primary leukemia cells from 444 patients newly diagnosed with ALL and found significantly higher expression of CASP1 (encoding caspase 1) and its activator NLRP3 in glucocorticoid-resistant leukemia cells, resulting from significantly lower somatic methylation of the CASP1 and NLRP3 promoters. Overexpression of CASP1 resulted in cleavage of the glucocorticoid receptor, diminished the glucocorticoid-induced transcriptional response and increased glucocorticoid resistance. Knockdown or inhibition of CASP1 significantly increased glucocorticoid receptor levels and mitigated glucocorticoid resistance in CASP1-overexpressing ALL. Our findings establish a new mechanism by which the NLRP3-CASP1 inflammasome modulates cellular levels of the glucocorticoid receptor and diminishes cell sensitivity to glucocorticoids. The broad impact on the glucocorticoid transcriptional response suggests that this mechanism could also modify glucocorticoid effects in other diseases.

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 ß-galactosidase (ß-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 ß-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.

Chaperone-mediated gene therapy with recombinant AAV-PPCA in a new mouse model of type I sialidosis.

The lysosomal storage disease sialidosis is caused by a primary deficiency of the sialidase N-acetyl-α-neuraminidase-1 (NEU1). Patients with type I sialidosis develop an attenuated, non-neuropathic form of the disease also named cherry red spot myoclonus syndrome, with symptoms arising during juvenile/ adult age. NEU1 requires binding to its chaperone, protective protein/cathepsin A (PPCA), for lysosomal compartmentalization, stability and catalytic activation. We have generated a new mouse model of type I sialidosis that ubiquitously expresses a NEU1 variant carrying a V54M amino acid substitution identified in an adult patient with type I sialidosis. Mutant mice developed signs of lysosomal disease after 1year of age, predominantly in the kidney, albeit low residual NEU1 activity was detected in most organs and cell types. We demonstrate that the activity of the mutant enzyme could be effectively increased in all systemic tissues by chaperone-mediated gene therapy with a liver-tropic recombinant AAV2/8 vector expressing PPCA. This resulted in clear amelioration of the disease phenotype. These results suggest that at least some of the NEU1 mutations associated with type I sialidosis may respond to PPCA-chaperone-mediated gene therapy.

LPS-induced cytokine production in human dendritic cells is regulated by sialidase activity.

Removal of sialic acid from glycoconjugates on the surface of monocytes enhances their response to bacterial LPS. We tested the hypothesis that endogenous sialidase activity creates a permissive state for LPS-induced cytokine production in human monocyte-derived DCs. Of the four genetically distinct sialidases (Neu1-4), Neu1, Neu3, and Neu4 are expressed in human monocytes, but only Neu1 and Neu3 are up-regulated as cells differentiate into DCs. Neu1 and Neu3 are present on the surface of monocytes and DCs and are also present intracellularly. DCs contain a greater amount of sialic acid than monocytes, but the amount of sialic acid/mg total protein declines during differentiation to DCs. This relative hyposialylation of cells does not occur in mature DCs grown in the presence of zanamivir, a pharmacologic inhibitor of Neu3 but not Neu1, or DANA, an inhibitor of Neu1 and Neu3. Inhibition of sialidase activity during differentiation to DCs causes no detectable change in cell viability or expression of DC surface markers. Differentiation of monocytes into DCs in the presence of zanamivir results in reduced LPS- induced expression of IL-6, IL-12p40, and TNF-α by mature DCs, demonstrating a role for Neu3 in cytokine production. A role for Neu3 is supported by inhibition of cytokine production by DANA in DCs from Neu1⁻/⁻ and WT mice. We conclude that sialidase-mediated change in sialic acid content of specific cell surface glycoconjugates in DCs regulates LPS-induced cytokine production, thereby contributing to development of adaptive immune responses.

Muscle degeneration in neuraminidase 1-deficient mice results from infiltration of the muscle fibers by expanded connective tissue.

Neuraminidase 1 (NEU1) regulates the catabolism of sialoglycoconjugates in lysosomes. Congenital NEU1 deficiency in children is the basis of sialidosis, a severe neurosomatic disorder in which patients experience a broad spectrum of clinical manifestations varying in the age of onset and severity. Osteoskeletal deformities and muscle hypotonia have been described in patients with sialidosis. Here we present the first comprehensive analysis of the skeletal muscle pathology associated with loss of Neu1 function in mice. In this animal model, skeletal muscles showed an expansion of the epimysial and perimysial spaces, associated with proliferation of fibroblast-like cells and abnormal deposition of collagens. Muscle fibers located adjacent to the expanded connective tissue underwent extensive invagination of their sarcolemma, which resulted in the infiltration of the fibers by fibroblast-like cells and extracellular matrix, and in their progressive cytosolic fragmentation. Both the expanded connective tissue and the juxtaposed infiltrated muscle fibers were strongly positive for lysosomal markers and displayed increased proteolytic activity of lysosomal cathepsins and metalloproteinases. These combined features could lead to abnormal remodeling of the extracellular matrix that could be responsible for sarcolemmal invagination and progressive muscle fiber degeneration, ultimately resulting in an overt atrophic phenotype. This unique pattern of muscle damage, which has never been described in any myopathy, might explain the neuromuscular manifestations reported in patients with the type II severe form of sialidosis. More broadly, these findings point to a potential role of NEU1 in cell proliferation and extracellular matrix remodeling.

Heterodimerization of the sialidase NEU1 with the chaperone protective protein/cathepsin A prevents its premature oligomerization.

Lysosomal neuraminidase-1 (NEU1) forms a multienzyme complex with beta-galactosidase and protective protein/cathepsin A (PPCA). Because of its association with PPCA, which acts as a molecular chaperone, NEU1 is transported to the lysosomal compartment, catalytically activated, and stabilized. However, the mode(s) of association between these two proteins both en route to the lysosome and in the multienzyme complex has remained elusive. Here, we have analyzed the hydrodynamic properties of PPCA, NEU1, and a complex of the two proteins and identified multiple binding sites on both proteins. One of these sites on NEU1 that is involved in binding to PPCA can also bind to other NEU1 molecules, albeit with lower affinity. Therefore, in the absence of PPCA, as in the lysosomal storage disease galactosialidosis, NEU1 self-associates into chain-like oligomers. Binding of PPCA can reverse self-association of NEU1 by causing the disassembly of NEU1-oligomers and the formation of a PPCA-NEU1 heterodimeric complex. The identification of binding sites between the two proteins allowed us to create innovative structural models of the NEU1 oligomer and the PPCA-NEU1 heterodimeric complex. The proposed mechanism of interaction between NEU1 and its accessory protein PPCA provides a rationale for the secondary deficiency of NEU1 in galactosialidosis.

Neuraminidase 1 is a negative regulator of lysosomal exocytosis.

Lysosomal exocytosis is a Ca2+-regulated mechanism that involves proteins responsible for cytoskeletal attachment and fusion of lysosomes with the plasma membrane. However, whether luminal lysosomal enzymes contribute to this process remains unknown. Here we show that neuraminidase NEU1 negatively regulates lysosomal exocytosis in hematopoietic cells by processing the sialic acids on the lysosomal membrane protein LAMP-1. In macrophages from NEU1-deficient mice, a model of the disease sialidosis, and in patients' fibroblasts, oversialylated LAMP-1 enhances lysosomal exocytosis. Silencing of LAMP-1 reverts this phenotype by interfering with the docking of lysosomes at the plasma membrane. In neu1-/- mice the excessive exocytosis of serine proteases in the bone niche leads to inactivation of extracellular serpins, premature degradation of VCAM-1, and loss of bone marrow retention. Our findings uncover an unexpected mechanism influencing lysosomal exocytosis and argue that exacerbations of this process form the basis for certain genetic diseases.

Short-term, high dose enzyme replacement therapy in sialidosis mice.

Given the success of enzyme replacement therapy (ERT) in treating the systemic manifestations in a number of lysosomal storage disorders (LSDs), we evaluated the effect of ERT on the mouse model of sialidosis. This glycoproteinosis, which affects primarily the reticuloendothelial (RE) system, is caused by deficiency of lysosomal neuraminidase (NEU1) and consequent accumulation of sialylated glycoconjugates. NEU1 lacks a functional mannose-6-phosphate recognition marker and is not endocytosed by mammalian cells. However, the enzyme produced in insect cells has features that allow its effective uptake by RE cells and macrophages via the mannose receptor, and therefore represent an alternative method of therapy. In this study we tested the therapeutic efficacy of baculovirus (BV) expressed mouse neuraminidase (Neu1) in sialidosis mice. Four-week-old Neu1-/- mice were first injected intravenously with a single dose of the recombinant enzyme for assessment of the half-life of mannosylated Neu1 in vivo. Afterwards, a short-term ERT with a total of five enzyme injections over a 2-week period was performed for evaluation of phenotype correction. Neu1 infused alone or co-administered with its associated protein, protective protein/cathepsin A (PPCA) was effectively taken up by resident macrophages in many tissues. Restored Neu1 activity persisted for up to 4 days, depending on the tissue, and resulted in a significant reduction of lysosomal storage. However, beyond 2 weeks of treatment, ERT mice developed a severe immune response towards the exogenous Neu1 enzyme. These results may have important implications for ERT in sialidosis patients.

Targeting macrophages with baculovirus-produced lysosomal enzymes: implications for enzyme replacement therapy of the glycoprotein storage disorder galactosialidosis.

Lysosomal storage diseases (LSDs) are monogenic disorders of metabolism caused by deficiency of hydrolytic enzymes. In several LSDs, cells of the reticuloendothelial (RE) system are the primary targets of the disease. Exogenous administration of recombinant enzymes overproduced in mammalian cells has proved effective for treating the systemic phenotype in nonneuropathic patients with LSDs. However, for the treatment of diseases with primary involvement of the RE system, the production of the therapeutic enzyme in insect cells could be an alternative and advantageous method because glycoproteins expressed in insect cells carry carbohydrates of the pauci-mannose or core-type. These recombinant enzymes are in principle already poised to be internalized by cells that express mannose receptors, including macrophages. Here, we demonstrate that three baculovirus-expressed enzymes, protective protein/cathepsin A (PPCA), neuraminidase (Neu1), and beta-glucosidase, were readily taken up and restored lysosomal function in enzyme-deficient mouse macrophages. The capacity of recombinant PPCA and Neu1 to clear the lysosomal storage in target cells was assessed in PPCA-/- mice, a model of galactosialidosis. Intravenously injected PPCA-/- mice efficiently internalized the corrective enzymes in resident macrophages of many organs. In addition, treated mice showed overall clearance of lysosomal storage in the most affected systemic organs, kidney, liver, and spleen. Our results suggest that ERT with baculovirus-expressed enzymes might be an effective treatment for nonneuropathic patients with galactosialidosis and possibly for others with LSDs that primarily involve the RE system.

Cathepsin A regulates chaperone-mediated autophagy through cleavage of the lysosomal receptor.

Protective protein/cathepsin A (PPCA) has a serine carboxypeptidase activity of unknown physiological function. We now demonstrate that this protease activity triggers the degradation of the lysosome-associated membrane protein type 2a (lamp2a), a receptor for chaperone-mediated autophagy (CMA). Degradation of lamp2a is important because its level in the lysosomal membrane is a rate-limiting step of CMA. Cells defective in PPCA show reduced rates of lamp2a degradation, higher levels of lamp2a and higher rates of CMA. Restoration of PPCA protease activity increases rates of lamp2a degradation, reduces levels of lysosomal lamp2a and reduces rates of CMA. PPCA associates with lamp2a on the lysosomal membrane and cleaves lamp2a near the boundary between the luminal and transmembrane domains. In addition to the well-studied role of PPCA in targeting and protecting two lysosomal glycosidases, we have defined a role for the proteolytic activity of this multifunctional protein.