Human Cathepsin Research Articles

Abstract 14232: The Cathepsin D-m6pr-erk1/2 Pathway Mediates Cellular Senescence and Osteogenic Activities in Human Aortic Valve Interstitial Cells

Background: Calcific aortic valve disease (CAVD) is one of the most prevalent cardiovascular disorders in the elderly, and aortic valve interstitial cells (AVICs) play a major role in valvular calcification associated with CAVD progression. Recent studies found that pharmacological or genetic clearance of senescent cells attenuates aortic valve calcification in animal models, indicating an important role of senescent cells in promoting valvular calcification. However, the mechanism underlying cellular senescence in aortic valves is unclear. Our pilot work found that greater densities of senescent cells in AVIC isolates from diseased human aortic valves are associated with elevated levels of matrilin 2 and cathepsin D in AVICs. We tested the hypothesis that matrilin 2 and cathepsin D mediate the senescence and osteogenic responses in human AVICs. Methods and Results: AVICs were isolated from normal and diseased aortic valves. Levels of P16 were assessed by immunoblotting, and senescent changes is examined using senescence-associated β-galactosidase (SA-β-gal) staining. AVICs from diseased valves displayed higher levels of P16 and greater SA-β-gal staining. Both recombinant human matrilin 2 (2.0 μg/ml) and recombinant human cathepsin D (40 nM) promoted P16 expression and enhanced SA-β-gal staining in normal AVICs, leading to greater calcium deposition. Knockdown of cathepsin D reduced matrilin-2-induced P16 expression, SA-β-gal staining and calcium deposition in normal AVICs. Moreover, recombinant cathepsin D activated the M6PR-ERK1/2 pathway in normal AVICs. Blocking or knockdown of M6PR (M6P, 10 mM or siRNA, 1.0 nM) or inhibition of ERK1/2 (PD98059, 25 μM) attenuated the effect of cathepsin D on AVIC senescent change and associated osteogenic activation. Conclusion: AVICs of aortic valves from patients with CAVD have higher levels of senescence activity. Cathepsin D mediates AVIC senescence and associated osteogenic activation through the M6PR-ERK1/2 pathway and is involved in the mechanism by which soluble matrilin 2 promotes cellular senescence and osteogenic activity in human AVICs. Targeting the cathepsin D-M6PR-ERK1/2 cascade may suppress AVIC senescence activity and attenuate aortic valve calcification.

Design, synthesis and stepwise optimization of nitrile-based inhibitors of cathepsins B and L

Human cathepsin B (CatB) is an important biological target in cancer therapy. In this work, we performed a knowledge-based design approach and the synthesis of a new set of 19 peptide-like nitrile-based cathepsin inhibitors. Reported compounds were assayed against a panel of human cysteine proteases: CatB, CatL, CatK, and CatS. Three compounds (7h, 7i, and 7j) displayed nanomolar inhibition of CatB and selectivity over CatK and CatL. The selectivity was achieved by using the combination of a para biphenyl ring at P3, halogenated phenylalanine in P2 and Thr-O-Bz group at P1. Likewise, compounds 7i and 7j showed selective CatB inhibition among the panel of enzymes studied. We have also described a successful example of bioisosteric replacement of the amide bond for a sulfonamide one [7e → 6b], where we observed an increase in affinity and selectivity for CatB while lowering the compound lipophilicity (ilogP). Our knowledge-based design approach and the respective structure–activity relationships provide insights into the specific ligand-target interactions for therapeutically relevant cathepsins.

Druggable Hot Spots in the Schistosomiasis Cathepsin B1 Target Identified by Functional and Binding Mode Analysis of Potent Vinyl Sulfone Inhibitors.

Schistosomiasis, a parasitic disease caused by blood flukes of the genus Schistosoma, is a global health problem with over 200 million people infected. Treatment relies on just one drug, and new chemotherapies are needed. Schistosoma mansoni cathepsin B1 (SmCB1) is a critical peptidase for the digestion of host blood proteins and a validated drug target. We screened a library of peptidomimetic vinyl sulfones against SmCB1 and identified the most potent SmCB1 inhibitors reported to date that are active in the subnanomolar range with second order rate constants (k2nd) of ∼2 × 105 M–1 s–1. High resolution crystal structures of the two best inhibitors in complex with SmCB1 were determined. Quantum chemical calculations of their respective binding modes identified critical hot spot interactions in the S1′ and S2 subsites. The most potent inhibitor targets the S1′ subsite with an N-hydroxysulfonic amide moiety and displays favorable functional properties, including bioactivity against the pathogen, selectivity for SmCB1 over human cathepsin B, and reasonable metabolic stability. Our results provide structural insights for the rational design of next-generation SmCB1 inhibitors as potential drugs to treat schistosomiasis.

Assessing the consequences of environmental exposures on the expression of the human receptor and proteases involved in SARS-CoV-2 cell-entry

The role of environmental condition on the infection by the novel pathogenic SARS-CoV-2 virus remains uncertain. In here, exploiting a large panel of publicly available genome-wide data, we investigated whether the human receptor ACE2 and human proteases TMPRSS2, FURIN and CATHEPSINs (B, L and V), which are involved in SARS-CoV-2 cell entry, are transcriptionally regulated by environmental cues. We report that more than 50 chemicals modulate the expression of ACE2 or human proteases important for SARS-CoV-2 cell entry. We further demonstrate that transcription factor AhR, which is commonly activated by pollutants, binds to the promoter of TMPRSS2 and enhancers and/or promoters of Cathepsin B, L and V encoding genes. Our exploratory study documents an influence of environmental exposures on the expression of genes involved in SARS-CoV-2 cell entry. These results could be conceptually and medically relevant to our understanding of the COVID-19 disease, and should be further explored in laboratory and epidemiologic studies.

Influence of lysosomal protease sensitivity in the immunogenicity of the antitumor biopharmaceutical asparaginase

L-asparaginase (ASNase) from Escherichia coli (EcAII) is used in the treatment of acute lymphoblastic leukaemia (ALL). EcAII activity in vivo has been described to be influenced by the human lysosomal proteases asparaginyl endopeptidase (AEP) and cathepsin B (CTSB); these hydrolases cleave and could expose epitopes associated with the immune response against EcAII. In this work, we show that ASNase resistance to CTSB and/or AEP influences the formation of anti-ASNase antibodies, one of the main causes of hypersensitivity reactions in patients. Error-prone polymerase chain reaction was used to produce variants of EcAII more resistant to proteolytic cleavage by AEP and CTSB. The variants with enzymatic activity and cytotoxicity levels equivalent to or better than EcAII WT were submitted to in vivo assays. Only one of the mutants presented increased serum half-life, so resistance to these proteases is not the only feature involved in EcAII stability in vivo. Our results showed alteration of the phenotypic profile of B cells isolated after animal treatment with different protease-resistant proteoforms. Furthermore, mice that were exposed to the protease-resistant proteoforms presented lower anti-asparaginase antibodies production in vivo. Our data suggest that modulating resistance to lysosomal proteases can result in less immunogenic protein drugs.

In Silico Identification of Potential Natural Product Inhibitors of Human Proteases Key to SARS-CoV-2 Infection.

Presently, there are no approved drugs or vaccines to treat COVID-19, which has spread to over 200 countries and at the time of writing was responsible for over 650,000 deaths worldwide. Recent studies have shown that two human proteases, TMPRSS2 and cathepsin L, play a key role in host cell entry of SARS-CoV-2. Importantly, inhibitors of these proteases were shown to block SARS-CoV-2 infection. Here, we perform virtual screening of 14,011 phytochemicals produced by Indian medicinal plants to identify natural product inhibitors of TMPRSS2 and cathepsin L. AutoDock Vina was used to perform molecular docking of phytochemicals against TMPRSS2 and cathepsin L. Potential phytochemical inhibitors were filtered by comparing their docked binding energies with those of known inhibitors of TMPRSS2 and cathepsin L. Further, the ligand binding site residues and non-covalent interactions between protein and ligand were used as an additional filter to identify phytochemical inhibitors that either bind to or form interactions with residues important for the specificity of the target proteases. This led to the identification of 96 inhibitors of TMPRSS2 and 9 inhibitors of cathepsin L among phytochemicals of Indian medicinal plants. Further, we have performed molecular dynamics (MD) simulations to analyze the stability of the protein-ligand complexes for the three top inhibitors of TMPRSS2 namely, qingdainone, edgeworoside C and adlumidine, and of cathepsin L namely, ararobinol, (+)-oxoturkiyenine and 3α,17α-cinchophylline. Interestingly, several herbal sources of identified phytochemical inhibitors have antiviral or anti-inflammatory use in traditional medicine. Further in vitro and in vivo testing is needed before clinical trials of the promising phytochemical inhibitors identified here.

Synthesis and matched molecular pair analysis of covalent reversible inhibitors of the cysteine protease CPB

Cysteine protease B (CPB) can be targeted by reversible covalent inhibitors that could serve as antileishmanial compounds. Here, sixteen dipeptidyl nitrile derivatives were synthesized, tested against CPB, and analyzed using matched molecular pairs to determine the effects of stereochemistry and p-phenyl substitution on enzyme inhibition. The compound (S)-2-(((S)-1-(4-bromophenyl)-2,2,2-trifluoroethyl)amino)-N-(1-cyanocyclopropyl)-3-phenylpropanamide (5) was the most potent CPB inhibitor (pKi = 6.82), which was also selective for human cathepsin B (pKi < 5). The inversion of the stereochemistry from S to R was more detrimental to potency when placed at the P2 position than at P3. The p-Br derivatives were more potent than the p-CH3 and p-OCH3 derivatives, probably due to intermolecular interactions with the S3 subsite.

Profiling selectivity of chagasin mutants towards cysteine proteases cruzain or cathepsin L through molecular dynamics simulations

Chagasin, an endogenous cysteine protease inhibitor from Trypanosoma cruzi, can control the activity of the parasitic cruzain and its homologous human cathepsin L. While chagasin inhibits both enzymes with similar potency, mutations have different effects on binding to these enzymes. Mutants T31A and T31A/T32A bind well to cathepsin L, but their affinity for cruzain drops ∼40 to 140–fold. On the other hand, the mutant W93A binds well to cruzain, but it loses potency against cathepsin L. Here, we employed molecular dynamics simulations to understand the selectivity in inhibition of cruzain or cathepsin L by chagasin mutants W93A, T31A, and T31A/T32A. Our results allowed profiling the nonbonded interactions in the interfaces of each mutant with these cysteine proteases. Additionally, we observed differences in the binding conformation of the chagasin loops L2 and L6 of the W93A mutant, favoring interactions with cruzain and reducing interactions with cathepsin L. These differences are associated with a partial dissociation of the W93A-cathepsin L complex, providing a likely cause for the selectivity of the mutant W93A towards cruzain. Communicated by Ramaswamy H. Sarma.

N-Sulfonyl dipeptide nitriles as inhibitors of human cathepsin S: In silico design, synthesis and biochemical characterization

A library of cathepsin S inhibitors of the dipeptide nitrile chemotype, bearing a bioisosteric sulfonamide moiety, was synthesized. Kinetic investigations were performed at four human cysteine proteases, i.e. cathepsins S, B, K and L. Compound 12 with a terminal 3-biphenyl sulfonamide substituent was the most potent (Ki = 4.02 nM; selectivity ratio cathepsin S/K = 5.8; S/L = 67) and 24 with a 4′-fluoro-4-biphenyl sulfonamide substituent the most selective cathepsin S inhibitor (Ki = 35.5 nM; selectivity ratio cathepsin S/K = 57; S/L = 31). In silico design and biochemical evaluation emphasized the impact of the sulfonamide linkage on selectivity and a possible switch of P2 and P3 substituents with respect to the occupation of the corresponding binding sites of cathepsin S.

Bauhinia Protease Inhibitors Attenuate Gastric Ulcer by Blocking Neutrophil Enzymes.

Proteases play a pivotal role in many signaling pathways; inhibitors of well-established proteases have shown a substantial therapeutic success. This study aimed to examine the in vivo effects of 3 protease inhibitors isolated from Bauhinia species: i) Bauhinia mollis elastase inhibitor, which blocks human neutrophil elastase (Kiapp 2.8 nM) and cathepsin G (Kiapp 1.0 nM) activities; ii) Bauhinia mollis trypsin inhibitor, a trypsin inhibitor (Kiapp 5.0 nM); and iii) Bauhinia bauhinioides cruzipain inhibitor, which inhibits elastase (Kiapp 2.6 nM), cathepsin G (Kiapp 160.0 nM), and the cysteine proteases cathepsin L (Kiapp 0.2 nM). Bauhinia bauhinioides cruzipain inhibitor, Bauhinia mollis elastase inhibitor, and Bauhinia mollis trypsin inhibitor were isolated using acetone and ammonium sulfate fractionations, DEAE-Sephadex, trypsin-Sepharose, and Resource-Q chromatographies. Mice and rats were treated intraperitoneally with 1 dose of inhibitor; gastric mucosal lesions were induced by cold-restraint stress. Oral pretreatment of mice with Bauhinia mollis elastase inhibitor or Bauhinia mollis trypsin inhibitor (1 - 10 mg/kg) did not show anti-ulcer effect, while Bauhinia bauhinioides cruzipain inhibitor (0.1 - 1.0 mg/kg) produced a similar reduction of the index of mucosal damage at all effective doses (30 to 33% < control). In rats at doses lower than those used in mice, Bauhinia mollis elastase inhibitor and Bauhinia bauhinioides cruzipain inhibitor reduced the index of mucosal damage by 66% and 54% of controls, respectively. The results indicate a protective effect against gastric mucosal lesions associated with elastase inhibition but not inhibition of trypsin activities. Moreover, the lack of Bauhinia mollis elastase inhibitor efficacy observed in mice may possibly be related to the reported structural differences of elastase in mice and rats.

Serpins in Fasciola hepatica: insights into host–parasite interactions

Protease inhibitors play crucial roles in parasite development and survival, modulating the immune responses of their vertebrate hosts. Members of the serpin family are irreversible inhibitors of serine proteases and regulate systems related to defence against parasites. Limited information is currently available on protease inhibitors from the liver fluke Fasciola hepatica. In this study, we characterised four serpins from F. hepatica (FhS-1–FhS-4). Biochemical characterisation revealed that recombinant FhS-2 (rFhS) inhibits the activity of human neutrophil cathepsin G, while rFhS-4 inhibits the activity of bovine pancreatic chymotrypsin and cathepsin G. Consistent with inhibitor function profiling data, rFhS-4 inhibited cathepsin G-activated platelet aggregation in a dose-responsive manner.Similar to other serpins, rFhS2 and rFhS-4 bind to heparin with high affinity. Tissue localisation demonstrated that these serpins have different spatial distributions. FhS-2 is localised in the ovary, while FhS-4 was found in gut cells. Both of them co-localised in the spines within the tegument. These findings provide the basis for study of functional roles of these proteins as part of an immune evasion mechanism in the adult fluke, and in protection of eggs to ensure parasite life cycle continuity. Further understanding of serpins from the liver fluke may lead to the discovery of novel anti-parasitic interventions.

Water Bridges Play a Key Role in Affinity and Selectivity for Malarial Protease Falcipain-2.

Falcipain-2 (FP-2) is hemoglobinase considered an attractive drug target of Plasmodium falciparum. Recently, it has been shown that peptidomimetic nitriles containing a 3-pyridyl (3Pyr) moiety at P2 display high affinity and selectivity for FP-2 with respect to human cysteine cathepsins (hCats), outperforming other P2-Pyr isomers and analogs. Further characterization demonstrated that certain P3 variants of these compounds possess micromolar inhibition of parasite growth in vitro and no cytotoxicity against human cell lines. However, the structural determinants underlying the selectivity of the 3Pyr-containing nitriles for FP-2 remain unknown. In this work, we conduct a thorough computational study combining MD simulations and free energy calculations to decipher the bases of the selectivity of the aforementioned nitriles. Our results reveal that water bridges involving the nitrogen and one carboxyl oxygen of I85 and D234 of FP-2, respectively, and the nitrogen of the neutral 3Pyr moiety, which are either less prevalent or nonexistent in the other complexes, explain the experimental activity profiles. The presence of crystallographic waters close to the bridging water positions in the studied proteases strongly supports the occurrence of such interactions. Overall, our findings suggest that selective FP-2 inhibitors can be designed by promoting water bridge formation at the bottom of the S2 subsite and/or by introducing complementary groups that displace the bridging water.

Green asymmetric synthesis of epoxypeptidomimetics and evaluation as human cathepsin K inhibitors

Cathepsin K (CatK) is a cysteine protease known for its potent collagenolytic activity, being recognized as an important target to the development of therapies for the treatment of bone disorders. Epoxypeptidomimetics have been reported as potent inhibitors of cathepsins, thus in this work we present a green synthesis of new peptidomimetics by using a one-pot asymmetric epoxidation/Ugi multicomponent reaction. The compounds were evaluated against CatK showing selectivity when compared with cathepsin L, with an inhibition profile in the low micromolar IC50 range. Investigation of the mechanism of action carried out for compounds LSPN428 and LSPN694 suggested a mixed inhibition mode and docking studies allowed a better understanding about interactions of inhibitors with the enzyme.

Structural and kinetic evidence of aging after organophosphate inhibition of human Cathepsin A

Human Cathepsin A (CatA) is a lysosomal serine carboxypeptidase of the renin-angiotensin system (RAS) and is structurally similar to acetylcholinesterase (AChE). CatA can remove the C-terminal amino acids of endothelin I, angiotensin I, Substance P, oxytocin, and bradykinin, and can deamidate neurokinin A. Proteomic studies identified CatA and its homologue, SCPEP1, as potential targets of organophosphates (OP). CatA could be stably inhibited by low µM to high nM concentrations of racemic sarin (GB), soman (GD), cyclosarin (GF), VX, and VR within minutes to hours at pH 7. Cyclosarin was the most potent with a kinetically measured dissociation constant (KI) of 2 µM followed by VR (KI = 2.8 µM). Bimolecular rate constants for inhibition by cyclosarin and VR were 1.3 × 103 M−1sec−1 and 1.2 × 103 M−1sec−1, respectively, and were approximately 3-orders of magnitude lower than those of human AChE indicating slower reactivity. Notably, both AChE and CatA bound diisopropylfluorophosphate (DFP) comparably and had KIDFP = 13 µM and 11 µM, respectively. At low pH, greater than 85% of the enzyme spontaneously reactivated after OP inhibition, conditions under which OP-adducts of cholinesterases irreversibly age. At pH 6.5 CatA remained stably inhibited by GB and GF and <10% of the enzyme spontaneously reactivated after 200 h. A crystal structure of DFP-inhibited CatA was determined and contained an aged adduct. Similar to AChE, CatA appears to have a “backdoor” for product release. CatA has not been shown previously to age. These results may have implications for: OP-associated inflammation; cardiovascular effects; and the dysregulation of RAS enzymes by OP.

Modular Solid-Phase Synthesis of Antiprotozoal Barnesin Derivatives.

Here, we applied and optimized a solid support (SP)-based Horner-Wadsworth-Emmons reagent to prepare SP-bound vinylogous amino acids. Subsequent SP-based peptide synthesis, global deprotection, and chemical modifications yielded 14 lipodipeptides carrying vinylogous amino acids, including the natural product barnesin A (1). Biological evaluation revealed that several synthesized derivatives show micromolar to nanomolar inhibitory activity against papain-like cysteine proteases, human cathepsin L, and rhodesain.

The Homology Modeling and Docking Investigation of Human Cathepsin B

Background: Cathepsin B comprises a group of lysosomal cysteine proteases belonging to the Papain family; it has an intracellular function in the process of protein catabolism, antigen processing in the immune response, and Alzheimer’s disease. In cancers, cathepsin B interferes with autophagy and intracellular catabolism, and breaks down extracellular matrix, decreases protease inhibitors expression, and ultimately helps to accelerate metastasis, tumor malignancy, and reduce immune resistance. Methods: In this study, the 3D structure of cathepsin B was constructed using modeler and Iterative Threading ASSEmbly Refinement (I-TASSER), based on similarity to the crystallographic model of procathepsin B (1PBH). Then, the predicted cathepsin B model was evaluated using PROCHECK and PROSA for quality and reliability. Molecular studies suggested that the amino acids cysteine 108, histidine 189, and histidine 190 form the envelope of the active site of cathepsin B. The docking studies of cathepsin B was performed with protease inhibitors cystatin C, E-64 and leupeptin. Results: The lowest binding energy was related to the cathepsin B-E-64 complex. Accordingly, it was found that E64 interacts with the amino acid cysteine 108 of the active site of cathepsin B. Leupeptin made 2 hydrogen bonds with cathepsin B, but none with the active site of cathepsin amino acids. Cystatin C established a hydrogen bond with the arginine 18 of cathepsin B and made electrostatic bonds with aspartate 148 of cathepsin B. Conclusion: Therefore, the bioinformatics and docking studies of cathepsin B with its inhibitors could be used as reliable identification, treatment, and alternative methods for selecting the inhibitors and controllers of cancer progression.

Mycotoxins from Fusarium proliferatum: new inhibitors of papain-like cysteine proteases.

Papain-like cysteine proteases (PLCPs) in plants are essential to prevent phytopathogen invasion. In order to search for cysteine protease inhibitors and to investigate compounds that could be associated to pineapple Fusarium disease, a chemistry investigation was performed on Fusarium proliferatum isolated from Ananas comosus (pineapple) and cultivated in Czapek medium. From F. proliferatum extracts, nine secondary metabolites were isolated and characterized by nuclear magnetic resonance spectroscopy and mass spectrometry experiments: beauvericin (1), fusaric acid (2), N-ethyl-3-phenylacetamide (3), N-acetyltryptamine (4), cyclo(L-Val-L-Pro) cyclodipeptide (5), cyclo(L-Leu-L-Pro) cyclodipeptide (6), cyclo(L-Leu-L-Pro) diketopiperazine (7), 2,4-dihydroxypyrimidine (8), and 1H-indole-3-carbaldehyde (9). Compounds 1, 3, and 6 showed significant inhibition of papain, with IC50 values of 25.3 ± 1.9, 39.4 ± 2.5, and 7.4 ± 0.5μM, respectively. Compound 1 also showed significant inhibition against human cathepsins V and B with IC50 of 46.0 ± 3.0 and 6.8 ± 0.7μM, respectively. The inhibition of papain by mycotoxins (fusaric acid and beauvericin) may indicate a mechanism of Fusarium in the roles of infection process.

Peptidomimetic Vinyl Heterocyclic Inhibitors of Cruzain Effect Antitrypanosomal Activity.

Cruzain, an essential cysteine protease of the parasitic protozoan, Trypanosoma cruzi, is an important drug target for Chagas disease. We describe here a new series of reversible but time-dependent inhibitors of cruzain, composed of a dipeptide scaffold appended to vinyl heterocycles meant to provide replacements for the irreversible reactive "warheads" of vinyl sulfone inactivators of cruzain. Peptidomimetic vinyl heterocyclic inhibitors (PVHIs) containing Cbz-Phe-Phe/homoPhe scaffolds with vinyl-2-pyrimidine, vinyl-2-pyridine, and vinyl-2-(N-methyl)-pyridine groups conferred reversible, time-dependent inhibition of cruzain (Ki* = 0.1-0.4 μM). These cruzain inhibitors exhibited moderate to excellent selectivity versus human cathepsins B, L, and S and showed no apparent toxicity to human cells but were effective in cell cultures of Trypanosoma brucei brucei (EC50 = 1-15 μM) and eliminated T. cruzi in infected murine cardiomyoblasts (EC50 = 5-8 μM). PVHIs represent a new class of cruzain inhibitors that could progress to viable candidate compounds to treat Chagas disease and human sleeping sickness.

Recombinant expression, characterization and phylogenetic studies of novels cystatins-like proteins of sweet orange (Citrus sinensis) and clementine (Citrus clementina)

Phytocystatins are plant cystatins that are related to several physiological processes regulating endogenous cysteine proteases involved in seed development and germination, programmed cell death and response to stress conditions. In addition, phytocystatins can act in plant defense against exogenous peptidases from herbivorous insects, pathogens and nematodes. Considering that Citrus fruits are important to human nutrition and represent a high value crop in worldwide agriculture, in the present work, we performed the identification of putative cystatins from Citrus sinensis and from Citrus clementine and submitted them to phylogenetic analysis. Six cystatins from each species were identified as orthologous and classified into three well supported phylogenetic groups. Five cystatins representative of the phylogenetic groups were recombinantly expressed and the in vitro studies revealed them to be potent inhibitors against the cysteine peptidases papain, legumain, human cathepsins (B, L, S, K) and a cathepsin B-like from Diaphorina citri (the Asian Citrus psyllid). Our findings provide the C. clementina and C. sinensis cystatins classification and an enzyme-inhibitor interactions profile, which may reflect an evolutionary process of Citrus cystatins related to gene functions as initial germination rates and seedlings development as well associated to plant defense against pathogens, as insects and nematodes.

Mfsd8 localizes to endocytic compartments and influences the secretion of Cln5 and cathepsin D in Dictyostelium

The neuronal ceroid lipofuscinoses (NCLs) are a family of neurodegenerative diseases that affect people of all ages and ethnicities, yet many of the associated genes/proteins are not well characterized. Mutations in MFSD8 (major facilitator superfamily domain-containing 8) cause an infantile form of NCL referred to as CLN7 disease. In this study, we revealed the localization and binding partners of an ortholog of human MFSD8 (Mfsd8) in the social amoeba Dictyostelium discoideum. Putative lysosomal targeting motifs are conserved in Dictyostelium Mfsd8, as are several residues mutated in CLN7 disease patients. Mfsd8 tagged with GFP localizes to endocytic compartments, which includes acidic intracellular vesicles and late endosomes. We pulled-down GFP-Mfsd8 and used mass spectrometry to reveal the Mfsd8 interactome during Dictyostelium growth and starvation. Among the identified hits were the Dictyostelium ortholog of human cathepsin D (CtsD), as well as proteins linked to the functions of the CLN3 (Cln3) and CLN5 (Cln5) orthologs in Dictyostelium. To study the function of Mfsd8, we validated a publically available mfsd8− cell line (GWDI Project) and then used this knockout cell line to show that Mfsd8 influences the secretion of Cln5 and CtsD. This information is then integrated into an emerging model describing the molecular networking of NCL proteins in Dictyostelium. In total, this study identifies Dictyostelium as a new model system for studying CLN7 disease.