Hyperglycosylation of prosaposin in tumor dendritic cells drives immune escape.

Tumors develop strategies to evade immunity by suppressing antigen presentation. In this work, we show that prosaposin (pSAP) drives CD8 T cell-mediated tumor immunity and that its hyperglycosylation in tumor dendritic cells (DCs) leads to cancer immune escape. We found that lysosomal pSAP and its single-saposin cognates mediated disintegration of tumor cell-derived apoptotic bodies to facilitate presentation of membrane-associated antigen and T cell activation. In the tumor microenvironment, transforming growth factor-ß (TGF-ß) induced hyperglycosylation of pSAP and its subsequent secretion, which ultimately caused depletion of lysosomal saposins. pSAP hyperglycosylation was also observed in tumor-associated DCs from melanoma patients, and reconstitution with pSAP rescued activation of tumor-infiltrating T cells. Targeting DCs with recombinant pSAP triggered tumor protection and enhanced immune checkpoint therapy. Our studies demonstrate a critical function of pSAP in tumor immunity and may support its role in immunotherapy.

Intranasal administration of recombinant prosaposin attenuates neuronal apoptosis through GPR37/PI3K/Akt/ASK1 pathway in MCAO rats.

Studies have reported that Prosaposin (PSAP) is neuroprotective in cerebrovascular diseases. We hypothesized that PSAP would reduce infarct volume by attenuating neuronal apoptosis and promoting cell survival through G protein-coupled receptor 37(GPR37)/PI3K/Akt/ASK1 pathway in middle cerebral artery occlusion (MCAO) rats. Two hundred and thirty-five male and eighteen female Sprague-Dawley rats were used. Recombinant human PSAP (rPSAP) was administered intranasally 1 h (h) after reperfusion. PSAP small interfering ribonucleic acid (siRNA), GPR37 siRNA, and PI3K specific inhibitor LY294002 were administered intracerebroventricularly 48 h before MCAO. Infarct volume, neurological score, immunofluorescence staining, Western blot, Fluoro-Jade C (FJC) and TUNEL staining were examined. The expression of endogenous PSAP and GPR37 were increased after MCAO. Intranasal administration of rPSAP reduced brain infarction, neuronal apoptosis, and improved both short- and long-term neurological function. Knockdown of endogenous PSAP aggravated neurological deficits. Treatment with exogenous rPSAP increased PI3K expression, Akt and ASK1 phosphorylation, and Bcl-2 expression; phosphorylated-JNK and Bax levels were reduced along with the number of FJC and TUNEL positive neurons. GPR37 siRNA and LY294002 abolished the anti-apoptotic effect of rPSAP at 24 h after MCAO. In conclusion, rPSAP attenuated neuronal apoptosis and improved neurological function through GPR37/PI3K/Akt/ASK1 pathway after MCAO in rats. Therefore, further exploration of PSAP as a potential treatment option in ischemic stroke is warranted.

Efferocytes release extracellular vesicles to resolve inflammation and tissue injury via prosaposin-GPR37 signaling.

Macrophages release soluble mediators following efferocytic clearance of apoptotic cells to facilitate intercellular communication and promote the resolution of inflammation. However, whether inflammation resolution is modulated by extracellular vesicles (EVs) and vesicular mediators released by efferocytes is not known. We report that efferocyte-derived EVs express prosaposin, which binds to macrophage GPR37 to increase expression of the efferocytosis receptor Tim4 via an ERK-AP1-dependent signaling axis, leading to increased macrophage efferocytosis efficiency and accelerated resolution of inflammation. Neutralization and knockdown of prosaposin or blocking GRP37 abrogates the pro-resolution effects of efferocyte-derived EVs in vivo. Administration of efferocyte-derived EVs in a murine model of atherosclerosis is associated with an increase in lesional macrophage efferocytosis efficiency and a decrease in plaque necrosis and lesional inflammation. Thus, we establish a critical role for efferocyte-derived vesicular mediators in increasing macrophage efferocytosis efficiency and accelerating the resolution of inflammation and tissue injury.

Proteomic characterization of Opisthorchis felineus exosome-like vesicles and their uptake by human cholangiocytes.

The epidemiologically important food-borne trematode Opisthorchis felineus infests the liver biliary tract of fish-eating mammals and causes disorders, including bile duct neoplasia. Many parasitic species release extracellular vesicles (EVs) that mediate host-parasite interaction. Currently, there is no information on O. felineus EVs. Using gel electrophoresis followed by liquid chromatography coupled with tandem mass spectrometry, we aimed to characterize the proteome of EVs released by the adult O. felineus liver fluke. Differential abundance of proteins between whole adult worms and EVs was assessed by semiquantitative iBAQ (intensity-based absolute quantification). Imaging, flow cytometry, inhibitor assays, and colocalization assays were performed to monitor the uptake of the EVs by H69 human cholangiocytes. The proteomic analysis reliably identified 168 proteins (at least two peptides matched a protein). Among major proteins of EVs were ferritin, tetraspanin CD63, helminth defense molecule 1, globin 3, saposin B type domain-containing protein, 60S ribosomal protein, glutathione S-transferase GST28, tubulin, and thioredoxin peroxidase. Moreover, as compared to the whole adult worm, EVs proved to be enriched with tetraspanin CD63, saposin B, helminth defense molecule 1, and Golgi-associated plant pathogenesis-related protein 1 (GAPR1). We showed that EVs are internalized by human H69 cholangiocytes via clathrin-dependent endocytosis, whereas phagocytosis and caveolin-dependent endocytosis do not play a substantial role in this process. Our study describes for the first time proteomes and differential abundance of proteins in whole adult O. felineus worms and EVs released by this food-borne trematode. Studies elucidating the regulatory role of individual components of EVs of liver flukes should be continued to determine which components of EV cargo play the most important part in the pathogenesis of fluke infection and in a closely linked pathology: bile duct neoplasia. SIGNIFICANCE: The food-borne trematode Opisthorchis felineus is a pathogen that causes hepatobiliary disorders in humans and animals. Our study describes for the first time the release of EVs by the liver fluke O. felineus, their microscopic and proteomic characterization, and internalization pathways by human cholangiocytes. Differential abundance of proteins between whole adult worms and EVs was assessed. EVs are enriched with canonical EV markers as well as parasite specific proteins, i.e. tetraspanin CD63, saposin B, helminth defense molecule 1, and others. Our findings will form the basis of the search for potential immunomodulatory candidates with therapeutic potential in the context of inflammatory diseases, as well as novel vaccine candidates.

Prosaposin PS18 reduces dopaminergic neurodegeneration in a 6-hydroxydopamine rat model of Parkinson's disease.

Saposin and its precursor prosaposin are endogenous proteins with neurotrophic and anti-apoptotic properties. Prosaposin or its analog prosaposin-derived 18-mer peptide (PS18) reduced neuronal damage in hippocampus and apoptosis in stroke brain. Its role in Parkinson's disease (PD) has not been well characterized. This study aimed to examine the physiological role of PS18 in 6-hydroxydopamine (6-OHDA) cellular and animal models of PD. We found that PS18 significantly antagonized 6-OHDA -mediated dopaminergic neuronal loss and TUNEL in rat primary dopaminergic neuronal culture. In SH-SY5Y cells overexpressing the secreted ER calcium-monitoring proteins, we found that PS18 significantly reduced thapsigargin and 6-OHDA-mediated ER stress. The expression of prosaposin and the protective effect of PS18 were next examined in hemiparkinsonian rats. 6-OHDA was unilaterally administered to striatum. The expression of prosaposin was transiently upregulated in striatum on D3 (day 3) after lesioning and returned below the basal level on D29. The 6-OHDA-lesioned rats developed bradykinesia and an increase in methamphetamine-mediated rotation, which was antagonized by PS18. Brain tissues were collected for Western blot, immunohistochemistry, and qRTPCR analysis. Tyrosine hydroxylase immunoreactivity was significantly reduced while the expressions of PERK, ATF6, CHOP, and BiP were upregulated in the lesioned nigra; these responses were significantly antagonized by PS18. Taken together, our data support that PS18 is neuroprotective in cellular and animal models of PD. The mechanisms of protection may involve anti-ER stress.

A saposin domain-containing protein of tongue sole Cynoglossus semilaevis: Antimicrobial activity and mechanism.

Prosaposin is a precursor that can be processed into four different saposins, designated as A, B, C, and D, which have multiple functions in mammals, including neuroprotection and immune modulation. The immune function of saposin in teleost remains largely unknown. In the present study, a saposin (SAP) domain-containing protein was identified in half-smooth tongue sole Cynoglossus semilaevis and named CsSDP. CsSDP harbors one SAP A domain and two SAP B domains. When expressed in HEK293T cells, CsSDP was specifically localized in the lysosome. When overexpressed in Escherichia coli, CsSDP markedly inhibited bacterial growth, and the inhibitory effect depended on two specific regions in the SAP A and SAP B domains. Two polypeptides (P32 and P30) derived from the above SAP A and B domains could bind to and inhibit the growth of both Gram-negative and Gram-positive bacteria. The ultrastructural analysis revealed that P32 and P30 killed target bacteria by disrupting the bacterial cell wall and inducing substantial release of cytoplasmic contents. These results shed new lights on the immune function of saposin domain-containing protein in teleost.

Emulation of the structure of the Saposin protein fold by a lung surfactant peptide construct of surfactant Protein B.

The three-dimensional structure of the synthetic lung Surfactant Protein B Peptide Super Mini-B was determined using an integrative experimental approach, including mass spectrometry and isotope enhanced Fourier-transform infrared (FTIR) spectroscopy. Mass spectral analysis of the peptide, oxidized by solvent assisted region-specific disulfide formation, confirmed that the correct folding and disulfide pairing could be facilitated using two different oxidative structure-promoting solvent systems. Residue specific analysis by isotope enhanced FTIR indicated that the N-terminal and C-terminal domains have well defined α-helical amino acid sequences. Using these experimentally derived measures of distance constraints and disulfide connectivity, the ensemble was further refined with molecular dynamics to provide a medium resolution, residue-specific structure for the peptide construct in a simulated synthetic lung surfactant lipid multilayer environment. The disulfide connectivity combined with the α-helical elements stabilize the peptide conformationally to form a helical hairpin structure that resembles critical elements of the Saposin protein fold of the predicted full-length Surfactant Protein B structure.

Skene's Gland Derivatives in the Female Genital Tract and Cervical Adenoid Basal Carcinoma are Consistently Positive With Prostatic Marker NKX3.1.

Cervical ectopic prostatic tissue and vaginal tubulosquamous polyp are rare lesions which exhibit variable, and often focal, immunohistochemical expression with traditional prostatic markers [prostate-specific antigen and prostatic acid phosphatase (PSAP)]. These lesions are thought to arise from periurethral Skene's glands, the female equivalent of prostatic glands in the male. Adenoid basal carcinoma is a rare and indolent cervical neoplasm. Expression of the prostatic marker NKX3.1 in ectopic prostatic tissue and tubulosquamous polyp has been reported but no studies have examined immunoreactivity with this marker in adenoid basal carcinoma. We stained 19 cases [adenoid basal carcinoma (n=6), cervical ectopic prostatic tissue (n=11), and vaginal tubulosquamous polyp (n=3); 1 case contained both adenoid basal carcinoma and ectopic prostatic tissue] with NKX3.1. In all cases, the glandular component of these lesions exhibited diffuse nuclear immunoreactivity while normal endocervical glands were negative. Prostate-specific antigen was positive in 4 of 9 and 0 of 3 cases of ectopic prostatic tissue and tubulosquamous polyp, respectively, while PSAP was positive in 3 of 4 and 2 of 2 cases of ectopic prostatic tissue and tubulosquamous polyp respectively; 3 of 5 cases of adenoid basal carcinoma tested were focally positive with PSAP and all 5 were negative with prostate-specific antigen. While the specificity of NKX3.1 should be investigated in future studies, positivity with this marker may be useful in diagnosing these uncommon lesions. NKX3.1 appears a more sensitive marker of ectopic prostatic tissue and tubulosquamous polyp than traditional prostatic markers and positive staining provides further support that these lesions exhibit "prostatic" differentiation and are of Skene's gland origin. NKX3.1 and PSAP positivity in adenoid basal carcinoma raises the possibility of an association with benign glandular lesions exhibiting prostatic differentiation and we critically discuss the possible association.

Prosaposin in the rat oviductal epithelial cells.

Prosaposin (PSAP) has two forms: a precursor and a secreted form. The secreted form has neurotrophic, myelinotrophic, and myotrophic properties. The precursor form is a precursor protein of saposins A-D. Although the distribution of PSAP in male reproductive organs is well known, its distribution in female reproductive organs, especially in the oviduct, is unclear. Immunoblots and immunohistochemistry of oviducts showed that oviductal tissues contain PSAP proteins, and a significant increase in PSAP was observed in the estrus-metestrus phase compared to the diestrus-proestrus phase in the ampulla. To identify PSAP trafficking in cells, double-immunostaining was performed with antibodies against PSAP in combination with sortilin, mannose 6 phosphate receptor (M6PR), or low-density lipoprotein receptor-related protein 1 (LRP1). PSAP and sortilin double-positive reactions were observed near the nuclei, as well as in the apical portion of microvillous epithelial cells, whereas these reactions were only observed near the nuclei of ciliated epithelial cells. PSAP and M6PR double-positive reactions were observed near the nuclei of microvillous and ciliated epithelial cells. PSAP and M6PR double-positive reactions were also observed in the apical portion of microvillous epithelial cells. PSAP and LRP1 double-positive reactions were observed in the plasma membrane and apical portion of both microvillous and ciliated epithelial cells. Immunoelectron staining revealed PSAP immunoreactive small vesicles with exocytotic features at the apical portion of microvillous epithelial cells. These findings suggest that PSAP is present in the oviductal epithelium and has a pivotal role during pregnancy in providing an optimal environment for gametes and/or sperm in the ampulla.

The role of disulfide bonds in a Solanum tuberosum saposin-like protein investigated using molecular dynamics.

The Solanum tuberosum plant specific insert (StPSI) has a defensive role in potato plants, with the requirements of acidic pH and anionic lipids. The StPSI contains a set of three highly conserved disulfide bonds that bridge the protein's helical domains. Removal of these bonds leads to enhanced membrane interactions. This work examined the effects of their sequential removal, both individually and in combination, using all-atom molecular dynamics to elucidate the role of disulfide linkages in maintaining overall protein tertiary structure. The tertiary structure was found to remain stable at both acidic (active) and neutral (inactive) pH despite the removal of disulfide linkages. The findings include how the dimer structure is stabilized and the impact on secondary structure on a residue-basis as a function of disulfide bond removal. The StPSI possesses an extensive network of inter-monomer hydrophobic interactions and intra-monomer hydrogen bonds, which is likely the key to the stability of the StPSI by stabilizing local secondary structure and the tertiary saposin-fold, leading to a robust association between monomers, regardless of the disulfide bond state. Removal of disulfide bonds did not significantly impact secondary structure, nor lead to quaternary structural changes. Instead, disulfide bond removal induces regions of amino acids with relatively higher or lower variation in secondary structure, relative to when all the disulfide bonds are intact. Although disulfide bonds are not required to preserve overall secondary structure, they may have an important role in maintaining a less plastic structure within plant cells in order to regulate membrane affinity or targeting.

SapC-DOPS - a Phosphatidylserine-targeted Nanovesicle for selective Cancer therapy.

Phosphatidylserine (PS) is normally located in the inner leaflet of the membrane bilayer of healthy cells, however it is expressed at high levels on the surface of cancer cells. This has allowed for the development of selective therapeutic agents against cancer cells (without affecting healthy cells). SapC-DOPS is a PS-targeting nanovesicle which effectively targets and kills several cancer types including pancreatic, lung, brain, and pediatric tumors. Our studies have demonstrated that SapC-DOPS selectively induces apoptotic cell death in malignant and metastatic cells, whereas untransformed cells remain unaffected due to low surface PS expression. Furthermore, SapC-DOPS can be used in combination with standard therapies such as irradiation and chemotherapeutic drugs to significantly enhance the antitumor efficacy of these treatments. While the PS-targeting nanovesicles are a promising selective therapeutic option for the treatment of cancers, more preclinical studies are needed to fully understand the mechanisms leading to non-apoptotic PS expression on the surface of viable cancer cells and to determine the effectiveness of SapC-DOPS in advanced metastatic disease. In addition, the completion of clinical studies will determine therapeutic effects and drug safety in patients. A phase I clinical trial using SapC-DOPS has been completed on patients with solid tumors and has demonstrated compelling patient outcomes with a strong safety profile. Results from this study are informing future studies with SapC-DOPS. Abstract video.

Characterization of lysosomal proteins Progranulin and Prosaposin and their interactions in Alzheimer's disease and aged brains: increased levels correlate with neuropathology.

Progranulin (PGRN) is a protein encoded by the GRN gene with multiple identified functions including as a neurotrophic factor, tumorigenic growth factor, anti-inflammatory cytokine and regulator of lysosomal function. A single mutation in the human GRN gene resulting in reduced PGRN expression causes types of frontotemporal lobar degeneration resulting in frontotemporal dementia. Prosaposin (PSAP) is also a multifunctional neuroprotective secreted protein and regulator of lysosomal function. Interactions of PGRN and PSAP affect their functional properties. Their roles in Alzheimer's disease (AD), the leading cause of dementia, have not been defined. In this report, we examined in detail the cellular expression of PGRN in middle temporal gyrus samples of a series of human brain cases (n = 45) staged for increasing plaque pathology. Immunohistochemistry showed PGRN expression in cortical neurons, microglia, cerebral vessels and amyloid beta (Aß) plaques, while PSAP expression was mainly detected in neurons and Aß plaques, and to a limited extent in astrocytes. We showed that there were increased levels of PGRN protein in AD cases and corresponding increased levels of PSAP. Levels of PGRN and PSAP protein positively correlated with amyloid beta (Aß), with PGRN levels correlating with phosphorylated tau (serine 205) levels in these samples. Although PGRN colocalized with lysosomal-associated membrane protein-1 in neurons, most PGRN associated with Aß plaques did not. Aß plaques with PGRN and PSAP deposits were identified in the low plaque non-demented cases suggesting this was an early event in plaque formation. We did not observe PGRN-positive neurofibrillary tangles. Co-immunoprecipitation studies of PGRN from brain samples identified only PSAP associated with PGRN, not sortilin or other known PGRN-binding proteins, under conditions used. Most PGRN associated with Aß plaques were immunoreactive for PSAP showing a high degree of colocalization of these proteins that did not change between disease groups. As PGRN supplementation has been considered as a therapeutic approach for AD, the possible involvement of PGRN and PSAP interactions in AD pathology needs to be further considered.

Functional Reconstitution of HlyB, a Type I Secretion ABC Transporter, in Saposin-A Nanoparticles.

Type I secretion systems (T1SS) are ubiquitous transport machineries in Gram-negative bacteria. They comprise a relatively simple assembly of three membrane-localised proteins: an inner-membrane complex composed of an ABC transporter and a membrane fusion protein, and a TolC-like outer membrane component. T1SS transport a wide variety of substrates with broad functional diversity. The ABC transporter hemolysin B (HlyB), for example, is part of the hemolysin A-T1SS in Escherichia coli. In contrast to canonical ABC transporters, an accessory domain, a C39 peptidase-like domain (CLD), is located at the N-terminus of HlyB and is essential for secretion. In this study, we have established an optimised purification protocol for HlyB and the subsequent reconstitution employing the saposin-nanoparticle system. We point out the negative influence of free detergent on the basal ATPase activity of HlyB, studied the influence of a lysolipid or lipid matrix on activity and present functional studies with the full-length substrate proHlyA in its folded and unfolded states, which both have a stimulatory effect on the ATPase activity.

Prosaposin is a biomarker of mesenchymal glioblastoma and regulates mesenchymal transition through the TGF-ß1/Smad signaling pathway.

Mesenchymal glioblastoma (GBM) is the most aggressive subtype of GBM. Our previous study found that neurotrophic factor prosaposin (PSAP) is highly expressed and secreted in glioma and can promote the growth of glioma. The role of PSAP in mesenchymal GBM is still unclear. In this study, bioinformatic analysis, western blotting and RT-qPCR were used to detect the expression of PSAP in different GBM subtypes. Human glioma cell lines and patient-derived glioma stem cells were studied in vitro and in vivo, revealing that mesenchymal GBM expressed and secreted the highest level of PSAP among four subtypes of GBM, and PSAP could promote GBM invasion and epithelial-mesenchymal transition (EMT)-like processes in vivo and in vitro. Bioinformatic analysis and western blotting showed that PSAP mainly played a regulatory role in GBM invasion and EMT-like processes via the TGF-ß1/Smad signaling pathway. In conclusion, the overexpression and secretion of PSAP may be an important factor causing the high invasiveness of mesenchymal GBM. PSAP is therefore a potential target for the treatment of mesenchymal GBM. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Prosaposin promotes the proliferation and tumorigenesis of glioma through toll-like receptor 4 (TLR4)-mediated NF-κB signaling pathway.

BACKGROUND: As a neurotrophic factor, prosaposin (PSAP) can exert neuroprotective and neurotrophic effects. It is involved in the occurrence and development of prostate and breast cancer. However, there is no research about the role of PSAP in glioma. METHODS: The PSAP overexpressed or silenced glioma cells or glioma stem cells were established based on Lentiviral vector transfection. Cell viability assay, Edu assay, neurosphere formation assay and xenograft experiments were used to detect the proliferative ability. Western blot, Elisa and luciferase reporter assays were used to detect the possible mechanism. FINDINGS: Our study firstly found that PSAP was highly expressed and secreted in clinical glioma specimens, glioma stem cells, and glioma cell lines. It was associated with poor prognosis. We found that PSAP significantly promoted the proliferation of glioma stem cells and cell lines. Moreover, PSAP promoted tumorigenesis in subcutaneous and orthotopic models of this disease. Furthermore, GSEA and KEGG analysis predicted that PSAP acts through the TLR4 and NF-κB signaling pathways, which was confirmed by western blot, immunoprecipitation, immunofluorescence, and use of the TLR4-specific inhibitor TAK-242. INTERPRETATION: The findings of this study suggest that PSAP can promote glioma cell proliferation via the TLR4/NF-κB signaling pathway and may be an important target for glioma treatment. FUND: This work was funded by National Natural Science Foundation of China (Nos. 81101917, 81270036, 81201802, 81673025), Program for Liaoning Excellent Talents in University (No. LR2014023), and Liaoning Province Natural Science Foundation (Nos. 20170541022, 20172250290). The funders did not play a role in manuscript design, data collection, data analysis, interpretation nor writing of the manuscript.

Glio- and neuro-protection by prosaposin is mediated by orphan G-protein coupled receptors GPR37L1 and GPR37.

Discovery of neuroprotective pathways is one of the major priorities for neuroscience. Astrocytes are natural neuroprotectors and it is likely that brain resilience can be enhanced by mobilizing their protective potential. Among G-protein coupled receptors expressed by astrocytes, two highly related receptors, GPR37L1 and GPR37, are of particular interest. Previous studies suggested that these receptors are activated by a peptide Saposin C and its neuroactive fragments (prosaptide TX14(A)), which were demonstrated to be neuroprotective in various animal models by several groups. However, pairing of Saposin C or prosaptides with GPR37L1/GPR37 has been challenged and presently GPR37L1/GPR37 have regained their orphan status. Here, we demonstrate that in their natural habitat, astrocytes, these receptors mediate a range of effects of TX14(A), including protection from oxidative stress. The Saposin C/GPR37L1/GPR37 pathway is also involved in the neuroprotective effect of astrocytes on neurons subjected to oxidative stress. The action of TX14(A) is at least partially mediated by Gi-proteins and the cAMP-PKA axis. On the other hand, when recombinant GPR37L1 or GPR37 are expressed in HEK293 cells, they are not functional and do not respond to TX14(A), which explains unsuccessful attempts to confirm the ligand-receptor pairing. Therefore, this study identifies GPR37L1/GPR37 as the receptors for TX14(A), and, by extension of Saposin C, and paves the way for the development of neuroprotective therapeutics acting via these receptors.

The Saposin-Like Protein AplD Displays Pore-Forming Activity and Participates in Defense Against Bacterial Infection During a Multicellular Stage of Dictyostelium discoideum.

Due to their archaic life style and microbivor behavior, amoebae may represent a source of antimicrobial peptides and proteins. The amoebic protozoon Dictyostelium discoideum has been a model organism in cell biology for decades and has recently also been used for research on host-pathogen interactions and the evolution of innate immunity. In the genome of D. discoideum, genes can be identified that potentially allow the synthesis of a variety of antimicrobial proteins. However, at the protein level only very few antimicrobial proteins have been characterized that may interact directly with bacteria and help in fighting infection of D. discoideum with potential pathogens. Here, we focus on a large group of gene products that structurally belong to the saposin-like protein (SAPLIP) family and which members we named provisionally Apls (amoebapore-like peptides) according to their similarity to a comprehensively studied antimicrobial and cytotoxic pore-forming protein of the protozoan parasite Entamoeba histolytica. We focused on AplD because it is the only Apl gene that is reported to be primarily transcribed further during the multicellular stages such as the mobile slug stage. Upon knock-out (KO) of the gene, aplD- slugs became highly vulnerable to virulent Klebsiella pneumoniae. AplD- slugs harbored bacterial clumps in their interior and were unable to slough off the pathogen in their slime sheath. Re-expression of AplD in aplD- slugs rescued the susceptibility toward K. pneumoniae. The purified recombinant protein rAplD formed pores in liposomes and was also capable of permeabilizing the membrane of live Bacillus megaterium. We propose that the multifarious Apl family of D. discoideum comprises antimicrobial effector polypeptides that are instrumental to interact with bacteria and their phospholipid membranes. The variety of its members would allow a complementary and synergistic action against a variety of microbes, which the amoeba encounters in its environment.

Structural basis for the activation of acid ceramidase.

Acid ceramidase (aCDase, ASAH1) hydrolyzes lysosomal membrane ceramide into sphingosine, the backbone of all sphingolipids, to regulate many cellular processes. Abnormal function of aCDase leads to Farber disease, spinal muscular atrophy with progressive myoclonic epilepsy, and is associated with Alzheimer's, diabetes, and cancer. Here, we present crystal structures of mammalian aCDases in both proenzyme and autocleaved forms. In the proenzyme, the catalytic center is buried and protected from solvent. Autocleavage triggers a conformational change exposing a hydrophobic channel leading to the active site. Substrate modeling suggests distinct catalytic mechanisms for substrate hydrolysis versus autocleavage. A hydrophobic surface surrounding the substrate binding channel appears to be a site of membrane attachment where the enzyme accepts substrates facilitated by the accessory protein, saposin-D. Structural mapping of disease mutations reveals that most would destabilize the protein fold. These results will inform the rational design of aCDase inhibitors and recombinant aCDase for disease therapeutics.

Phosphatidylserine: A cancer cell targeting biomarker.

Cancer is a leading cause of mortality and morbidity globally. Many prominent cancer-associated molecules have been identified over the recent years which include EGFR, CD44, TGFbRII, HER2, miR-497, NMP22, BTA, Fibrin/FDP etc. These biomarkers are often used for screening, detection, diagnosis, prognosis, prediction and monitoring of cancer development. Phosphatidylserine (PS) is an essential component in all human cells which is present on the inner leaflet of the cell membrane. The oxidative stress causes exposure of PS on the surface of the vascular endothelium in the cancer cells (lung, breast, pancreatic, bladder, skin, brain metastasis, rectal adenocarcinoma etc.) but not on the normal cells. The external PS is regulated by calcium-dependent flippase activity. Cancer cell lines with high surface PS have low flippase activity and high intracellular calcium content. Human Annexin-V, PS targeting antibodies (PGN635 and bavituximab and mch1N11), lysosomal protein, phospholipid Saposin C dioleoylphosphatidylserine (SapC-DOPS), peptide-peptoid hybrid PPS1, PS-binding 14-mer peptide (PSBP-6) and hexapeptide (E3) have been reported to target PS present on cancer cell surface. High expression of CD47 inhibits tumor cell phagocytosis by macrophages. The PS cancer biomarker has also been used to target the drugs to cancer cells specifically without affecting other healthy cells. Currently, the fusion protein (FP) consisting of L-methionase linked to human Annexin-V has been reported to target the cancer cells. The FP catalyzes the conversion of non-toxic prodrug selenomethionine into toxic methyl selenol which thus also prevents the methionine (essential amino acid) supplementation to the cancer cells.

Co-localization of cystatin C and prosaposin in cultured neurons and in anterior horn neurons with amyotrophic lateral sclerosis.

Cystatin C (CST3) is a cysteine protease inhibitor that regulates lysosomal enzyme activity and is reported to be involved in the process of neurodegeneration. In the present study, we investigated whether CST3 interacts with other proteins and affects neurodegeneration in vitro and under disease conditions. We intended to identify any protein that interacts with CST3 by using a yeast two-hybrid system, and found prosaposin (PSAP) as a candidate protein. The binding of CST3 and PSAP was confirmed using an immunoprecipitation-based in vitro assay. An enzyme activity assay revealed that PSAP ameliorated CST3-mediated inhibition of cathepsin B activity. To investigate further, CST3 and PSAP were co-expressed in HeLa cells and in a human neuronal cell line (A1). Subsequent immunocytochemical studies demonstrated that they were co-localized mainly in the lysosomes. In spinal motor neurons of autopsy cases, both proteins showed a granular staining pattern. However, the staining intensities of CST3 and PSAP decreased in Bunina body-positive motor neurons of patients with amyotrophic lateral sclerosis (ALS). Further analysis with immunofluorescence staining revealed that CST3 was immunopositive in the inclusions of ALS motor neurons, where it was closely associated, and sometimes co-localized, with PSAP. CST3 immunoreactivity is recognized as a marker for Bunina bodies in ALS, suggesting that PSAP might also be included in Bunina bodies. The interaction of CST3 and PSAP may alter their functions, leading to motor neuron degeneration in ALS.