•LipA from Pseudomonas aeruginosa is fixed on polypropylene with ethanol as additive.•Immobilization of LipA hyperactivated with ethanol increased its performance.•Thermal tolerance is achieved ...depending on the amount of ethanol used.•Immobilized enzymes were more tolerant to pure solvents.•Catalytic chemoselectivity changed from long-chain to short-chain fatty acids.
Immobilization is practical to upgrade enzymes, increasing their performance and expanding their applications. The recombinant, solvent tolerant lipase LipA PSA01 from Pseudomonas aeruginosa was immobilized on polypropylene Accurel® MP1004 to improve its performance. We investigated the effect of ethanol as an additive during the immobilization process at three concentrations (20%, 25%, and 30%) on the operational behavior of the enzyme. The immobilization efficiency was higher than 92%, and the immobilized enzymes showed hyperactivation and thermal resistance depending on the concentration of ethanol. For example, at 70 °C, the free enzyme lost the activity, while the prepared one with ethanol 25% conserved a residual activity of up to 73.3% (∆ T1550 = 27.1 °C). LipA immobilized had an optimal pH value lower than that of the free enzyme, and the organic solvent tolerance of the immobilized enzymes depended on the ethanol used. Hence, the immobilized enzyme with ethanol 25% showed hyperactivation to more solvents than the soluble enzyme. Remarkable stability towards methanol (up to 8 folds) was evidenced in all the immobilized preparations. The immobilized enzyme changed their chemo preference, and it hydrolyzed oils preferentially with short-chain than those with long-chain. LipA had a notable shelf-life after one year, keeping its activity up to 87%. Ethanol facilitated the access of the enzyme to the hydrophobic support and increased its activity and stability according to the amount of ethanol added.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Lysosomal acid lipase deficiency (LAL‐D) is a multi‐organ autosomal recessive disease caused by mutations in LIPA. We reviewed data from 681 samples (white blood cells WBC n = 625, fibroblasts = 30, ...liver = 4, amniocytes = 13, chorionic villus = 9) received for analysis of lysosomal acid lipase (LAL) activity over a 15‐year period. LIPA sequencing was performed in 49 patients with reduced (n = 26) or deficient (n = 23) LAL activity. The Exome Aggregation Consortium and Genome Aggregation Database dataset were used for LAL‐D prevalence calculations. LAL WBC activity was reduced in 67 patients (10.72%) and deficient in 37 (5.92%). The average of LAL activity ± margin of error (CI 95%) was 19.32 ± 0.86 pmol/min/mg for reduced activity patients and 5.90 ± 1.42 pmol/min/mg for deficient patients. The average age at diagnosis for LAL‐D was 23.6 years with several patients older than age 30. The correlation between the age at diagnosis and LAL activity showed a significant moderate direct correlation (Pearson's r = 0.46, P < 0.005). Homozygous or compound heterozygous mutations were identified in 9 out of 23 patients with deficient results (detection rate 39.1%). The average LAL activity in molecularly confirmed patients was 4.02 ± 2.02 pmol/min/mg protein, while in molecularly negative patients was 13.886 ± 1.49 pmol/min/mg (P < 0.0001). Twenty‐two different mutations were identified including two novel variants (c.309C>A and c.856G>C). A carrier frequency of approximately 1 in 350 was inferred. LAL activity in WBC is a validated tool for LAL‐D diagnosis. Higher residual enzymatic activity might result in a milder phenotype leading to diagnosis delay. A cut‐off below 12 pmol/min/mg protein might be useful to discriminate patients with LIPA mutations.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Lipolysis is a metabolic process by which triglycerides (TGs) are hydrolyzed into free fatty acids (FFAs) and glycerol to serve as fuels during fasting or cold-induced nonshivering thermogenesis. ...Cytoplasmic lipases are thought to be the predominant mechanism of liberating FFA from lipid stores. However, lysosomes also contain lipolytic activity via lysosomal acid lipase (Lipa). In adipocytes, the primary lipid-storing cell in the body with significant involvement in metabolism, the roles of Lipa and lysosomal lipolysis are unclear. We found that Lipa expression increases in the adipose tissue of mice in response to fasting, cold exposure, and CL316,243 (CL) treatment, a mimic of cold-induced lipolysis. Similar results were obtained in cultured adipocytes with nutrient-depletion or isoproterenol treatment, mimicking fasting or cold, respectively. Treatment of C57BL/6J mice with Lalistat (Lali), a specific inhibitor of Lipa, impaired thermogenesis, as evidenced by suppression of cold- and fasting-enhanced increases in plasma FFAs levels during cold exposure or CL treatment. Lipolysis was inhibited by Lali treatment in cultured adipocytes and fresh adipose tissue of C57BL/6J mice under fasting- or cold-mimic conditions. To conduct more detailed in vivo studies, we developed adipose-specific Lipa knockout (A-Lipa) mice, which exhibited lower plasma FFAs when challenged with fasting, cold exposure, or CL treatment. Furthermore, A-Lipa mice were significantly thermogenesis-impaired with exposure to cold or treatment with CL. To determine whether autophagy is involved in these phenomena, adipose-specific Atg5 knockout (A-Atg5) mice were challenged with fasting, cold exposure, or CL treatment. As was found in A-Lipa mice, A-Atg5 mice had lower levels of plasma FFAs and obliteration of stress-induced thermogenesis. Overall, our data suggest a previously unrecognized contribution of lysosomal lipolysis in adipocytes, possibly involving autophagy-related lipophagy.
Disclosure
Y.Yeh: None. B.Razani: None.
Funding
American Heart Association (897628); National Institutes of Health (DK131188)
Lysosomal acid lipase (LAL) is a necessary enzyme for the hydrolysis of both triglycerides (TGs) and cholesteryl esters (CEs) in the lysosome. Deficiency of this enzyme encoded by the lipase A (
LIPA
...) gene leads to LAL deficiency (LAL-D). A severe disease subtype of LAL-D is known as Wolman disease (WD), present with diarrhea, hepatosplenomegaly, and adrenal calcification. Untreated patients do not survive more than a year. The aim of this study was to assess the clinical and molecular characterizations of WD patients in Egypt. A total of seven patients (from five unrelated Egyptian families) were screened by targeted next-generation sequencing (NGS), and the co-segregation of causative variants was analyzed using Sanger sequencing. Furthermore, multiple in silico analyses were performed to assess the pathogenicity of the candidate variants. Overall, we identified three diseases causing variants harbored in the
LIPA
gene. One of these variants is a novel missense variant (NM_000235.4: c.1122 T > G; p. His374Gln), which was classified as a likely pathogenic variant. All variants were predicted to be disease causing using in silico analyses. Our findings expand the spectrum of variants involved in WD which may help to investigate phenotype-genotype correlation and assist genetic counseling. To the best of our knowledge, this is the first clinico-genetic study carried out on Egyptian patients affected with WD.
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EMUNI, FIS, FZAB, GEOZS, GIS, IJS, IMTLJ, KILJ, KISLJ, MFDPS, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, SBMB, SBNM, UKNU, UL, UM, UPUK, VKSCE, ZAGLJ
Lysosomal acid lipase (LAL) deficiency is an autosomal recessive disorder caused by LIPA gene mutations that disrupt LAL activity. We performed in vitro functional testing of 149
LIPA variants to ...increase the understanding of the variant effects on LAL deficiency and to improve disease prevalence estimates. Chosen variants had been reported in literature or population databases. Functional testing was done by plasmid transient transfection and LAL activity assessment. We assembled a set of 165 published LAL deficient patient genotypes to evaluate this assay's effectiveness to recapitulate genotype/phenotype relationships. Rapidly progressive LAL deficient patients showed negligible enzymatic activity (<1%), whereas patients with childhood/adult LAL deficiency typically have 1–7% average activity. We benchmarked six in silico variant effect prediction algorithms with these functional data. PolyPhen‐2 was shown to have a superior area under the receiver operating curve performance. We used functional data along with Genome Aggregation Database (gnomAD) allele frequencies to estimate LAL deficiency birth prevalence, yielding a range of 3.45–5.97 cases per million births in European‐ancestry populations. The low estimate only considers functionally assayed variants in gnomAD. The high estimate computes allele frequencies for variants absent in gnomAD, and uses in silico scores for unassayed variants. Prevalence estimates are lower than previously published, underscoring LAL deficiency's rarity.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
(HPV) is the main cause of genital warts and some anogenital cancers in male and female subjects which is commonly transmitted by sexual contacts. The objective of this cross-sectional study was to ...examine the prevalence of HPV genotypes in 10,266 Iranian male and female population, according to their age.
Samples were collected from the penile and anal sites of male subjects and the vagina and cervix of female subjects in a time period between 2011 and 2016. HPV DNA was detected in PCR using the MY09 and MY11 primers, and the INNO-LiPA assay was applied for HPV genotyping. To investigate the relevance of HPV infection and age, the samples were classified into 4 age groups (13-29, 30-44, 45-59, and 60-74).
Totally, the most common low risk HPV genotypes detected in the studied male and female subjects were HPV-6 (77.7% and 43.3%) and HPV-11 (13.7% and 11.4%), and more frequent high risk HPV genotypes were HPV-16 (5.5% and 16.6%) and HPV-52 (3.2% and 9.6%), respectively. High burden of the HPV infection was observed at ranges of 30 and 44 years (51.8%) with a peak at ranges between 30 and 32 years. No considerable statistically significant correlation was found between HPV infection and age (
=1).
This study gave an epidemiological overview of circulating HPV genotypes in Iranian population to develop future vaccination policies, though the findings of prevalent HPV genotypes in female subjects were inconsistent with the previous studies reported in Iran.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Lipid droplets (LDs) , organelles important for intracellular lipid metabolism, are actively formed in rat and human adult beta cells and affect islet function and health. Acute mobilization of LDs ...by adipose triglyceride lipase in beta cells is known to support insulin secretion. As LD degradation by lysosomal acid lipase (LIPA) through lipophagy is an alternative pathway for LD catabolism, we addressed whether LIPA regulates beta cell function. LDs in human beta cells are associated with autophagy (LC3) and lysosomal (LAMP1) markers. LIPA suppression via siRNA and LIPA inhibitor (lalistat2) in INS1 cells increased LD size (DMSO 0.5 vs. Lali2 1.5 µm2/cell, p<0.05, p value by Student’s t test unless specified otherwise) and number (Scr 2/cell vs. siLIPA 14/cell, p<0.05; DMSO 4.5/cell vs. Lali2 16/cell, p<0.05) . Rat and human beta cells treated with Lali2 also showed increased in LD area and/or number of LDs per cell (p<0.05) . In all three models, LIPA suppression increased TG contents confirming constitutively active lipophagy. While acute inhibition of LIPA by Lali2 had little effect on glucose-stimulated insulin secretion (GSIS) , chronic (over 48 h) LIPA suppression by Lali2 or siRNA reduced GSIS from INS1 cells, rat islets, and human islets. Lentiviral shRNA transduction in human pseudoislets decreased insulin secretion in response to glucose (41 ± 10.5% of Scr, p<0.05) and to KCl (61.3 ±16.1% of Scr, p<0.05) in vitro. When Scr and shLIPA human pseudoislets were transplanted under kidney capsules of NSG mice on Western diet, human insulin secretion in response to intraperitoneal glucose load was blunted in mice carrying shLIPA islets compared with shScr control (2-Way ANOVA, p<0.05) .
In summary, lipophagy is constitutively active in human and rat beta cells and serves as a pathway for LD catabolism that is acutely insensitive to glucose. However, LIPA mediated LD catabolism is needed for beta cell function in a long-term.
Disclosure
S.Liu: None. B.R.Brennecke: None. U.Yang: None. J.A.Ankrum: None. Y.Imai: None.
Funding
National Institute of Diabetes andDigestive and Kidney Diseases (R01=DK090490) U.S. Department of Veterans Affairs (IBX005107)
Human papillomavirus (HPV) epidemiological and vaccine studies require highly sensitive HPV detection systems. The widely used broad-spectrum SPF
10
-DEIA-LiPA
25
(SPF
10
method) has reduced ...sensitivity toward HPV-45 and -59. Therefore, anogenital samples from the PASSYON study were retrospectively analyzed with type-specific (TS) HPV-45 and -59 real-time quantitative PCR (qPCR) assays. The SPF
10
method missed 51.1% of HPV-45 and 76.1% of HPV-59 infections that were detected by the TS qPCR assays.
Human papillomavirus (HPV) epidemiological and vaccine studies require highly sensitive HPV detection systems. The widely used broad-spectrum SPF
10
-DEIA-LiPA
25
(SPF
10
method) has reduced sensitivity toward HPV-45 and -59. Therefore, anogenital samples from the PASSYON study were retrospectively analyzed with type-specific (TS) HPV-45 and -59 real-time quantitative PCR (qPCR) assays. The SPF
10
method missed 51.1% of HPV-45 and 76.1% of HPV-59 infections that were detected by the TS qPCR assays. The viral copy number (VCn) of SPF
10
-missed HPV-45 and -59 was significantly lower than SPF
10
-detected HPV-45 and -59 (
P
< 0.0001 for both HPV types). Sanger sequencing showed no phylogenetic distinction between SPF
10
-missed and SPF
10
-detected HPV-59 variants, but variants bearing the A6562G single-nucleotide polymorphism (SNP) in the SPF
10
target region were more likely to be missed (
P
= 0.0392). HPV cooccurrence slightly influenced the detection probability of HPV-45 and -59 with the SPF
10
method. Moreover, HPV-59 detection with the SPF
10
method was hampered more in nonvaccinated women than vaccinated women, likely due to a stronger masking effect by increased HPV cooccurrence in the former group. Consequently, the SPF
10
method led to a strong negative vaccine effectiveness (VE) of –84.6% against HPV-59, while the VE based on TS qPCR was 3.1%. For HPV-45, the relative increase in detection in nonvaccinated women compared vaccinated women was more similar, resulting in comparable VE estimates. In conclusion, this study shows that HPV-45 and -59 detection with the SPF
10
method is dependent on factors including VCn, HPV cooccurrence, and vaccination, thereby showing that knowledge of the limitations of the HPV detection method used is of great importance.
Background and Objective
Aggressive periodontitis (AgP) is characterized by general health and rapid destruction of periodontal tissue. The familial aggregation of this disease highlights the ...involvement of genetic factors in its pathogeny. We conducted a genome‐wide association study (GWAS) to identify AgP‐related genes in a Japanese population, and the lipid metabolism‐related gene, lipase‐a, lysosomal acid type (LIPA), was suggested as an AgP candidate gene. However, there is no report about the expression and function(s) of LIPA in periodontal tissue. Hence, we studied the involvement of how LIPA and its single‐nucleotide polymorphism (SNP) rs143793106 in AgP by functional analyses of LIPA and its SNP in human periodontal ligament (HPDL) cells.
Materials and Methods
GWAS was performed using the genome database of Japanese AgP patients, and the GWAS result was confirmed using Sanger sequencing. We examined the mRNA expression level of LIPA and the protein expression level of the encoded protein lysosomal acid lipase (LAL) in periodontium‐composing cells using conventional and real‐time polymerase chain reaction (PCR) and western blotting, respectively. Lentiviral vectors expressing LIPA wild‐type (LIPA WT) and LIPA SNP rs143793106 (LIPA mut) were transfected into HPDL cells. Western blotting was performed to confirm the transfection. LAL activity of transfected HPDL cells was determined using the lysosomal acid lipase activity assay. Transfected HPDL cells were cultured in mineralization medium. During the cytodifferentiation of transfected HPDL cells, mRNA expression of calcification‐related genes, alkaline phosphatase (ALPase) activity and calcified nodule formation were assessed using real‐time PCR, ALPase assay, and alizarin red staining, respectively.
Results
The GWAS study identified 11 AgP‐related candidate genes, including LIPA SNP rs143793106. The minor allele frequency of LIPA SNP rs143793106 in AgP patients was higher than that in healthy subjects. LIPA mRNA and LAL protein were expressed in HPDL cells; furthermore, they upregulated the cytodifferentiation of HPDL cells. LAL activity was lower in LIPA SNP‐transfected HPDL cells during cytodifferentiation than that in LIPA WT‐transfected HPDL cells. In addition, ALPase activity, calcified nodule formation, and calcification‐related gene expression levels were lower during cytodifferentiation in LIPA SNP‐transfected HPDL cells than those in LIPA WT‐transfected HPDL cells.
Conclusion
LIPA, identified as an AgP‐related gene in a Japanese population, is expressed in HPDL cells and is involved in regulating cytodifferentiation of HPDL cells. LIPA SNP rs143793106 suppressed cytodifferentiation of HPDL cells by decreasing LAL activity, thereby contributing to the development of AgP.
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CMK, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Ovarian cancer (OCa) is the most lethal form of gynecologic cancer, and the tumor heterogeneities at the molecular, cellular, and tissue levels fuel tumor resistance to standard therapies and pose a ...substantial clinical challenge. Here, we tested the hypothesis that the heightened basal endoplasmic reticulum stress (ERS) observed in OCa represents an exploitable vulnerability and may overcome tumor heterogeneity. Our recent studies identified LIPA as a novel target to induce ERS in cancer cells using the small molecule ERX-41. However, the role of LIPA and theutility of ERX-41 to treat OCa remain unknown. Expression analysis using the TNMplot web tool, TCGA data sets, and immunohistochemistry analysis using a tumor tissue array showed that LIPA is highly expressed in OCa tissues, compared to normal tissues. ERX-41 treatment significantly reduced the cell viability and colony formation ability and promoted the apoptosis of OCa cells. Mechanistic studies revealed a robust and consistent induction of ERS markers, including CHOP, elF2α, PERK, and ATF4, upon ERX-41 treatment. In xenograft and PDX studies, ERX-41 treatment resulted in a significant reduction in tumor growth. Collectively, our results suggest that ERX-41 is a novel therapeutic agent that targets the LIPA with a unique mechanism of ERS induction, which could be exploited to treat heterogeneity in OCa.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
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