Glucose controls lipolysis through Golgi PtdIns4P-mediated regulation of ATGL.

Metabolic crosstalk of the major nutrients glucose, amino acids and fatty acids (FAs) ensures systemic metabolic homeostasis. The coordination between the supply of glucose and FAs to meet various physiological demands is especially important as improper nutrient levels lead to metabolic disorders, such as diabetes and metabolic dysfunction-associated steatohepatitis (MASH). In response to the oscillations in blood glucose levels, lipolysis is thought to be mainly regulated hormonally to control FA liberation from lipid droplets by insulin, catecholamine and glucagon. However, whether general cell-intrinsic mechanisms exist to directly modulate lipolysis via glucose sensing remains largely unknown. Here we report the identification of such an intrinsic mechanism, which involves Golgi PtdIns4P-mediated regulation of adipose triglyceride lipase (ATGL)-driven lipolysis via intracellular glucose sensing. Mechanistically, depletion of intracellular glucose results in lower Golgi PtdIns4P levels, and thus reduced assembly of the E3 ligase complex CUL7FBXW8 in the Golgi apparatus. Decreased levels of the E3 ligase complex lead to reduced polyubiquitylation of ATGL in the Golgi and enhancement of ATGL-driven lipolysis. This cell-intrinsic mechanism regulates both the pool of intracellular FAs and their extracellular release to meet physiological demands during fasting and glucose deprivation. Moreover, genetic and pharmacological manipulation of the Golgi PtdIns4P-CUL7FBXW8-ATGL axis in mouse models of simple hepatic steatosis and MASH, as well as during ex vivo perfusion of a human steatotic liver graft leads to the amelioration of steatosis, suggesting that this pathway might be a promising target for metabolic dysfunction-associated steatotic liver disease and possibly MASH.

Phenotypic and genetic characterization of a lipolytic Ralstonia mannitolilytica isolate from petroleum-contaminated soil in Iraq.

BACKGROUND: Lipases play a crucial role in various industrial applications, and microbial lipases, particularly those from bacteria, possess significant properties. With increasing concerns about the environmental and health impacts of hydrocarbons from pipelines and refineries, there is a growing need to mitigate the risks associated with these compounds. METHODS: In this study, 40 bacterial isolates were recovered from contaminated soil samples collected from multiple refineries across Iraq. Using the Vitek system, bacterial isolates were identified up to the species level, revealing that only 12 isolates exhibited lipase-producing capabilities. RESULTS: Among the lipase-producing isolates, Ralstonia mannitolilytica demonstrated the highest extracellular lipase activity, as determined by an olive oil plate assay supplemented with rhodamine B. Confirmation of the species identity was achieved through 16S rRNA gene sequencing, with the obtained sequence deposited under accession number LC772176.1. Further sequence analysis revealed single nucleotide polymorphisms (SNPs) in the genome of Ralstonia mannitolilytica strain H230303-10_N19_7x_R2 (CP011257.1, positions 1,311,102 and 1,311,457). Additionally, the presence of the lipase gene was confirmed through amplification and sequencing using a thermocycler PCR. Sequence analysis of the gene, aligned using Geneious Prime software, identified SNPs (CP010799, CP049132, AY364601, CP011257, and CP023537), and a phylogenetic tree was constructed based on genetic characterization. CONCLUSION: Our findings highlight the potential of Ralstonia mannitolilytica as a promising candidate for lipase production and contribute to our understanding of its genetic diversity and biotechnological applications in hydrocarbon degradation and industrial processes.

LET-767 determines lipid droplet protein targeting and lipid homeostasis.

Lipid droplets (LDs) are composed of a core of neutral lipids wrapped by a phospholipid (PL) monolayer containing several hundred proteins that vary between different cells or organisms. How LD proteins target to LDs is still largely unknown. Here, we show that RNAi knockdown or gene mutation of let-767, encoding a member of hydroxysteroid dehydrogenase (HSD), displaced the LD localization of three well-known LD proteins: DHS-3 (dehydrogenase/reductase), PLIN-1 (perilipin), and DGAT-2 (diacylglycerol O-acyltransferase 2), and also prevented LD growth in Caenorhabditis elegans. LET-767 interacts with ARF-1 (ADP-ribosylation factor 1) to prevent ARF-1 LD translocation for appropriate LD protein targeting and lipid homeostasis. Deficiency of LET-767 leads to the release of ARF-1, which further recruits and promotes translocation of ATGL-1 (adipose triglyceride lipase) to LDs for lipolysis. The displacement of LD proteins caused by LET-767 deficiency could be reversed by inhibition of either ARF-1 or ATGL-1. Our work uncovers a unique LET-767 for determining LD protein targeting and maintaining lipid homeostasis.

Mutational scanning pinpoints distinct binding sites of key ATGL regulators in lipolysis.

ATGL is a key enzyme in intracellular lipolysis and plays an important role in metabolic and cardiovascular diseases. ATGL is tightly regulated by a known set of protein-protein interaction partners with activating or inhibiting functions in the control of lipolysis. Here, we use deep mutational protein interaction perturbation scanning and generate comprehensive profiles of single amino acid variants that affect the interactions of ATGL with its regulatory partners: CGI-58, G0S2, PLIN1, PLIN5 and CIDEC. Twenty-three ATGL amino acid variants yield a specific interaction perturbation pattern when validated in co-immunoprecipitation experiments in mammalian cells. We identify and characterize eleven highly selective ATGL switch mutations which affect the interaction of one of the five partners without affecting the others. Switch mutations thus provide distinct interaction determinants for ATGL's key regulatory proteins at an amino acid resolution. When we test triglyceride hydrolase activity in vitro and lipolysis in cells, the activity patterns of the ATGL switch variants trace to their protein interaction profile. In the context of structural data, the integration of variant binding and activity profiles provides insights into the regulation of lipolysis and the impact of mutations in human disease.

The deleterious effects of sofosbuvir and ribavirin (antiviral drugs against hepatitis C virus) on different body systems in male albino rats regarding reproductive, hematological, biochemical, hepatic, and renal profiles and histopathological changes.

Sofosbuvir is one of the crucial drugs used in the treatment of chronic hepatitis C virus (HCV) in adults and children with compensated liver disease, including cirrhosis. It may be used alone or with other drugs. Ribavirin is an antiviral medication used to treat HCV infection. It is not effective when used alone and must be used in combination with other medications, such as sofosbuvir. This study pertains to a comprehensive assessment of the deleterious effects of sofosbuvir (an antiviral drug against chronic HCV) or sofosbuvir combined with ribavirin (an antiviral drug against RNA and DNA viruses) on several biological activities of the body, including hematological, hormonal, biochemical, histological, and immunohistochemical examinations during a long-standing period on male healthy rats. In addition, fertility assessments were performed, including sperm collections and semen parameter investigations. This study was conducted on 21 male rats divided into three equal groups. Group I (control group) received distilled water; group II (sofosbuvir group) received sofosbuvir (4 mg/kg); and group III (sofosbuvir + ribavirin) received sofosbuvir (4 mg/kg) plus ribavirin (30 ml/kg). All groups received the specific drug for six months. Blood and tissue samples were collected for hematological, hormonal, biochemical, histological, and immunohistochemical examinations. In addition, sperm collection and assessments of semen parameters were performed. Results revealed that sofosbuvir causes a highly significant decrease in the mean of most hematological, immunological, hormonal, and biochemical parameters, except for a few numbers of parameters such as neutrophils, monocytes, basophils, cortisol, GOT, and lipase, which exhibit a significant increase. The same occurred in the sofosbuvir + ribavirin group, but at much higher levels, as most hematological, immunological, hormonal, and biochemical parameters exhibit a highly significant decrease except for monocytes, triglyceride, and lipase, which exhibit a significant increase. When compared to the sofosbuvir group alone, the sofosbuvir + ribavirin group demonstrated a highly significant decline in the mean of most hematological, immunological, hormonal, and biochemical parameters except lymphocytes and triglycerides, which exhibit a substantial increase. For the reproductive parameters, both groups exhibit a significant decrease in the total sperm motility percentage. Finally, it can be concluded that sofosbuvir causes acute pancreatitis and combined immunodeficiency. Ribavirin is associated with hormonal deficiency, which indicates the occurrence of hypopituitarism. Moreover, sofosbuvir and ribavirin synergistically affect myelosuppression and cause iron-deficiency anemia. However, sofosbuvir, or its combination with ribavirin, is associated with a reduced risk of hepatocellular carcinoma. Besides, adding ribavirin to be combined with sofosbuvir improved the immunodeficiency caused by sofosbuvir; this confirms that using ribavirin with sofosbuvir reduces the side effects of both alone.

Identification of cheese rancidity-related lipases in Aspergillus oryzae AHU 7139.

The adjunct product with enzymatic activity from Aspergillus oryzae is beneficial for flavor enrichment in the ripened cheese. However, an excessive lipolytic reaction leads to the release of volatile free fatty acids. Accordingly, a strong off-flavor (i.e., rancidity) has been detected when A. oryzae AHU 7139 is used. To identify the rancidity-related lipase from this strain, we evaluated the substrate specificity and lipase distribution using five mutants cultured on a whey-based solid medium under different initial pH conditions. The results showed a higher diacylglycerol lipase activity than triacylglycerol lipase activity. Moreover, an initial pH of 6.5 for the culture resulted in higher lipolytic activity than a pH of 4.0, and most of the activity was found in the extracellular fraction. Based on the gene expression analysis by real-time polymerase chain reaction and location and substrate specificity, five genes (No. 1, No. 19, mdlB, tglA, and cutL) were selected among 25 annotated lipase genes to identify the respective knockout strains. Because ΔtglA and ΔmdlB showed an outstanding involvement in the release of free fatty acids, these strains were applied to in vitro cheese curd experiments. In conclusion, we posit that triacylglycerol lipase (TglA) plays a key role as the trigger of rancidity and the resulting diglycerides have to be exposed to diacylglycerol lipase (MdlB) to stimulate rancidity in cheese made with A. oryzae AHU 7139. This finding could help screen suitable A.oryzae strains as cheese adjuncts to prevent the generation of the rancid-off flavor.

Impaired lysosomal acidity maintenance in acid lipase-deficient cells leads to defective autophagy.

The lysosome is an acid organelle that contains a variety of hydrolytic enzymes and plays a significant role in intracellular degradation to maintain cellular homeostasis. Genetic variants in lysosome-related genes can lead to severe congenital diseases, such as lysosomal storage diseases. In the present study, we investigated the impact of depleting lysosomal acid lipase A (LIPA), a lysosomal esterase that metabolizes esterified cholesterol or triglyceride, on lysosomal function. Under nutrient-rich conditions, LIPA gene KO (LIPAKO) cells exhibited impaired autophagy, whereas, under starved conditions, they showed normal autophagy. The cause underlying the differential autophagic activity was increased sensitivity of LIPAKO cells to ammonia, which was produced from l-glutamine in the medium. Further investigation revealed that ammonia did not affect upstream signals involved in autophagy induction, autophagosome-lysosome fusion, and hydrolytic enzyme activities in LIPAKO cells. On the other hand, LIPAKO cells showed defective lysosomal acidity upon ammonia loading. Microscopic analyses revealed that lysosomes of LIPAKO cells enlarged, whereas the amount of lysosomal proton pump V-ATPase did not proportionally increase. Since the enlargement of lysosomes in LIPAKO cells was not normalized under starved conditions, this is the primary change that occurred in the LIPAKO cells, and autophagy was affected by impaired lysosomal function under the specific conditions. These findings expand our comprehension of the pathogenesis of Wolman's disease, which is caused by a defect in the LIPA gene, and suggest that conditions, such as hyperlipidemia, may easily disrupt lysosomal functions.

Endothelial lipase variant T111I does not alter inhibition by angiopoietin-like proteins.

High levels of HDL-C are correlated with a decreased risk of cardiovascular disease. HDL-C levels are modulated in part by the secreted phospholipase, endothelial lipase (EL), which hydrolyzes the phospholipids of HDL and decreases circulating HDL-C concentrations. A 584C/T polymorphism in LIPG, the gene which encodes EL, was first identified in individuals with increased HDL levels. This polymorphism results in a T111I point mutation the EL protein. The association between this variant, HDL levels, and the risk of coronary artery disease (CAD) in humans has been extensively studied, but the findings have been inconsistent. In this study, we took a biochemical approach, investigating how the T111I variant affected EL activity, structure, and stability. Moreover, we tested whether the T111I variant altered the inhibition of phospholipase activity by angiopoietin-like 3 (ANGPTL3) and angiopoietin-like 4 (ANGPTL4), two known EL inhibitors. We found that neither the stability nor enzymatic activity of EL was altered by the T111I variant. Moreover, we found no difference between wild-type and T111I EL in their ability to be inhibited by ANGPTL proteins. These data suggest that any effect this variant may have on HDL-C levels or cardiovascular disease are not mediated through alterations in these functions.

Synergic kinetic and physiological control to improve the efficiency of Komagataella phaffii recombinant protein production bioprocesses.

The yeast Komagataella phaffii (Pichia pastoris) is currently considered a versatile and highly efficient host for recombinant protein production (RPP). Interestingly, the regulated application of specific stress factors as part of bioprocess engineering strategies has proven potential for increasing the production of recombinant products. This study aims to evaluate the impact of controlled oxygen-limiting conditions on the performance of K. phaffii bioprocesses for RPP in combination with the specific growth rate (µ) in fed-batch cultivations. In this work, Candida rugosa lipase 1 (Crl1) production, regulated by the constitutive GAP promoter, growing at different nominal µ (0.030, 0.065, 0.100 and 0.120 h-1 ) under both normoxic and hypoxic conditions in carbon-limiting fed-batch cultures is analysed. Hypoxic fermentations were controlled at a target respiratory quotient (RQ) of 1.4, with excellent performance, using an innovative automated control based on the stirring rate as the manipulated variable developed during this study. The results conclude that oxygen limitation positively affects bioprocess efficiency under all growing conditions compared. The shift from respiratory to respiro-fermentative metabolism increases bioprocess productivity by up to twofold for the specific growth rates evaluated. Moreover, the specific product generation rate (qp ) increases linearly with µ, regardless of oxygen availability. Furthermore, this hypoxic boosting effect was also observed in the production of Candida antarctica lipase B (CalB) and pro-Rhizopus oryzae lipase (proRol), thus proving the synergic effect of kinetic and physiological stress control. Finally, the Crl1 production scale-up was conducted successfully, confirming the strategy's scalability and the robustness of the results obtained at the bench-scale level.

Shifting the pH profiles of Staphylococcus epidermidis lipase (SEL) and Staphylococcus hyicus lipase (SHL) through generating chimeric lipases by DNA shuffling strategy.

Enzymes are often required to function in a particular reaction condition by the industrial procedure. In order to identify critical residues affecting the optimum pH of Staphylococcal lipases, chimeric lipases from homologous lipases were generated via a DNA shuffling strategy. Chimeric 1 included mutations of G166S, K212E, T243A, H271Y. Chimeric 2 consisted of substitutions of K212E, T243A, H271Y. Chimeric 3 contained substitutions of K212E, R359L. From the screening results, the pH profiles for chimeric 1 and 2 lipases were shifted from pH 7 to 6. While the pH of chimeric 3 was shifted to 8. It seems the mutation of K212E in chimeric 1 and 2 decreased the pH to 6 by changing the electrostatic potential surface. Furthermore, chimeric 3 showed 10 ˚C improvement in the optimum temperature due to the rigidification of the catalytic loop through the hydrophobic interaction network. Moreover, the substrate specificity of chimeric 1 and 2 was increased towards the longer carbon length chains due to the mutation of T243A adjacent to the lid region through increasing the flexibility of the lid. Current study illustrated that directed evolution successfully modified lipase properties including optimum pH, temperature and substrate specificity through mutations, especially near catalytic and lid regions.

Evaluation of the Structure-Function Relationship of SGNH Lipase from Streptomyces rimosus by Site-Directed Mutagenesis and Computational Approach.

Streptomyces rimosus extracellular lipase (SrL) is a multifunctional hydrolase belonging to the SGNH family. Here site-directed mutagenesis (SDM) was used for the first time to investigate the functional significance of the conserved amino acid residues Ser10, Gly54, Asn82, Asn213, and His216 in the active site of SrL. The hydrolytic activity of SrL variants was determined using para-nitrophenyl (pNP) esters with C4, C8, and C16 fatty acid chains. Mutation of Ser10, Asn82, or His216, but not Gly54, to Ala abolished lipase activity for all substrates. In contrast, the Asn213Ala variant showed increased enzymatic activity for C8 and C16 pNP esters. Molecular dynamics (MD) simulations showed that the interactions between the long alkyl chain substrate (C16) and Ser10 and Asn82 were strongest in Asn213Ala SrL. In addition to Asn82, Gly54, and Ser10, several new constituents of the substrate binding site were recognized (Lys28, Ser53, Thr89, and Glu212), as well as strong electrostatic interactions between Lys28 and Glu212. In addition to the H bonds Ser10-His216 and His216-Ser214, Tyr11 interacted strongly with Ser10 and His216 in all complexes with an active enzyme form. A previously unknown strong H bond between the catalytically important Asn82 and Gly54 was uncovered, which stabilizes the substrate in an orientation suitable for the enzyme reaction.

Identification and characterization of GLYCEROLIPASE A1 for wound-triggered JA biosynthesis in Nicotiana benthamiana leaves.

Although many important discoveries have been made regarding the jasmonate signaling pathway, how jasmonate biosynthesis is initiated is still a major unanswered question in the field. Previous evidences suggest that jasmonate biosynthesis is limited by the availability of fatty acid precursor, such as ⍺-linolenic acid (⍺-LA). This indicates that the lipase responsible for releasing α-LA in the chloroplast, where early steps of jasmonate biosynthesis take place, is the key initial step in the jasmonate biosynthetic pathway. Nicotiana benthamiana glycerol lipase A1 (NbGLA1) is homologous to N. attenuata GLA1 (NaGLA1) which has been reported to be a major lipase in leaves for jasmonate biosynthesis. NbGLA1 was studied for its potential usefulness in a species that is more common in laboratories. Virus-induced gene silencing of both NbGLA1 and NbGLA2, another homolog, resulted in more than 80% reduction in jasmonic acid (JA) biosynthesis in wounded leaves. Overexpression of NbGLA1 utilizing an inducible vector system failed to increase JA, indicating that transcriptional induction of NbGLA1 is insufficient to trigger JA biosynthesis. However, co-treatment with wounding in addition to NbGLA1 induction increased JA accumulation several fold higher than the gene expression or wounding alone, indicating an enhancement of the enzyme activity by wounding. Domain-deletion of a 126-bp C-terminal region hypothesized to have regulatory roles increased NbGLA1-induced JA level. Together, the data show NbGLA1 to be a major lipase for wound-induced JA biosynthesis in N. benthamiana leaves and demonstrate the use of inducible promoter-driven construct of NbGLA1 in conjunction with its transient expression in N. benthamiana as a useful system to study its protein function.

Dynamic metabolism of endothelial triglycerides protects against atherosclerosis in mice.

Blood vessels are continually exposed to circulating lipids, and elevation of ApoB-containing lipoproteins causes atherosclerosis. Lipoprotein metabolism is highly regulated by lipolysis, largely at the level of the capillary endothelium lining metabolically active tissues. How large blood vessels, the site of atherosclerotic vascular disease, regulate the flux of fatty acids (FAs) into triglyceride-rich (TG-rich) lipid droplets (LDs) is not known. In this study, we showed that deletion of the enzyme adipose TG lipase (ATGL) in the endothelium led to neutral lipid accumulation in vessels and impaired endothelial-dependent vascular tone and nitric oxide synthesis to promote endothelial dysfunction. Mechanistically, the loss of ATGL led to endoplasmic reticulum stress-induced inflammation in the endothelium. Consistent with this mechanism, deletion of endothelial ATGL markedly increased lesion size in a model of atherosclerosis. Together, these data demonstrate that the dynamics of FA flux through LD affects endothelial cell homeostasis and consequently large vessel function during normal physiology and in a chronic disease state.

Conjugated Linoleic Acid Reduces Lipid Accumulation via Down-regulation Expression of Lipogenic Genes and Up-regulation of Apoptotic Genes in Grass Carp (Ctenopharyngodon idella) Adipocyte In Vitro.

The relationship between conjugated linoleic acid (CLA) and lipogenesis has been extensively studied in mammals and some cell lines, but it is relatively rare in fish, and the potential mechanism of action of CLA reducing fat mass remains unclear. The established primary culture model for studying lipogenesis in grass carp (Ctenopharyngodon idella) preadipocytes was used in the present study, and the objective was to explore the effects of CLA on intracellular lipid and TG content, fatty acid composition, and mRNA levels of adipogenesis transcription factors, lipase, and apoptosis genes in grass carp adipocytes in vitro. The results showed that CLA reduced the size of adipocyte and lipid droplet and decreased the content of intracellular lipid and TG, which was accompanied by a significant down-regulation of mRNA abundance in transcriptional regulators including peroxisome proliferator-activated receptor (PPAR) γ, CCAAT/enhancer-binding protein (C/EBP) α, sterol regulatory element-binding protein (SREBP) 1c, lipase genes including fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), lipoprotein lipase (LPL). Meanwhile, it decreased the content of saturated fatty acids (SFAs) and n - 6 polyunsaturated fatty acid (n-6 PUFA) and increased the content of monounsaturated fatty acid (MUFA) and n - 3 polyunsaturated fatty acid (n-3 PUFA) in primary grass carp adipocyte. In addition, CLA induced adipocyte apoptosis through downregulated anti-apoptotic gene B-cell CLL/lymphoma 2 (Bcl-2) mRNA level and up-regulated pro-apoptotic genes tumor necrosis factor-α (TNF-α), Bcl-2-associated X protein (Bax), Caspase-3, and Caspase-9 mRNA level in a dose-dependent manner. These findings suggest that CLA can act on grass carp adipocytes through various pathways, including decreasing adipocyte size, altering fatty acid composition, inhibiting adipocyte differentiation, promoting adipocyte apoptosis, and ultimately decreasing lipid accumulation.

Neutral lipid storage disease with myopathy: clinicopathological and genetic features of nine Iranian patients.

The rare disorder known as Neutral Lipid Storage Disease with Myopathy presents with a variety of clinical manifestations, including myopathy, cardiac dysfunction, and other organ complications. Early diagnosis is crucial due to the increased risk of cardiomyopathy. We describe the clinical, histopathological, muscle imaging, and genetic findings of nine neutral lipid storage myopathy patients. Proximal weakness and asymmetric involvement may suggest lipid storage myopathy. While skeletal muscle weakness was the main manifestation in our patients, one case presented only with hyperCKemia. Additionally, three patients had fertility issues, two suffered from diabetes mellitus, two had cardiomyopathy, and one had a history of hypothyroidism. Muscle histopathology revealed lipid depositions and rimmed vacuoles, prompting peripheral blood smears to detect Jordan Anomalies. All muscle biopsies and peripheral blood smear showed lipid droplets, rimmed vacuoles, and Jordan anomaly. Identifying PNPLA2 gene mutations is important for diagnosing neutral lipid storage myopathy; our cases showed some novel mutations. This study highlights the importance of early diagnosis and comprehensive evaluation in managing neutral lipid storage myopathy cases.

Deficiency of Brummer lipase disturbs lipid mobilization and locomotion, and impairs reproduction due to defects in the eggshell ultrastructure in the insect vector Rhodnius prolixus.

Rhodnius prolixus is a hematophagous insect, which feeds on large and infrequent blood meals, and is a vector of trypanosomatids that cause Chagas disease. After feeding, lipids derived from blood meal are stored in the fat body as triacylglycerol, which is recruited under conditions of energy demand by lipolysis, where the first step is catalyzed by the Brummer lipase (Bmm), whose orthologue in mammals is the adipose triglyceride lipase (ATGL). Here, we investigated the roles of Bmm in adult Rhodnius prolixus under starvation, and after feeding. Its gene (RhoprBmm) was expressed in all the analyzed insect organs, and its transcript levels in the fat body were not altered by nutritional status. RNAi-mediated knockdown of RhoprBmm caused triacylglycerol retention in the fat body during starvation, resulting in larger lipid droplets and lower ATP levels compared to control females. The silenced females showed decreased flight capacity and locomotor activity. When RhoprBmm knockdown occurred before the blood meal and the insects were fed, the females laid fewer eggs, which collapsed and showed low hatching rates. Their hemolymph had reduced diacylglycerol content and vitellogenin concentration. The chorion (eggshell) of their eggs had no difference in hydrocarbon amounts or in dityrosine crosslinking levels compared to control eggs. However, it showed ultrastructural defects. These results demonstrated that Bmm activity is important not only to guarantee lipid mobilization to maintain energy homeostasis during starvation, but also for the production of viable eggs after a blood meal, by somehow contributing to the right formation of the egg chorion.

Adipose Triglyceride Lipase Is a Therapeutic Target in Advanced Prostate Cancer That Promotes Metabolic Plasticity.

Lipid metabolism plays a central role in prostate cancer. To date, the major focus has centered on de novo lipogenesis and lipid uptake in prostate cancer, but inhibitors of these processes have not benefited patients. A better understanding of how cancer cells access lipids once they are created or taken up and stored could uncover more effective strategies to perturb lipid metabolism and treat patients. Here, we identified that expression of adipose triglyceride lipase (ATGL), an enzyme that controls lipid droplet homeostasis and a previously suspected tumor suppressor, correlates with worse overall survival in men with advanced, castration-resistant prostate cancer (CRPC). Molecular, genetic, or pharmacologic inhibition of ATGL impaired human and murine prostate cancer growth in vivo and in cell culture or organoids under conditions mimicking the tumor microenvironment. Mass spectrometry imaging demonstrated that ATGL profoundly regulates lipid metabolism in vivo, remodeling membrane composition. ATGL inhibition induced metabolic plasticity, causing a glycolytic shift that could be exploited therapeutically by cotargeting both metabolic pathways. Patient-derived phosphoproteomics identified ATGL serine 404 as a target of CAMKK2-AMPK signaling in CRPC cells. Mutation of serine 404 did not alter the lipolytic activity of ATGL but did decrease CRPC growth, migration, and invasion, indicating that noncanonical ATGL activity also contributes to disease progression. Unbiased immunoprecipitation/mass spectrometry suggested that mutation of serine 404 not only disrupts existing ATGL protein interactions but also leads to new protein-protein interactions. Together, these data nominate ATGL as a therapeutic target for CRPC and provide insights for future drug development and combination therapies. SIGNIFICANCE: ATGL promotes prostate cancer metabolic plasticity and progression through both lipase-dependent and lipase-independent activity, informing strategies to target ATGL and lipid metabolism for cancer treatment.

Structure-directed bioengineering the lid1 of cold-adapted Pseudomonas sp. TB11 esterase to boost catalytic capacity.

Structure-guided bioengineering enzymes has been an efficient strategy to obtain biocatalyst with desirable properties. In this study, the cold-adapted esterase from Pseudomonas sp. (CPE) was optimized through bioinformatic-based structured-guided bioengineering on lid1 region. Substitutions of non-conserved Q55 led to noticeable increase in hydrolysis without sacrificing enzyme thermostability, activating effects of Ca2+ and organic solvents. Compared to the wild type, both of Q55V and Q55N among the constructed variants exhibited about a 2.0-fold and 6.5-fold higher hydrolytic activity toward short-chain and long-chain substrates, respectively. In contrast, lid swapping with the lid of Thermomyces lanuginosus lipase reduced the activity and thermostability of CPE. Catalytic kinetics revealed that substitution of Q55 with Y, V, N and R enhanced the substrate affinity of CPE. Hydrolysis by Q55V remarkedly enriched the characteristic flavor components of single cream. The study sheds light on structure-guided bioengineering of lid tailoring cold-adapted esterases with desired catalytic performance to meet the demand from biotechnological applications.

OsGELP77, a QTL for broad-spectrum disease resistance and yield in rice, encodes a GDSL-type lipase.

Lipids and lipid metabolites have essential roles in plant-pathogen interactions. GDSL-type lipases are involved in lipid metabolism modulating lipid homeostasis. Some plant GDSLs modulate lipid metabolism altering hormone signal transduction to regulate host-defence immunity. Here, we functionally characterized a rice lipase, OsGELP77, promoting both immunity and yield. OsGELP77 expression was induced by pathogen infection and jasmonic acid (JA) treatment. Overexpression of OsGELP77 enhanced rice resistance to both bacterial and fungal pathogens, while loss-of-function of osgelp77 showed susceptibility. OsGELP77 localizes to endoplasmic reticulum and is a functional lipase hydrolysing universal lipid substrates. Lipidomics analyses demonstrate that OsGELP77 is crucial for lipid metabolism and lipid-derived JA homeostasis. Genetic analyses confirm that OsGELP77-modulated resistance depends on JA signal transduction. Moreover, population genetic analyses indicate that OsGELP77 expression level is positively correlated with rice resistance against pathogens. Three haplotypes were classified based on nucleotide polymorphisms in the OsGELP77 promoter where OsGELP77Hap3 is an elite haplotype. Three OsGELP77 haplotypes are differentially distributed in wild and cultivated rice, while OsGELP77Hap3 has been broadly pyramided for hybrid rice development. Furthermore, quantitative trait locus (QTL) mapping and resistance evaluation of the constructed near-isogenic line validated OsGELP77, a QTL for broad-spectrum disease resistance. In addition, OsGELP77-modulated lipid metabolism promotes JA accumulation facilitating grain yield. Notably, the hub defence regulator OsWRKY45 acts upstream of OsGELP77 by initiating the JA-dependent signalling to trigger immunity. Together, OsGELP77, a QTL contributing to immunity and yield, is a candidate for breeding broad-spectrum resistant and high-yielding rice.

Metagenomic discovery of lipases with predicted structural similarity to Candida antarctica lipase B.

Here we employed sequence-based and structure-based screening for prospecting lipases that have structural homolog to Candida antarctica lipase B (CalB). CalB, a widely used biocatalyst, was used as structural template reference because of its enzymatic properties. Structural homolog could aid in the discovery of novel wild-type enzymes with desirable features and serve as a scaffold for further biocatalyst design. The available metagenomic data isolated from various environments was leveraged as a source for bioprospecting. We identified two bacteria lipases that showed high structural similarity to CalB with <40% sequence identity. Partial purification was conducted. In comparison to CalB, the enzymatic characteristics of two potential lipases were examined. A candidate exhibited optimal pH of 8 and temperature of 50°C similar to CalB. The second lipase candidate demonstrated an optimal pH of 8 and a higher optimal temperature of 55°C. Notably, this candidate sustained considerable activity at extreme conditions, maintaining high activity at 70°C or pH 9, contrasting with the diminished activity of CalB under similar conditions. Further comprehensive experimentation is warranted to uncover and exploit these novel enzymatic properties for practical biotechnological purposes.