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Using inhibition kinetics and computational simulation, we studied the reversible inhibition of tyrosinase by isophthalic acid (IPA). IPA inhibited tyrosinase in a complex manner with K(i)=17.8 ± 1.8mM. Measurements of intrinsic and ANS-binding fluorescence showed that IPA induced no changes in tertiary protein structure. For further insight, we predicted the 3D structure of tyrosinase and used a docking algorithm to simulate binding between tyrosinase and IPA. Simulation was successful (binding energies for Dock6.3: -25.19 kcal/mol and for AutoDock4.2: -4.28 kcal/mol), suggesting that IPA interacts with PRO175 or VAL190. This strategy of predicting tyrosinase inhibition based on hydroxyl group number and orientation may prove useful for the screening of potential tyrosinase inhibitors.
Alpha-ketoglutaric acid (AKG) is naturally found in organisms and is a well-known intermediate in the production of ATP or GTP in the Krebs cycle. We elucidated the effects of AKG on tyrosinase activity and conformation via methods of inhibition kinetics integrated with molecular dynamics (MD) simulations. AKG was found to be a reversible inhibitor of tyrosinase (IC50=15±0.5mM) and induced parabolic slope mixed-type inhibition. Based on our newly established equation, the dissociation constant (Kislope) was determined to be 7.93±0.31mM. The spectrofluorimetry studies showed that AKG mainly induced regional changes in the active site of tyrosinase, which reflects the flexibility of the active site. The computational docking and molecular dynamics (MD) simulations further demonstrated that AKG could interact with several residues near the substrate-binding site located in the tyrosinase active site pocket. Our study provides insight into the mechanism by which energy-producing intermediates such as AKG inhibit tyrosinase through its ketone groups. Also, AKG could be a potential natural antipigmentation agent due to its non-toxic property.
Aberrant mitophagy has been implicated in a broad spectrum of disorders. PINK1, Parkin, and ubiquitin have pivotal roles in priming mitophagy. However, the entire regulatory landscape and the precise control mechanisms of mitophagy remain to be elucidated. Here, we uncover fundamental mitophagy regulation involving PINK1 and a non-canonical role of the mitochondrial Tu translation elongation factor (TUFm). The mitochondrion-cytosol dual-localized TUFm interacts with PINK1 biochemically and genetically, which is an evolutionarily conserved Parkin-independent route toward mitophagy. A PINK1-dependent TUFm phosphoswitch at Ser222 determines conversion from activating to suppressing mitophagy. PINK1 modulates differential translocation of TUFm because p-S222-TUFm is restricted predominantly to the cytosol, where it inhibits mitophagy by impeding Atg5-Atg12 formation. The self-antagonizing feature of PINK1/TUFm is critical for the robustness of mitophagy regulation, achieved by the unique kinetic parameters of p-S222-TUFm, p-S65-ubiquitin, and their common kinase PINK1. Our findings provide new mechanistic insights into mitophagy and mitophagy-associated disorders.
Fatty acid uptake is the first step in fatty acid utilization, but it remains unclear how the process is regulated. Protein palmitoylation is a fatty acyl modification that plays a key regulatory role in protein targeting and trafficking; however, its function in regulating fatty acid metabolism is unknown. Here, we show that two of the Asp-His-His-Cys (DHHC) motif-containing palmitoyl acyltransferases, DHHC4 and DHHC5, regulate fatty acid uptake. DHHC4 and DHHC5 function at different subcellular localizations to control the palmitoylation, plasma membrane localization, and fatty acid uptake activity of the scavenger receptor CD36. Depletion of either DHHC4 or DHHC5 in cells disrupts CD36-dependent fatty acid uptake. Furthermore, both Dhhc4-/- and adipose-specific Dhhc5 knockout mice show decreased fatty acid uptake activity in adipose tissues and develop severe hypothermia upon acute cold exposure. These findings demonstrate a critical role of DHHC4 and DHHC5 in regulating fatty acid uptake.
Single-nucleotide polymorphisms (SNPs) are common genetic material changes that often occur naturally. SNPs can cause amino acid replacements that may lead to severe diseases, such as the well-known sickle-cell anemia. We constructed eight SNP mutants of human brain-type creatine kinase (CKB) based on bioinformatics predictions. The biochemical and biophysical characteristics of these SNP mutants were determined and compared to those of the wild-type creatine kinase to explore the potential molecular mechanisms of possible creatine kinase SNP-induced diseases. While the reactivation of six SNP mutants after heat shock dropped more than 45%, only three of them showed notable increases in ANS fluorescence intensity and decreases in catalytic efficiency. Among them, H26Y and P36T bind substrates as well as the wild-type form does, but the melting temperatures (T(m)) dropped below body temperature, while the T59I mutant exhibited decreased catalytic activity that was most likely due to the much reduced binding affinity of this mutant for substrates. These findings indicate that SNPs such as H26Y, P36T and T59I have the potential to induce genetic diseases by different mechanisms.
Tyrosinase is a ubiquitous enzyme with diverse physiologic roles related to pigment production. Tyrosinase inhibition has been well studied for cosmetic, medicinal, and agricultural purposes. We simulated the docking of tyrosinase and D-(-)-arabinose and found a binding energy of -4.5 kcal/mol for theup-formof D-(-)-arabinose and -4.4 kcal/mol for thedown-form of D-(-)-arabinose. The results of molecular dynamics simulation suggested that D-(-)-arabinose interacts mostly with HIS85, HIS259, and HIS263, which are believed to be in the active site. Our kinetic study showed that D-(-)-arabinose is a reversible, mixed-type inhibitor of tyrosinase (α-value = 6.11 ± 0.98, K(i) = 0.21 ± 0.19 M). Measurements of intrinsic fluorescence showed that D-(-)-arabinose induced obvious tertiary changes to tyrosinase (binding constant K = 1.58 ± 0.02 M(-1), binding number n = 1.49 ± 0.06). This strategy of predicting tyrosinase inhibition based on specific interactions of aldehyde and hydroxyl groups with the enzyme may prove useful for screening potential tyrosinase inhibitors.
Improved mesothelioma patient survival will require development of novel and more effective pharmacological interventions. TP53 genomic mutations are uncommon in mesothelioma, and recent data indicate that p53 remains functional, and therefore is a potential therapeutic target in these cancers. In addition, the tumour suppressor NF2 is inactivated by genomic mechanisms in more than 80% of mesothelioma, causing upregulation of FAK activity. Because FAK is a negative regulator of p53, NF2 regulation of FAK-p53-MDM2 signalling loops were evaluated.
Different types of cells uptake fatty acids in response to different stimuli or physiological conditions; however, little is known about context-specific regulation of fatty acid uptake. Here, we show that muscle injury induces fatty acid uptake in muscle stem cells (MuSCs) to promote their proliferation and muscle regeneration. In humans and mice, fatty acids are mobilized after muscle injury. Through CD36, fatty acids function as both fuels and growth signals to promote MuSC proliferation. Mechanistically, injury triggers the translocation of CD36 in MuSCs, which relies on dynamic palmitoylation of STX11. Palmitoylation facilitates the formation of STX11/SNAP23/VAMP4 SANRE complex, which stimulates the fusion of CD36- and STX11-containing vesicles. Restricting fatty acid supply, blocking fatty acid uptake, or inhibiting STX11 palmitoylation attenuates muscle regeneration in mice. Our studies have identified a critical role of fatty acids in muscle regeneration and shed light on context-specific regulation of fatty acid sensing and uptake.
Creatine kinase (CK) helps maintain homeostasis of intracellular ATP level by catalyzing the reversible phosphotransfer between ATP and phosphocreatine. In humans, there are two cytosolic CK isoforms, the muscle-type (M) and the brain-type (B), which frequently function as homodimers (hMMCK and hBBCK). Interestingly, these isoenzymes exhibit significantly different thermostabilities, despite high similarity in amino acid sequences and tertiary structures. In order to investigate the mechanism of this phenomenon, in this work, we first used domain swapping and site-directed mutagenesis to search for the key residues responsible for the isoenzyme-specific thermostability. Strikingly, the difference in thermostability was found to principally arise from one single residue substitution at position 36 (Pro in hBBCK vs. Leu in hMMCK). We then engaged the molecular dynamics simulations to study the molecular mechanism. The calculations imply that the P36L substitution introduces additional local interactions around residue 36 and thus further stabilizes the dimer interface through a complex interaction network, which rationalizes the observation that hMMCK is more resistant to thermal inactivation than hBBCK. We finally confirmed this molecular explanation through thermal inactivation assays on Asp36 mutants that were proposed to devastate the local interactions and thus the dimer associations in both isoenzymes.
Lung cancer is a common cancer and the leading cause of cancer-related death worldwide. SIX3 is a human homologue of the highly conserved sine oculis gene family essential during embryonic development in vertebrates, and encodes a homeo-domain containing transcription factor. Little is known about the role of SIX3 in human tumorigenesis. This study is to assess the expression/function of SIX3 and the significance of SIX3 as a prognostic biomarker in lung adenocarcinoma.
How complex interactions of genetic, environmental factors and aging jointly contribute to dopaminergic degeneration in Parkinson's disease (PD) is largely unclear. Here, we applied frequent gene co-expression analysis on human patient substantia nigra-specific microarray datasets to identify potential novel disease-related genes. In vivo Drosophila studies validated two of 32 candidate genes, a chromatin-remodeling factor SMARCA4 and a biliverdin reductase BLVRA. Inhibition of SMARCA4 was able to prevent aging-dependent dopaminergic degeneration not only caused by overexpression of BLVRA but also in four most common Drosophila PD models. Furthermore, down-regulation of SMARCA4 specifically in the dopaminergic neurons prevented shortening of life span caused by α-synuclein and LRRK2. Mechanistically, aberrant SMARCA4 and BLVRA converged on elevated ERK-ETS activity, attenuation of which by either genetic or pharmacological manipulation effectively suppressed dopaminergic degeneration in Drosophila in vivo. Down-regulation of SMARCA4 or drug inhibition of MEK/ERK also mitigated mitochondrial defects in PINK1 (a PD-associated gene)-deficient human cells. Our findings underscore the important role of epigenetic regulators and implicate a common signaling axis for therapeutic intervention in normal aging and a broad range of age-related disorders including PD.
Fumaric acid (FA), which is naturally found in organisms, is a well known intermediate of the citric acid cycle. We evaluated the effects of FA on tyrosinase activity and structure via enzyme kinetics and computational simulations. FA was found to be a reversible inhibitor of tyrosinase and its induced mechanism was the parabolic non-competitive inhibition type with IC50=13.7±0.25mM and Kislope=12.64±0.75mM. We newly established the equation for the dissociation constant (Kislope) for the parabolic inhibition type in this study. Kinetic measurements and spectrofluorimetry studies showed that FA induced regional changes in the active site of tyrosinase. One possible binding site for FA was identified under the condition without L-DOPA. The computational docking simulations further revealed that FA can interact with HIS263 and HIS85 at the active site. Furthermore, four important hydrogen bonds were found to be involved with the docking of FA on tyrosinase. Our study provides insight into the mechanism by which dicarboxylic acids such as FA inhibit tyrosinase. By inhibiting tyrosinase and its central role in pigment production, FA is a potential natural antipigmentation agent.
Similarly to the Wnt protein palmitoyltransferase, porcupine (PPN) is essential to the activation of the Wnt/β-catenin signaling pathway. However, little is known about the role of PPN activity in human gastric cancer, one of the most common causes of cancer-related mortality. Real-time quantitative PCR was used to detect the expression levels of PPN in paired gastric cancer tissues. Cell proliferation, migration and invasion assays were performed following treatment using a newly developed small molecule PPN inhibitor (inhibitors of Wnt production, IWP-2) in the gastric cancer MKN28 cell line. Expression of downstream target genes and transcriptional activity of the Wnt/β-catenin signaling pathway were examined following IWP-2 treatment in MKN28. We identified that PPN was overexpressed in human gastric cancer tissue samples and cell lines. Following treatment of the gastric cancer cell line MKN28 with IWP-2, we detected that IWP-2 decreased MKN28 cell proliferation, migration and invasion, and elevated caspase 3/7 activity. Further analysis demonstrated that IWP-2 downregulated the transcriptional activity of the Wnt/β-catenin signaling pathway and downregulated the expression levels of downstream Wnt/β-catenin target genes in MKN28 cells. As current Wnt pathway-targeting strategies used for anticancer therapy have mainly focused on Wnt-receiving cells, our data shed light on the potential use of Wnt palmitoyltransferase PPN inhibitors to abrogate Wnt production in Wnt-producing cells, thus providing a potential therapeutic option for gastric cancer.
Site-specific DNA double-strand breaks have been used to generate knock-in through the homology-dependent or -independent pathway. However, low efficiency and accompanying negative impacts such as undesirable indels or tumorigenic potential remain problematic. In this study, we present an enhanced reduced-risk genome editing strategy we named as NEO, which used either site-specific trans or cis double-nicking facilitated by four bacterial recombination factors (RecOFAR). In comparison to currently available approaches, NEO achieved higher knock-in (KI) germline transmission frequency (improving from zero to up to 10% efficiency with an average of 5-fold improvement for 8 loci) and 'cleaner' knock-in of long DNA fragments (up to 5.5 kb) into a variety of genome regions in zebrafish, mice and rats. Furthermore, NEO yielded up to 50% knock-in in monkey embryos and 20% relative integration efficiency in non-dividing primary human peripheral blood lymphocytes (hPBLCs). Remarkably, both on-target and off-target indels were effectively suppressed by NEO. NEO may also be used to introduce low-risk unrestricted point mutations effectively and precisely. Therefore, by balancing efficiency with safety and quality, the NEO method reported here shows substantial potential and improves the in vivo gene-editing strategies that have recently been developed.
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