Palmitoyl Protein Thioesterase Research Articles

Binding of hydroxychloroquine and chloroquine dimers to palmitoyl-protein thioesterase 1 (PPT1) and its glycosylated forms: a computational approach

The lysosomal enzyme palmitoyl-protein thioesterase 1 (PPT1) removes thioester-linked fatty acid groups from membrane-bound proteins to facilitate their proteolysis. A lack of PPT1 (due to gene mutations) causes the progressive death of cortical neurons and is responsible for infantile neural ceroid lipofuscinosis (INCL), a severe neurodegenerative disorder in children. Conversely, PPT1 is often over-expressed in cancer, and considered as a valid target to control tumor growth. Potent and selective inhibitors of PPT1 have been designed, in particular 4-amino-7-chloro-quinoline derivatives such as hydroxychloroquine (HCQ) and the dimeric analogues Lys05 and DC661. We have modeled the interaction of these three compounds with the enzyme, taking advantage of the PPT1 crystallographic structure. The molecules can fit into the palmitate site of the protein, with the dimeric compounds forming more stable complexes than the monomer. But the molecular modeling suggests that the most favorable binding sites are located outside the active site. Two sites centered on residues Met112 and Gln144 were identified, offering suitable cavities for drug binding. According to the calculated empirical energies of interaction (ΔE), the dimer DC661 forms the most stable complex at site Met112 of palmitate-bound PPT1. N-glycosylated forms of PPT1 were elaborated. Paucimannosidic glycans (M2FA and M3F) and a bulkier tetra-antennary complex glycan were introduced at asparagine residues N197, N212 and N232. These N-glycans do not impede drug binding, thus suggesting that all glycoforms of PPT1 can be targeted with these compounds. Communicated by Ramaswamy H. Sarma

Management of CLN1 Disease: International Clinical Consensus

BackgroundCLN1 disease (neuronal ceroid lipofuscinosis type 1) is a rare, genetic, neurodegenerative lysosomal storage disorder caused by palmitoyl-protein thioesterase 1 (PPT1) enzyme deficiency. Clinical features include developmental delay, psychomotor regression, seizures, ataxia, movement disorders, visual impairment, and early death. In general, the later the age at symptom onset, the more protracted the disease course. We sought to evaluate current evidence and to develop expert practice consensus to support clinicians who have not previously encountered patients with this rare disease. MethodsWe searched the literature for guidelines and evidence to support clinical practice recommendations. We surveyed CLN1 disease experts and caregivers regarding their experiences and recommendations, and a meeting of experts was conducted to ascertain points of consensus and clinical practice differences. ResultsWe found a limited evidence base for treatment and no clinical management guidelines specific to CLN1 disease. Fifteen CLN1 disease experts and 39 caregivers responded to the surveys, and 14 experts met to develop consensus-based recommendations. The resulting management recommendations are uniquely informed by family perspectives, due to the inclusion of caregiver and advocate perspectives. A family-centered approach is supported, and individualized, multidisciplinary care is emphasized in the recommendations. Ascertainment of the specific CLN1 disease phenotype (infantile-, late infantile-, juvenile-, or adult-onset) is of key importance in informing the anticipated clinical course, prognosis, and care needs. Goals and strategies should be periodically reevaluated and adapted to patients' current needs, with a primary aim of optimizing patient and family quality of life.

Ligand-induced transition in conformations of vicinal cysteine disulfides in proteins.

Vicinal cysteine disulfides are thought to be associated with specific conformations of cysteine disulfides due to the restricted rotation of single bonds in an eight-membered cyclic disulfide loop. Conformations of vicinal cysteine disulfides are analyzed using χ1 , χ2 , χ3 , χ2 ', χ1 ' torsion angles in the crystal structures of proteins retrieved from Protein Data Bank (PDB). 85% of vicinal disulfides have (+, -)LHStaple conformation with trans configuration of the peptide bond and 9% have (-, -)RHStaple conformation with cis configured peptide bond. Conformational analysis of dipeptide Cys-Cys vicinal disulfide by density functional theory (DFT) further supported (+, -)LHStaple, (-, -)RHStaple, and (+, +)RHStaple as the preferred conformations of vicinal disulfides. Interestingly, the rare conformations of vicinal disulfides are observed in the ligand-bound forms of proteins and have higher disulfide strain energy. Conformations of vicinal disulfides in palmitoyl protein thioesterase 1, AChBP, and α7 nicotinic receptor are changed from preferred (+, -)LHStaple to rare (+, -)AntiLHHook/(+, -)AntiRHHook/(+, +)RHStaple conformation due to binding of ligands. Surprisingly, ligands are proximal to the vicinal disulfides in protein complexes that exhibited rare conformations of vicinal disulfides. The report has identified (+, -) LHStaple/(-, -) RHStaple as the hallmark conformations of vicinal disulfides and unraveled ligand-induced transition in conformations of vicinal cysteine disulfides in proteins.

GFAP hyperpalmitoylation exacerbates astrogliosis and neurodegenerative pathology in PPT1-deficient mice

The homeostasis of protein palmitoylation and depalmitoylation is essential for proper physiological functions in various tissues, in particular the central nervous system (CNS). The dysfunction of PPT1 (PPT1-KI, infantile neuronal ceroid lipofuscinosis [INCL] mouse model), which catalyze the depalmitoylation process, results in serious neurodegeneration accompanied by severe astrogliosis in the brain. Endeavoring to determine critical factors that might account for the pathogenesis in CNS by palm-proteomics, glial fibrillary acidic protein (GFAP) was spotted, indicating that GFAP is probably palmitoylated. Questions concerning if GFAP is indeed palmitoylated in vivo and how palmitoylation of GFAP might participate in neural pathology remain unexplored and are waiting to be investigated. Here we show that GFAP is readily palmitoylated in vitro and in vivo; specifically, cysteine-291 is the unique palmitoylated residue in GFAP. Interestingly, it was found that palmitoylated GFAP promotes astrocyte proliferation in vitro. Furthermore, we showed that PPT1 depalmitoylates GFAP, and the level of palmitoylated GFAP is overwhelmingly up-regulated in PPT1-knockin mice, which lead us to speculate that the elevated level of palmitoylated GFAP might accelerate astrocyte proliferation in vivo and ultimately led to astrogliosis in INCL. Indeed, blocking palmitoylation by mutating cysteine-291 into alanine in GFAP attenuate astrogliosis, and remarkably, the concurrent neurodegenerative pathology in PPT1-knockin mice. Together, these findings demonstrate that hyperpalmitoylated GFAP plays critical roles in regulating the pathogenesis of astrogliosis and neurodegeneration in the CNS, and most importantly, pinpointing that cysteine-291 in GFAP might be a valuable pharmaceutical target for treating INCL and other potential neurodegenerative diseases.

A case of juvenile CLN1– challenge in diagnosis and epilepsy treatment

ABSTRACT Introduction Classic onset of CLN1 disease is within the first year of life with developmental arrest, epilepsy and rapid progression. In an atypical variant of CLN1 disease onset is later in the juvenile epoch. Although epilepsy in the juvenile form of CLN1 often is less severe than in typical CLN1, treatment of seizures and status epilepticus may be challenging. Case presentation The clinical course, misdiagnosis and epilepsy phenotype are presented in a girl with juvenile CLN1. Cognitive and neurologic regression started at age 5.5 years. Epilepsy was a major clinical issue as the patient suffered from focal seizures, recurrent status epilepticus and epilepsia partialis continua. In one episode of refractory status epilepticus, the patient had significant bradycardia associated with the intravenous infusion of levetiracetam. Diagnosis was made at the age of 12 years, based on palmitoyl protein-thioesterase (PPT) enzyme deficiency and genetic testing that documented a homozygous exon missense mutation in the CLN1 gene (PPT1, c.541G>A, p.Val181Met). Discussion Epilepsy in all NCL patients is a major clinical issue and presumed related to neuronal excitation and epileptogenesis. The treatment of status epilepticus, in juvenile CLN1 patients, presents a particular challenge and requires monitoring of potential serious pharmacologic side effects of therapy.

Neuronal Ceroid Lipofuscinoses in Children.

Background:The neuronal ceroid lipofuscinoses (NCL) constitute a group of gray matter neurodegenerative disorders characterized by the accumulation of ceroid lipopigment in lysosomes in neurons and other cell types. There are very few published studies on NCL from India, especially in children.Methods:A retrospective study of confirmed patients of NCL diagnosed over a period of 10 years from January 2019 to December 2019.Results:Fifty children had a definitive diagnosis of NCL based on enzymatic studies or genetic testing using next-generation sequencing. Around 15 children were diagnosed to have CLN-1 (ceroid lipofuscinoses, neuronal-1) based on palmitoyl protein thioesterase-1 deficiency; 24 children were diagnosed with CLN2 (ceroid lipofuscinoses, neuronal-2) based on deficient tripeptidyl-peptidase-1 activity; three patients were diagnosed as CLN6, five patients as CLN7, one case each of CLN8, CLN11, and CLN14 based on genetic testing. Clinical presentation was quite varied and included refractory seizures, developmental delay/regression, and abnormal movements. Visual failure was not common in the present case series. Neuroimaging patterns in different types of NCL were different. All children had a progressive downhill course resulting in death in many over a period of 5–10 years of disease onset.Conclusion:NCL is not uncommon and diagnosis can be suspected based on clinical investigations and neuroimaging findings. Diagnosis can be confirmed by enzymatic assays or genetic testing.

Human INCL fibroblasts display abnormal mitochondrial and lysosomal networks and heightened susceptibility to ROS-induced cell death.

Infantile Neuronal Ceroid Lipofuscinosis (INCL) is a pediatric neurodegenerative disorder characterized by progressive retinal and central nervous system deterioration during infancy. This lysosomal storage disorder results from a deficiency in the Palmitoyl Protein Thioesterase 1 (PPT1) enzyme—a lysosomal hydrolase which cleaves fatty acid chains such as palmitate from lipid-modified proteins. In the absence of PPT1 activity, these proteins fail to be degraded, leading to the accumulation of autofluorescence storage material in the lysosome. The underlying molecular mechanisms leading to INCL pathology remain poorly understood. A role for oxidative stress has been postulated, yet little evidence has been reported to support this possibility. Here we present a comprehensive cellular characterization of human PPT1-deficient fibroblast cells harboring Met1Ile and Tyr247His compound heterozygous mutations. We detected autofluorescence storage material and observed distinct organellar abnormalities of the lysosomal and mitochondrial structures, which supported previous postulations about the role of ER, mitochondria and oxidative stress in INCL. An increase in the number of lysosomal structures was found in INCL patient fibroblasts, which suggested an upregulation of lysosomal biogenesis, and an association with endoplasmic reticulum stress response. The mitochondrial network also displayed abnormal spherical punctate morphology instead of normal elongated tubules with extensive branching, supporting the involvement of mitochondrial and oxidative stress in INCL cell death. Autofluorescence accumulation and lysosomal pathologies can be mitigated in the presence of conditioned wild type media suggesting that a partial restoration via passive introduction of the enzyme into the cellular environment may be possible. We also demonstrated, for the first time, that human INCL fibroblasts have a heightened susceptibility to exogenous reactive oxygen species (ROS)-induced cell death, which suggested an elevated basal level of endogenous ROS in the mutant cell. Collectively, these findings support the role of intracellular organellar networks in INCL pathology, possibly due to oxidative stress.

Electrophysiological Profile Remodeling via Selective Suppression of Voltage-Gated Currents by CLN1/PPT1 Overexpression in Human Neuronal-Like Cells.

CLN1 disease (OMIM #256730) is an inherited neurological disorder of early childhood with epileptic seizures and premature death. It is associated with mutations in CLN1 coding for Palmitoyl-Protein Thioesterase 1 (PPT1), a lysosomal enzyme which affects the recycling and degradation of lipid-modified (S-acylated) proteins by removing palmitate residues. Transcriptomic evidence from a neuronal-like cellular model derived from differentiated SH-SY5Y cells disclosed the potential negative roles of CLN1 overexpression, affecting the elongation of neuronal processes and the expression of selected proteins of the synaptic region. Bioinformatic inquiries of transcriptomic data pinpointed a dysregulated expression of several genes coding for proteins related to voltage-gated ion channels, including subunits of calcium and potassium channels (VGCC and VGKC). In SH-SY5Y cells overexpressing CLN1 (SH-CLN1 cells), the resting potential and the membrane conductance in the range of voltages close to the resting potential were not affected. However, patch-clamp recordings indicated a reduction of Ba2+ currents through VGCC of SH-CLN1 cells; Ca2+ imaging revealed reduced Ca2+ influx in the same cellular setting. The results of the biochemical and morphological investigations of CACNA2D2/α2δ-2, an accessory subunit of VGCC, were in accordance with the downregulation of the corresponding gene and consistent with the hypothesis that a lower number of functional channels may reach the plasma membrane. The combined use of 4-AP and NS-1643, two drugs with opposing effects on Kv11 and Kv12 subfamilies of VGKC coded by the KCNH gene family, provides evidence for reduced functional Kv12 channels in SH-CLN1 cells, consistent with transcriptomic data indicating the downregulation of KCNH4. The lack of compelling evidence supporting the palmitoylation of many ion channels subunits investigated in this study stimulates inquiries about the role of PPT1 in the trafficking of channels to the plasma membrane. Altogether, these results indicate a reduction of functional voltage-gated ion channels in response to CLN1/PPT1 overexpression in differentiated SH-SY5Y cells and provide new insights into the altered neuronal excitability which may underlie the severe epileptic phenotype of CLN1 disease. It remains to be shown if remodeling of such functional channels on plasma membrane can occur as a downstream effect of CLN1 disease.

Enzymatic diagnosis of neuronal lipofuscinoses in dried blood spots using substrates for concomitant tandem mass spectrometry and fluorimetry.

Neuronal ceroid lipofuscinoses (NCLs) are a group of neurodegenerative diseases predominantly in childhood that are characterized by psychomotor deterioration, epilepsy, and early death of patients. The NCLs analyzed in the present study are caused by defects of the specific enzymes, CLN1 (palmitoyl protein thioesterase 1; PPT1), CLN2 (tripeptidyl peptidase 1; TPP1), and CLN10 (cathepsin D). Specific and sensitive diagnostic assays of NCLs were the main goal of this study. They are of increasing importance, particularly since enzyme replacement therapy (ERT) for NCL2 has recently become available for clinical treatment, and ERTs for further NCLs are under development. Here, we report specific and sensitive determinations for CLN1, CLN2, and CLN10 on dried blood spots by tandem mass spectrometry using multiple reaction monitoring mass spectrometry (MRM-MS). Identical substrates suitable for (i) fluorimetric determination of single enzymes and (ii) for MRM-MS determination of multiple enzymes were synthesized by chemical coupling of alkyl-umbelliferone building blocks with the corresponding peptidyl-substrate groups recognized by the target enzyme. Enzymatic determinations were performed both by fluorimetry and MRM-MS in patients with NCL1, NCL2, and NCL10 and showed good agreement in single assays. Moreover, duplex and triplex determinations were successfully performed for NCL1, NCL2, and NCL10. Specific peptidyl-(4-alkyl-umbelliferone) substrates were also synthesized for mass spectrometric determinations of different cathepsins (cathepsins-D, -F, and -B), to provide a differentiation of proteolytic specificities.

Protein Delivery by Peptide-Based Stealth Liposomes: A Biomolecular Insight into Enzyme Replacement Therapy.

Infantile neural ceroid lipofuscinosis (INCL) is a lysosomal storage disorder characterized by mutations in the CLN1 gene that leads to lack of the lysosomal enzyme palmitoyl-protein thioesterase-1 (PPT1), which causes the progressive death of cortical neurons. Enzyme replacement therapy (ERT) is one of the most promising treatments, but its translation toward a clinical use is hampered by the need to deliver the enzyme to the central nervous system and a more detailed understanding of its capability to restore physiologic conditions at the biochemical and protein level, beyond the simple regulation of enzymatic activity. Targeted nanoparticles can promote protein delivery to the central nervous system and affect biological pathways inside cells. Here, we describe an innovative peptide-based stealth nanoparticle that inhibits serum protein adsorption exploiting transferrin-driven internalization to convey the PPT1 enzyme to transferrin receptor-mediated pathways (endocytosis in this work, or transcytosis, in perspective, in vivo). These enzyme-loaded nanoparticles were able to restore stable levels of enzymatic activity in CLN1 patient's fibroblasts, comparable with the free enzyme, demonstrating that delivery after encapsulation in the nanocarrier does not alter uptake or intracellular trafficking. We also investigate, for the first time, dysregulated pathways of proteome and palmitoylome and their alteration upon enzyme delivery. Our nanoparticles were able of halving palmitoylated protein levels restoring conditions similar to the normal cells. From proteomic analysis, we also highlighted the reduction of the different groups of proteins after treatments with the free or encapsulated enzyme. In conclusion, our system is able to deliver the enzyme to a model of CLN1 disease restoring normal conditions in cells. Investigation of molecular details of pathologic state and enzyme-based correction reveals dysregulated pathways with unprecedented details for CLN1. Finally, we unveil for the first time the dysregulation landscape of palmitoylome and proteome in primary patient-derived fibroblasts and their modifications in response to enzyme administration. These findings will provide a guideline for the validation of future therapeutic strategies based on enzyme replacement therapy or acting at different metabolic levels.

Sex- and region-biased depletion of microglia/macrophages attenuates CLN1 disease in mice

BackgroundThe neuronal ceroid lipofuscinoses (CLN diseases) are fatal lysosomal storage diseases causing neurodegeneration in the CNS. We have previously shown that neuroinflammation comprising innate and adaptive immune reactions drives axonal damage and neuron loss in the CNS of palmitoyl protein thioesterase 1-deficient (Ppt1−/−) mice, a model of the infantile form of the diseases (CLN1). Therefore, we here explore whether pharmacological targeting of innate immune cells modifies disease outcome in CLN1 mice.MethodsWe applied treatment with PLX3397 (150 ppm in the chow), a potent inhibitor of the colony stimulating factor-1 receptor (CSF-1R) to target innate immune cells in CLN1 mice. Experimental long-term treatment was non-invasively monitored by longitudinal optical coherence tomography and rotarod analysis, as well as analysis of visual acuity, myoclonic jerks, and survival. Treatment effects regarding neuroinflammation, neural damage, and neurodegeneration were subsequently analyzed by histology and immunohistochemistry.ResultsWe show that PLX3397 treatment attenuates neuroinflammation in CLN1 mice by depleting pro-inflammatory microglia/macrophages. This leads to a reduction of T lymphocyte recruitment, an amelioration of axon damage and neuron loss in the retinotectal system, as well as reduced thinning of the inner retina and total brain atrophy. Accordingly, long-term treatment with the inhibitor also ameliorates clinical outcomes in CLN1 mice, such as impaired motor coordination, visual acuity, and myoclonic jerks. However, we detected a sex- and region-biased efficacy of CSF-1R inhibition, with male microglia/macrophages showing higher responsiveness toward depletion, especially in the gray matter of the CNS. This results in a better treatment outcome in male Ppt1−/− mice regarding some histopathological and clinical readouts and reflects heterogeneity of innate immune reactions in the diseased CNS.ConclusionsOur results demonstrate a detrimental impact of innate immune reactions in the CNS of CLN1 mice. These findings provide insights into CLN pathogenesis and may guide in the design of immunomodulatory treatment strategies.

Palmitoyl Protein Thioesterase 1 Is Essential for Myogenic Autophagy of C2C12 Skeletal Myoblast.

Skeletal muscle differentiation is an essential process for the maintenance of muscle development and homeostasis. Reactive oxygen species (ROS) are critical signaling molecules involved in muscle differentiation. Palmitoyl protein thioesterase 1 (PPT1), a lysosomal enzyme, is involved in removing thioester-linked fatty acid groups from modified cysteine residues in proteins. However, the role of PPT1 in muscle differentiation remains to be elucidated. Here, we found that PPT1 plays a critical role in the differentiation of C2C12 skeletal myoblasts. The expression of PPT1 gradually increased in response to mitochondrial ROS (mtROS) during muscle differentiation, which was attenuated by treatment with antioxidants. Moreover, we revealed that PPT1 transactivation occurs through nuclear factor erythroid 2-regulated factor 2 (Nrf2) binding the antioxidant response element (ARE) in its promoter region. Knockdown of PPT1 with specific small interference RNA (siRNA) disrupted lysosomal function by increasing its pH. Subsequently, it caused excessive accumulation of autophagy flux, thereby impairing muscle fiber formation. In conclusion, we suggest that PPT1 is factor a responsible for myogenic autophagy in differentiating C2C12 myoblasts.

Comparative proteomic profiling reveals mechanisms for early spinal cord vulnerability in CLN1 disease

CLN1 disease is a fatal inherited neurodegenerative lysosomal storage disease of early childhood, caused by mutations in the CLN1 gene, which encodes the enzyme Palmitoyl protein thioesterase-1 (PPT-1). We recently found significant spinal pathology in Ppt1-deficient (Ppt1−/−) mice and human CLN1 disease that contributes to clinical outcome and precedes the onset of brain pathology. Here, we quantified this spinal pathology at 3 and 7 months of age revealing significant and progressive glial activation and vulnerability of spinal interneurons. Tandem mass tagged proteomic analysis of the spinal cord of Ppt1−/−and control mice at these timepoints revealed a significant neuroimmune response and changes in mitochondrial function, cell-signalling pathways and developmental processes. Comparing proteomic changes in the spinal cord and cortex at 3 months revealed many similarly affected processes, except the inflammatory response. These proteomic and pathological data from this largely unexplored region of the CNS may help explain the limited success of previous brain-directed therapies. These data also fundamentally change our understanding of the progressive, site-specific nature of CLN1 disease pathogenesis, and highlight the importance of the neuroimmune response. This should greatly impact our approach to the timing and targeting of future therapeutic trials for this and similar disorders.

PPT1 inhibition enhances the antitumor activity of anti-PD-1 antibody in melanoma.

New strategies are needed to enhance the efficacy of anti–programmed cell death protein antibody (anti–PD-1 Ab) in cancer. Here, we report that inhibiting palmitoyl-protein thioesterase 1 (PPT1), a target of chloroquine derivatives like hydroxychloroquine (HCQ), enhances the antitumor efficacy of anti–PD-1 Ab in melanoma. The combination resulted in tumor growth impairment and improved survival in mouse models. Genetic suppression of core autophagy genes, but not Ppt1, in cancer cells reduced priming and cytotoxic capacity of primed T cells. Exposure of antigen-primed T cells to macrophage-conditioned medium derived from macrophages treated with PPT1 inhibitors enhanced melanoma-specific killing. Genetic or chemical Ppt1 inhibition resulted in M2 to M1 phenotype switching in macrophages. The combination was associated with a reduction in myeloid-derived suppressor cells in the tumor. Ppt1 inhibition by HCQ, or DC661, induced cyclic GMP-AMP synthase/stimulator of interferon genes/TANK binding kinase 1 pathway activation and the secretion of interferon-β in macrophages, the latter being a key component for augmented T cell–mediated cytotoxicity. Genetic Ppt1 inhibition produced similar findings. These data provide the rationale for this combination in melanoma clinical trials and further investigation in other cancers.

Neuronal Ceroid Lipofuscinosis: Clinical and Laboratory Profile in Children from Tertiary Care Centre in South India.

Neuronal ceroid Lipofuscinosis (NCL), inherited disorders of lysosomal storage disorders, constitute the most common progressive encephalopathies with an incidence of 1.3 to 7 in 100,000 live births. We reported clinical, electrophysiological, radiological, ultrastructural, and molecular genetic features of NCL. This is a retrospective review, in a tertiary care center from January 2016 to December 2019. All children with clinical features of NCL and confirmed by pathogenic mutation and/or enzyme assay were included. A total of 60 children (male:female = 3:1) were studied. The commonest type was CLN 2 (41.7%). Neuroregression, seizures, and ataxia were present in all cases. Retinal arterial attenuation was seen in 38.33% cases. Magnetic resonance imaging (MRI) brain was abnormal in all patients, thalamic and caudate nucleus atrophy common in CLN1 (62%). Electroencephalography was abnormal in all children, but photoparoxysmal response at low intermittent photic stimulation frequencies was seen in four children of CLN2. Electron microscopy done in 43 children revealed abnormal inclusions in 20 (46.52%) children. Enzyme study showed low levels in 36 (78%) out of 46 cases. Of these, 21 had low tripeptidyl peptidase and 15 had low palmitoyl protein thioesterase levels. Molecular testing done in 26 cases showed pathogenic variant in 23 (88%) cases. Infantile onset with thalamic atrophy on MRI is common in CLN1 and refractory epilepsy, visual impairment and specific EEG changes are common in CLN2. These features are helpful in selecting enzyme assay for CLN1 versus CLN2. Electron microscopy helped in the diagnosis and genetic testing in subtyping. Thus, a multimode approach played a role in the diagnosis of NCL.

Cln1-mutations suppress Rab7-RILP interaction and impair autophagy contributing to neuropathology in a mouse model of infantile neuronal ceroid lipofuscinosis.

Infantile neuronal ceroid lipofuscinosis (INCL) is a devastating neurodegenerative lysosomal storage disease (LSD) caused by inactivating mutations in the CLN1 gene. CLN1 encodes palmitoyl-protein thioesterase-1 (PPT1), a lysosomal enzyme that catalyzes the deacylation of S-palmitoylated proteins to facilitate their degradation and clearance by lysosomal hydrolases. Despite the discovery more than two decades ago that CLN1 mutations causing PPT1-deficiency underlies INCL, the precise molecular mechanism(s) of pathogenesis has remained elusive. Here, we report that autophagy is dysregulated in Cln1-/- mice, which mimic INCL and in postmortem brain tissues as well as cultured fibroblasts from INCL patients. Moreover, Rab7, a small GTPase, critical for autophagosome-lysosome fusion, requires S-palmitoylation for trafficking to the late endosomal/lysosomal membrane where it interacts with Rab-interacting lysosomal protein (RILP), essential for autophagosome-lysosome fusion. Notably, PPT1-deficiency in Cln1-/- mice, dysregulated Rab7-RILP interaction and preventing autophagosome-lysosome fusion, which impaired degradative functions of the autolysosome leading to INCL pathogenesis. Importantly, treatment of Cln1-/- mice with a brain-penetrant, PPT1-mimetic, small molecule, N-tert (butyl)hydroxylamine (NtBuHA), ameliorated this defect. Our findings reveal a previously unrecognized role of CLN1/PPT1 in autophagy and suggest that small molecules functionally mimicking PPT1 may have therapeutic implications.

Cloning and Expression of Palmitoyl Protein Thioesterases and Acyl Protein Thioesterases for Use in Depalmitoylation Reactions

Palmitoylation is a dynamic process for regulating protein function and localization. It involves the attachment of palmitate to protein cysteine residues by palmitoyl transferases and its removal by palmitoyl protein thioesterase (PPTs) or acyl protein thioesterases (APTs). Techniques to study palmitoylation are difficult to perform and use harsh depalmitoylating reagents like hydroxylamine (HAM), often leading to destruction of the protein being studied. Using recombinant or over‐expressed PPT/APTs could be used as an alternative to HAM for studying protein palmitoylation/depalmitoylation dynamics, as well as investigating PPT/APT substate specificities. We have cloned human APT and PPT cDNA for the production of GST or MBP‐tagged fusion proteins to be used in acyl‐biotin exchange assays. We have also produced mCherry‐tagged versions for co‐expression with a target protein of interest to evaluate their activities. In these experiments, we will focus on the Rho GTPase TCL/RhoJ, which was recently shown to associate with the plasma membrane through palmitoylation. Cherry‐tagged APT/PPTs will be co‐transfected with YFP‐tagged TCL to determine if TCL membrane association diminishes. Overall, the goal of these experiments is to improve techniques for studying protein palmitoylation/depalmitoylation.Support or Funding InformationThis work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).

Characterization of Heterogeneous Metabolism in Hepatocellular Carcinoma Identifies New Therapeutic Target and Treatment Strategy

Background: Metabolic reprogramming is a well-known hallmark of cancer. Systematical identification of clinically relevant metabolic subtypes of Hepatocellular carcinoma (HCC) is critical to understand tumor heterogeneity and develop efficient treatment strategies. Methods: We performed an integrative analysis of genomic, transcriptomic, and clinical data from an HCC patient cohort in The Cancer Genome Atlas (TCGA). In vitro experiments were carried out for preliminary validation. Findings: Four metabolic subtypes were defined: mHCC1, mHHC2, mHCC3, and mHCC4. These subtypes had distinct differences in mutations profiles, activities of metabolic pathways, prognostic metabolism genes, and immune features. The mHCC1 was associated with poorest outcome and was characterized by extensive metabolic alterations, abundant immune infiltration, and increased expression of immunosuppressive checkpoints. The mHHC2 displayed lowest metabolic alteration level and was associated with most significant improvement in overall survival in response to high CD8+ T cell infiltration. The mHHC3 was a “cold-tumor” with low immune infiltration and few metabolic alterations. The mHCC4 presented a medium degree of metabolic alteration and high CTNNB1 mutation rate. Based on our HCC classification and in vitro study, we identified palmitoyl-protein thioesterase 1 (PPT1) was a specific prognostic gene and therapeutic target for mHCC1. Interpretation: Our study highlighted mechanistic differences among metabolic subtypes and identified potential therapeutic targets for subtype-specific treatment strategies targeting unique metabolic vulnerabilities. The immune heterogeneities across metabolic subtypes may help further clarify the association between metabolism and immune environment, and guide the development of novel strategies through targeting both unique metabolic vulnerabilities and immunosuppressive triggers. Funding Statement: This work was financially supported by the Outstanding Youth Scholars Project of Guangdong Provincial People's Hospital (No. KJ012019096). Declaration of Interests: All financial interests are unrelated to this study. All authors declare no competing interests.

The effects of chronic cadmium exposure on Bufo gargarizans larvae: Histopathological impairment, gene expression alteration and fatty acid metabolism disorder in the liver

Cadmium (Cd) a highly toxic metal to human and wildlife health and it is hazardous to both terrestrial and aquatic life. In this study, we used RNA sequencing analysis to examine the effects of chronic cadmium exposure on liver lipid metabolism of Bufo gargarizans larvae. Tadpoles were exposed to cadmium concentrations at 0, 5, 10, 50, 100 and 200 μg L−1 from Gosner stage 26–42 of metamorphic climax. The results showed high dose cadmium (50, 100 and 200 μg L−1) caused obvious histological changes characterized by hepatocytes deformation, nuclear pyknosis, increasing melanomacrophage centers (MMCs) and aggregated lipid droplets. Moreover, transcriptome analysis showed that liver function was seriously affected by cadmium exposure. Furthermore, high dose cadmium significantly upregulated the mRNA expression of elongation of very-long-chain fatty acids 1 (ELOVL1), Mitochondrial trans-2-enoyl-CoA reductase (MECR), Trans-2, 3-enoyl-CoA reductase (TER) and Hydroxysteroid (17β) dehydrogenase type 12 (HSD17B12) which are related with fatty acid synthesis. Meanwhile, mRNA levels of genes related with fat acid oxidation such as acetyl-CoA acyltransferase 2 (ACAA2) and enoyl-coenzyme A (CoA) hydratase short chain 1 (ECHS1) were significantly upregulated while the expression of Acyl-coA thioesterase 1 (ACOT1), 3-hydroxyacyl-CoA dehydrogenase (HADH), Palmitoyl-protein thioesterase 1(PPT1) and Acetyl-CoA acyltransferase 1(ACAA1) was significantly downregulated by high dose cadmium exposure. Furthermore, the mRNA level of ATP-binding cassette subfamily B member 11 (ABCB11) related with bile secretion was significantly decreased exposed to high dose cadmium. Our results suggested cadmium can cause liver dysfunction by inducing histopathological damages, genetic expression alterations and fatty acid metabolism disorder.

Overexpression of PPT2 Represses the Clear Cell Renal Cell Carcinoma Progression by Reducing Epithelial-to-mesenchymal Transition.

Clear cell renal cell carcinoma (ccRCC) is one of the most common malignant tumors of the urinary system and has a poor response to radiotherapy and chemotherapy. To date, it is urgent to find effective biomarkers for the prevention and treatment of ccRCC. The occurrence and development of ccRCC is closely related to metabolic disturbances. Palmitoyl protein thioesterase 2 (PPT2) is a lysosomal thioesterase which is highly associated with metabolism, and it has never been studied in ccRCC. In this study, we first revealed PPT2 is significantly downregulated in ccRCC, and its expression level is highly correlated with clinicopathological parameters of ccRCC patients. Our ROC curve analyses evaluated the potential of PPT2 as a novel diagnostic marker and prognostic factor. Functional experiment results showed overexpression of PPT2 represses the proliferation, migration and invasion of ccRCC cells in vitro. Mechanistic investigations demonstrated that overexpression of PPT2 represses the ccRCC progression by reducing epithelial-to-mesenchymal transition (EMT). In conclusion, PPT2 is downregulated in ccRCC. Decreased PPT2 expression may be considered as a novel diagnostic marker and prognostic factor and serve as a therapeutic target for ccRCC.