Investigating USP42 Mutation as Underlying Cause of Familial Non-Medullary Thyroid Carcinoma.

In a family with Familial Non-Medullary Thyroid Carcinoma (FNMTC), our investigation using Whole-Exome Sequencing (WES) uncovered a novel germline USP42 mutation [p.(Gly486Arg)]. USP42 is known for regulating p53, cell cycle arrest, and apoptosis, and for being reported as overexpressed in breast and gastric cancer patients. Recently, a USP13 missense mutation was described in FNMTC, suggesting a potential involvement in thyroid cancer. Aiming to explore the USP42 mutation as an underlying cause of FNMTC, our team validated the mutation in blood and tissue samples from the family. Using immunohistochemistry, the expression of USP42, Caspase-3, and p53 was assessed. The USP42 gene was silenced in human thyroid Nthy-Ori 3-1 cells using siRNAs. Subsequently, expression, viability, and morphological assays were conducted. p53, Cyclin D1, p21, and p27 proteins were evaluated by Western blot. USP42 protein was confirmed in all family members and was found to be overexpressed in tumor samples, along with an increased expression of p53 and cleaved Caspase-3. siRNA-mediated USP42 downregulation in Nthy-Ori 3-1 cells resulted in reduced cell viability, morphological changes, and modifications in cell cycle-related proteins. Our results suggest a pivotal role of USP42 mutation in thyroid cell biology, and this finding indicates that USP42 may serve as a new putative target in FNMTC.

Disruption of lysosomal nutrient sensing scaffold contributes to pathogenesis of a fatal neurodegenerative lysosomal storage disease.

The ceroid lipofuscinosis neuronal 1 (CLN1) disease, formerly called infantile neuronal ceroid lipofuscinosis, is a fatal hereditary neurodegenerative lysosomal storage disorder. This disease is caused by loss-of-function mutations in the CLN1 gene, encoding palmitoyl-protein thioesterase-1 (PPT1). PPT1 catalyzes depalmitoylation of S-palmitoylated proteins for degradation and clearance by lysosomal hydrolases. Numerous proteins, especially in the brain, require dynamic S-palmitoylation (palmitoylation-depalmitoylation cycles) for endosomal trafficking to their destination. While 23 palmitoyl-acyl transferases in the mammalian genome catalyze S-palmitoylation, depalmitoylation is catalyzed by thioesterases such as PPT1. Despite these discoveries, the pathogenic mechanism of CLN1 disease has remained elusive. Here, we report that in the brain of Cln1-/- mice, which mimic CLN1 disease, the mechanistic target of rapamycin complex-1 (mTORC1) kinase is hyperactivated. The activation of mTORC1 by nutrients requires its anchorage to lysosomal limiting membrane by Rag GTPases and Ragulator complex. These proteins form the lysosomal nutrient sensing scaffold to which mTORC1 must attach to activate. We found that in Cln1-/- mice, two constituent proteins of the Ragulator complex (vacuolar (H+)-ATPase and Lamtor1) require dynamic S-palmitoylation for endosomal trafficking to the lysosomal limiting membrane. Intriguingly, Ppt1 deficiency in Cln1-/- mice misrouted these proteins to the plasma membrane disrupting the lysosomal nutrient sensing scaffold. Despite this defect, mTORC1 was hyperactivated via the IGF1/PI3K/Akt-signaling pathway, which suppressed autophagy contributing to neuropathology. Importantly, pharmacological inhibition of PI3K/Akt suppressed mTORC1 activation, restored autophagy, and ameliorated neurodegeneration in Cln1-/- mice. Our findings reveal a previously unrecognized role of Cln1/Ppt1 in regulating mTORC1 activation and suggest that IGF1/PI3K/Akt may be a targetable pathway for CLN1 disease.

USP11 promotes glycolysis by regulating HIF-1α stability in hepatocellular carcinoma.

Understanding the mechanisms underlying metastasis in hepatocellular carcinoma (HCC) is crucial for developing new therapies against this fatal disease. Deubiquitinase ubiquitin-specific protease 11 (USP11) belongs to the deubiquitinating family and has previously been reported to play a critical role in cancer pathogenesis. Although it has been established that USP11 can facilitate the metastasis and proliferation ability of HCC, the underlying regulatory mechanisms are poorly understood. The primary objective of this research was to reveal hitherto undocumented functions of USP11 during HCC progression, especially those related to metabolism. Under hypoxic conditions, USP11 was found to significantly impact the glycolysis of HCC cells, as demonstrated through various techniques, including RNA-Seq, migration and colony formation assays, EdU and co-immunoprecipitation. Interestingly, we found that USP11 interacted with the HIF-1α complex and maintained HIF-1α protein stability by removing ubiquitin. Moreover, USP11/HIF-1α could promote glycolysis through the PDK1 and LDHA pathways. In general, our results demonstrate that USP11 promotes HCC proliferation and metastasis through HIF-1α/LDHA-induced glycolysis, providing new insights and the experimental basis for developing new treatments for this patient population.

Repurposing the inhibitors of MMP-9 and SGLT-2 against ubiquitin specific protease 30 in Parkinson's disease: computational modelling studies.

Ubiquitin specific protease 30 (USP30) has been attributed to mitochondrial dysfunction and impediment of mitophagy in Parkinson's disease (PD). This happens once ubiquitin that supposed to bind with deformed mitochondria at the insistence of Parkin, it's been recruited by USP30 via the distal ubiquitin binding domain. This is a challenge when PINK1 and Parkin loss their functions due to mutation. Although, there are reports on USP30s' inhibitors but no study on the repurposing of inhibitors approved against MMP-9 and SGLT-2 as potential inhibitors of USP30 in PD. Thus, the highlight therein, is to repurpose approved inhibitors of MMP-9 and SGLT-2 against USP30 in PD using extensive computational modelling framework. 3D structures of Ligands and USP30 were obtained from PubChem and protein database (PDB) servers respectively, and were subjected to molecular docking, ADMET evaluation, DFT calculation, molecular dynamics simulation (MDS) and free energy calculations. Out of the 18 drugs, 2 drugs showed good binding affinity to the distal ubiquitin binding domain, moderate pharmacokinetic properties and good stability. The findings showed canagliflozin and empagliflozin as potential inhibitors of USP30. Thus, we present these drugs as repurposing candidates for the treatment of PD. However, the findings in this current study needs to be validated experimentally.Communicated by Ramaswamy H. Sarma.

USP11 potentiates HGF/AKT signaling and drives metastasis in hepatocellular carcinoma.

USP11 is a member of the ubiquitin-specific protease family and plays a crucial role in tumor progression in various cancers. However, the precise mechanism by which USP11 promotes EMT and metastasis in hepatocellular carcinoma (HCC) is not fully understood. In this study, we demonstrated that the USP11 expression was dramatically upregulated in HCC tissues and cell lines. Increased USP11 expression was closely associated with tumor number, vascular invasion, and poor prognosis. Functional experiments demonstrated that USP11 markedly promoted metastasis and EMT in HCC via induction of the transcription factor Snail. Mechanistically, USP11 interacted with and deubiquitinated eEF1A1 on Lys439, thereby inhibiting its ubiquitin-mediated degradation. Subsequently, the elevated expression of eEF1A1 resulted in its binding to SP1, which in turn drove the binding of SP1 to its target HGF gene promoter to increase its transcription. This led to an enhanced expression of HGF and the activation of the downstream PI3K/AKT signaling pathway. We demonstrated that USP11 promotes EMT and metastasis in HCC via eEF1A1/SP1/HGF dependent-EMT. Our findings suggest that the USP11/ eEF1A1/SP1/HGF axis contributes to metastasis in HCC, and therefore, could be considered as a potential therapeutic target for the treatment of HCC.

Knockout or inhibition of USP30 protects dopaminergic neurons in a Parkinson's disease mouse model.

Mutations in SNCA, the gene encoding α-synuclein (αSyn), cause familial Parkinson's disease (PD) and aberrant αSyn is a key pathological hallmark of idiopathic PD. This α-synucleinopathy leads to mitochondrial dysfunction, which may drive dopaminergic neurodegeneration. PARKIN and PINK1, mutated in autosomal recessive PD, regulate the preferential autophagic clearance of dysfunctional mitochondria ("mitophagy") by inducing ubiquitylation of mitochondrial proteins, a process counteracted by deubiquitylation via USP30. Here we show that loss of USP30 in Usp30 knockout mice protects against behavioral deficits and leads to increased mitophagy, decreased phospho-S129 αSyn, and attenuation of SN dopaminergic neuronal loss induced by αSyn. These observations were recapitulated with a potent, selective, brain-penetrant USP30 inhibitor, MTX115325, with good drug-like properties. These data strongly support further study of USP30 inhibition as a potential disease-modifying therapy for PD.

Hepatic palmitoyl-proteomes and acyl-protein thioesterase protein proximity networks link lipid modification and mitochondria.

Acyl-protein thioesterases 1 and 2 (APT1 and APT2) reverse S-acylation, a potential regulator of systemic glucose metabolism in mammals. Palmitoylation proteomics in liver-specific knockout mice shows that APT1 predominates over APT2, primarily depalmitoylating mitochondrial proteins, including proteins linked to glutamine metabolism. miniTurbo-facilitated determination of the protein-protein proximity network of APT1 and APT2 in HepG2 cells reveals APT proximity networks encompassing mitochondrial proteins including the major translocases Tomm20 and Timm44. APT1 also interacts with Slc1a5 (ASCT2), the only glutamine transporter known to localize to mitochondria. High-fat-diet-fed male mice with dual (but not single) hepatic deletion of APT1 and APT2 have insulin resistance, fasting hyperglycemia, increased glutamine-driven gluconeogenesis, and decreased liver mass. These data suggest that APT1 and APT2 regulation of hepatic glucose metabolism and insulin signaling is functionally redundant. Identification of substrates and protein-protein proximity networks for APT1 and APT2 establishes a framework for defining mechanisms underlying metabolic disease.

Progress on lysosomal PPT1-mediated regulation of cellular homeostasis and pathogenesis.

Palmitoyl protein thioesterase 1(PPT1) is a lysosomal enzyme that catalyzes the protein depalmitoylation. It is considered to play a crucial role in regulating lysosomes, mitochondria and lipid metabolism. PPT1 has been reported to play an important role in the occurrence and progression of diseases, such as neurological diseases and cancers. However, the regulatory mechanisms remain unknown. In this review, we summarize the progress of PPT1 function and mechanisms in neurological disorders and cancers, which will provide as reference and guidance for exploring the regulatory mechanisms of PPT1 and developing new drugs for treating related diseases in the future.

Loss of Depalmitoylation Disrupts Homeostatic Plasticity of AMPARs in a Mouse Model of Infantile Neuronal Ceroid Lipofuscinosis.

Protein palmitoylation is the only reversible post-translational lipid modification. Palmitoylation is held in delicate balance by depalmitoylation to precisely regulate protein turnover. While over 20 palmitoylation enzymes are known, depalmitoylation is conducted by fewer enzymes. Of particular interest is the lack of the depalmitoylating enzyme palmitoyl-protein thioesterase 1 (PPT1) that causes the devastating pediatric neurodegenerative condition infantile neuronal ceroid lipofuscinosis (CLN1). While most of the research on Ppt1 function has centered on its role in the lysosome, recent findings demonstrated that many Ppt1 substrates are synaptic proteins, including the AMPA receptor (AMPAR) subunit GluA1. Still, the impact of Ppt1-mediated depalmitoylation on synaptic transmission and plasticity remains elusive. Thus, the goal of the present study was to use the Ppt1 -/- mouse model (both sexes) to determine whether Ppt1 regulates AMPAR-mediated synaptic transmission and plasticity, which are crucial for the maintenance of homeostatic adaptations in cortical circuits. Here, we found that basal excitatory transmission in the Ppt1 -/- visual cortex is developmentally regulated and that chemogenetic silencing of the Ppt1 -/- visual cortex excessively enhanced the synaptic expression of GluA1. Furthermore, triggering homeostatic plasticity in Ppt1 -/- primary neurons caused an exaggerated incorporation of GluA1-containing, calcium-permeable AMPARs, which correlated with increased GluA1 palmitoylation. Finally, Ca2+ imaging in awake Ppt1 -/- mice showed visual cortical neurons favor a state of synchronous firing. Collectively, our results elucidate a crucial role for Ppt1 in AMPAR trafficking and show that impeded proteostasis of palmitoylated synaptic proteins drives maladaptive homeostatic plasticity and abnormal recruitment of cortical activity in CLN1.SIGNIFICANCE STATEMENT Neuronal communication is orchestrated by the movement of receptors to and from the synaptic membrane. Protein palmitoylation is the only reversible post-translational lipid modification, a process that must be balanced precisely by depalmitoylation. The significance of depalmitoylation is evidenced by the discovery that mutation of the depalmitoylating enzyme palmitoyl-protein thioesterase 1 (Ppt1) causes severe pediatric neurodegeneration. In this study, we found that the equilibrium provided by Ppt1-mediated depalmitoylation is critical for AMPA receptor (AMPAR)-mediated plasticity and associated homeostatic adaptations of synaptic transmission in cortical circuits. This finding complements the recent explosion of palmitoylation research by emphasizing the necessity of balanced depalmitoylation.

RBM15 m6 A modification-mediated OTUB2 upregulation promotes cervical cancer progression via the AKT/mTOR signaling.

Cervical cancer (CC) is a deadly gynecological tumor worldwide. Otubain 2 (OTUB2) has been recently identified as an oncogene in human malignancies. However, its expression and function remain unclear. This work aims to explore the role of OTUB2 in CC progression. Herein, The Cancer Genome Atlas data revealed that OTUB2 expression was significantly upregulated in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) and gradually increased with CESC progression; moreover, OTUB2 expression predicted poor outcomes of CESC patients. Then, RT-qPCR and Western blotting were applied to determine mRNA and protein expression in CC and normal cells. Our results confirmed that OTUB2 was highly expressed in CC cell lines. As indicated by CCK-8, Transwell, and flow cytometry results, OTUB2 silencing attenuated proliferative and metastatic capacities of CC cells but promoted CC cell apoptosis. Then, RBM15, an N6-methyladenosine (m6 A) methyltransferase "writer," was also demonstrated to be upregulated in CESC and CC cells. Mechanistically, m6 A RNA immunoprecipitation (Me-RIP) results showed that RBM15 inhibition reduced the m6 A methylation level of OTUB2 in CC cells, leading to the decline of OTUB2 expression. In addition, OTUB2 inhibition deactivated the AKT/mTOR signaling in CC cells. Furthermore, SC-79 (AKT/mTOR activator) partially abated the inhibitory effects of OTUB2 knockdown on the AKT/mTOR signaling pathway and the malignant phenotypes of CC cells. In summary, this work showed that RBM15-mediated m6 A modification led to OTUB2 upregulation, thereby promoting malignant behaviors of CC cells via the AKT/mTOR signaling pathway.

Identification of PPT1 as a lysosomal core gene with prognostic value in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most frequent cancer worldwide with a poor prognosis. Unfortunately, there are few reports on effective biomarkers for HCC, identification of novel cancer targets is urgently needed. Lysosomes are central organelles for degradation and recycling processes in cells, and how lysosome-related genes are involved in the progression of hepatocellular carcinoma remains unclear. The aim of the present study was to identify key lysosome-related genes affecting HCC. In the present study, lysosome-related genes involved in HCC progression were screened based on the TCGA (The Cancer Genome Atlas) dataset. Differentially expressed genes (DEGs) were screened, and core lysosomal genes were obtained in combination with prognostic analysis and protein interaction networks. Two genes were associated with survival, and their prognostic value was validated by prognostic profiling. After mRNA expression validation and IHC, the palmitoyl protein thioesterase 1 (PPT1) gene was identified as an important lysosomal-related gene. We demonstrated that PPT1 promotes the proliferation of HCC cells in vitro. In addition, quantitative proteomics and bioinformatics analysis confirmed that PPT1 acts by affecting the metabolism, localization, and function of various macromolecular proteins. The present study reveals that PPT1 could be a promising therapeutic target for the treatment of HCC. These findings provided new insights into HCC and identified candidate gene prognosis signatures for HCC.

YY1-induced LncRNA-TUG1 elevates YOD1 to promote cell proliferation and inhibit bortezomib sensitivity in multiple myeloma.

Taurine upregulated gene 1 (TUG1) has been implicated in the onset and progression of various malignancies. The current study aimed to evaluate the biological function and potential mechanisms of TUG1 in multiple myeloma (MM) progression. TUG1 knockdown in MM cells was investigated in vitro and in vivo to evaluate the role of TUG1. We also predicted the transcription factor (TF) that bound to TUG1 together with the downstream target genes of the TUG1-TF interaction, and evaluated the regulatory mechanism of TUG1 in cell assays. TUG1 knockdown reduced the cell's proliferative and migratory capabilities while increasing apoptosis and bortezomib sensitivity in vitro and inhibiting tumorigenesis in vivo. TUG1 was found in the nucleus of MM cells and was found to be positively regulated by the TF-YY1. Further in vitro mechanistic investigations indicated that the YY1-TUG1 complex targeted YOD1 to regulate MM progression.

Evaluation of strategies to narrow the product chain-length distribution of microbially synthesized free fatty acids.

The dominant strategy for tailoring the chain-length distribution of free fatty acids (FFA) synthesized by heterologous hosts is expression of a selective acyl-acyl carrier protein (ACP) thioesterase. However, few of these enzymes can generate a precise (greater than 90% of a desired chain-length) product distribution when expressed in a microbial or plant host. The presence of alternative chain-lengths can complicate purification in situations where blends of fatty acids are not desired. We report the assessment of several strategies for improving the dodecanoyl-ACP thioesterase from the California bay laurel to exhibit more selective production of medium-chain free fatty acids to near exclusivity. We demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) was an effective library screening technique for identification of thioesterase variants with favorable shifts in chain-length specificity. This strategy proved to be a more effective screening technique than several rational approaches discussed herein. With this data, we isolated four thioesterase variants which exhibited a more selective FFA distribution over wildtype when expressed in the fatty acid accumulating E. coli strain, RL08. We then combined mutations from the MALDI isolates to generate BTE-MMD19, a thioesterase variant capable of producing free fatty acids consisting of 90% of C12 products. Of the four mutations which conferred a specificity shift, we noted that three affected the shape of the binding pocket, while one occurred on the positively charged acyl carrier protein landing pad. Finally, we fused the maltose binding protein (MBP) from E. coli to the N - terminus of BTE-MMD19 to improve enzyme solubility and achieve a titer of 1.9 g per L of twelve-carbon fatty acids in a shake flask.

High-content phenotypic screen to identify small molecule enhancers of Parkin-dependent ubiquitination and mitophagy.

Mitochondrial dysfunction and aberrant mitochondrial homeostasis are key aspects of Parkinson's disease (PD) pathophysiology. Mutations in PINK1 and Parkin proteins lead to autosomal recessive PD, suggesting that defective mitochondrial clearance via mitophagy is key in PD etiology. Accelerating the identification and/or removal of dysfunctional mitochondria could therefore provide a disease-modifying approach to treatment. To that end, we performed a high-content phenotypic screen (HCS) of ∼125,000 small molecules to identify compounds that positively modulate mitochondrial accumulation of the PINK1-Parkin-dependent mitophagy initiation marker p-Ser65-Ub in Parkin haploinsufficiency (Parkin +/R275W) human fibroblasts. Following confirmatory counter-screening and orthogonal assays, we selected compounds of interest that enhance mitophagy-related biochemical and functional endpoints in patient-derived fibroblasts. Identification of inhibitors of the ubiquitin-specific peptidase and negative regulator of mitophagy USP30 within our hits further validated our approach. The compounds identified in this work provide a novel starting point for further investigation and optimization.

An angiogenesis­related lncRNA signature for the prognostic prediction of patients with bladder cancer and LINC02321 promotes bladder cancer progression via the VEGFA signaling pathway.

The mechanism underlying bladder cancer metastasis is associated with tumor angiogenesis. The present study aimed to evaluate the predictive role and value of an angiogenesis­associated long non­coding (lnc)RNA signature in patients with bladder cancer and the role of long intergenic non­coding RNA (LINC)02321 in the progression of this malignancy. Angiogenesis­related lncRNAs were screened using Pearson correlation analysis and the signaturewas constructed using Cox regression analysis and evaluated using the receiver operating characteristic curve. LINC02321, which expressed the largest difference in bladder cancer, was screened using reverse transcription­quantitative PCR. The role of LINC02321 in the malignant progression of bladder cancer was evaluated using Transwell, wound healing and Cell Counting Kit 8 assays. A total of six angiogenesis­associated lncRNAs (USP30­AS1, LINC02321, PSMB8­AS1, KRT7­AS, LINC01767 and OCIAD1­AS1) were identified as candidates for the prognostic signature using Cox regression analysis. The overall survival of patients in the low­risk group was significantly longer compared with that in the high­risk group, with the highest area under the curve value being 0.807. A nomogram was constructed based on the traditional clinical indicators (age, sex, grade, American Joint Committee on Cancer stage) and risk score of patients. Compared with the traditional clinical indicators, the risk score demonstrated better clinical prediction capacity for predicting the prognosis of patients with bladder cancer. The Cancer Genome Atlas prediction and RT­qPCR experimental results demonstrated that only LINC02321 was highly expressed in bladder cancer tissue and promoted the proliferation, invasion, migration and cisplatin resistance of the malignancy. Gene set enrichment, Pearson's correlation analysis and experimental results demonstrated that the VEGFA signalling pathway may be involved in the LINC02321­regulated progression of bladder cancer. In conclusion, the six angiogenesis­associated lncRNA signatures reported in the present study may be used to predict the prognosis of patients with bladder cancer, and LINC02321 promoted malignant progression of bladder cancer via the VEGFA signalling pathway.

Identification and Functional Characterization of Acyl-ACP Thioesterases B (GhFatBs) Responsible for Palmitic Acid Accumulation in Cotton Seeds.

In spite of increasing use in the food industry, high relative levels of palmitic acid (C16:0) in cottonseed oil imposes harmful effects on human health when overconsumed in the diet. The limited understanding of the mechanism in controlling fatty acid composition has become a significant obstacle for breeding novel cotton varieties with high-quality oil. Fatty acyl-acyl carrier protein (ACP) thioesterase B (FatBs) are a group of enzymes which prefer to hydrolyze the thioester bond from saturated acyl-ACPs, thus playing key roles in controlling the accumulation of saturated fatty acids. However, FatB members and their roles in cotton are largely unknown. In this study, a genome-wide characterization of FatB members was performed in allotetraploid upland cotton, aiming to explore the GhFatBs responsible for high accumulations of C16:0 in cotton seeds. A total of 14 GhFatB genes with uneven distribution on chromosomes were identified from an upland cotton genome and grouped into seven subfamilies through phylogenetic analysis. The six key amino acid residues (Ala, Trys, Ile, Met, Arg and Try) responsible for substrate preference were identified in the N-terminal acyl binding pocket of GhFatBs. RNA-seq and qRT-PCR analysis revealed that the expression profiles of GhFatB genes varied in multiple cotton tissues, with eight GhFatBs (GhA/D-FatB3, GhA/D-FatB4, GhA/D-FatB5, and GhA/D-FatB7) having high expression levels in developing seeds. In particular, expression patterns of GhA-FatB3 and GhD-FatB4 were positively correlated with the dynamic accumulation of C16:0 during cotton seed development. Furthermore, heterologous overexpression assay of either GhA-FatB3 or GhD-FatB4 demonstrated that these two GhFatBs had a high substrate preference to 16:0-ACP, thus contributing greatly to the enrichment of palmitic acid in the tested tissues. Taken together, these findings increase our understanding on fatty acid accumulation and regulation mechanisms in plant seeds. GhFatBs, especially GhA-FatB3 and GhD-FatB4, could be molecular targets for genetic modification to reduce palmitic acid content or to optimize fatty acid profiles in cotton and other oil crops required for the sustainable production of healthy edible oil.

X-linked ubiquitin-specific peptidase 11 increases tauopathy vulnerability in women.

Although women experience significantly higher tau burden and increased risk for Alzheimer's disease (AD) than men, the underlying mechanism for this vulnerability has not been explained. Here, we demonstrate through in vitro and in vivo models, as well as human AD brain tissue, that X-linked ubiquitin specific peptidase 11 (USP11) augments pathological tau aggregation via tau deubiquitination initiated at lysine-281. Removal of ubiquitin provides access for enzymatic tau acetylation at lysines 281 and 274. USP11 escapes complete X-inactivation, and female mice and people both exhibit higher USP11 levels than males. Genetic elimination of usp11 in a tauopathy mouse model preferentially protects females from acetylated tau accumulation, tau pathology, and cognitive impairment. USP11 levels also strongly associate positively with tau pathology in females but not males. Thus, inhibiting USP11-mediated tau deubiquitination may provide an effective therapeutic opportunity to protect women from increased vulnerability to AD and other tauopathies.

Transcription factor AP2 enhances malignancy of non-small cell lung cancer through upregulation of USP22 gene expression.

BACKGROUND: Ubiquitin-specific protease 22 (USP22), a putative cancer stem cell marker, is frequently upregulated in cancers, and USP22 overexpression is associated with aggressive growth, metastasis, and therapy resistance in various human cancers including lung cancer. However, USP22 gene amplification seldom occurs, and the mechanism underlying USP22 upregulation in human cancers remains largely unknown. METHODS: A luciferase reporter driven by a promoter region of USP22 gene was selectively constructed to screen against a customized siRNA library targeting 89 selected transcription factors to identify potential transcription factors (TFs) that regulate USP22 expression in human non-small cell lung cancers (NSCLC). Association of identified TFs with USP22 and potential role of the TFs were validated and explored in NSCLC by biological assays and immunohistochemistry analysis. RESULTS: Luciferase reporter assays revealed that SP1 and activating transcription factor 3 (ATF3) inhibit USP22 transcription, while transcription factor AP-2 Alpha/Beta (TFAP2A/2B) and c-Myc promote USP22 transcription. Binding site-directed mutagenesis and chromosome immunoprecipitation (ChIP) assays validated AP2α and AP2ß are novel TFs of USP22. Furthermore, overexpression of AP2A and AP2B significantly upregulates USP22 expression, and its target: Cyclin D1, concurrently enhances the proliferation, migration, and invasion of NSCLC A549 and H1299 cells in a partially USP22-dependent manner. Moreover, AP2 protein level correlated with USP22 protein in human NSCLC tissues. CONCLUSION: Our findings indicate AP2α and AP2ß are important transcription factors driving USP22 gene expression to promote the progression of NSCLC, and further support USP22 as a potential biomarker and therapeutic target for lung cancer. Video Abstract.

Computational and structural investigation of Palmitoyl-Protein Thioesterase 1 (PPT1) protein causing Neuronal Ceroid Lipofuscinoses (NCL).

The Neuronal Ceroid Lipofuscinoses (NCL) are a group of progressive neurodegenerative disorders, associated with 14 Ceroid Lipofuscinosis Neuronal genes (CLN1-14). The mutations in the Palmitoyl-Protein Thioesterase 1 (PPT1) protein serve as one of the major reasons for the causative of NCL. The PPT1 involves degrading and modifying cysteine residues in proteins or peptides by removing thioester-linked fatty acyl groups like palmitate prefers acyl chains of 14-18 carbons in length. In this study, we have analyzed the impact of PPT1 mutations on the deleteriousness, stability, conservative nature of amino acid, and impact of mutations on the protein structure. We have also used molecular dynamics simulations using GROMACS to perceive the alteration in the dynamic behavior of the PPT1 at the residual level. In this study, we have retrieved 23 PPT1 mutations from the UniProt database, and these were subjected to a series of analyses using varied computer algorithms. From these analyses, out of 23 mutations, 16 mutations were identified as deleterious. Among 16, eight mutations were identified to destabilize the protein structure, and finally, two mutations (W38C and L222P) were found to be positioned in the highly conserved region. The structural impact study observed that the mutant proline could disrupt the alpha helix formed by the leucine at position 222. Finally, from the molecular dynamics simulations, we observed that due to the mutations (W38C and L222P), the protein had experienced higher deviation, fluctuation, and lower compactness. These structural changes elucidate that these mutations can impact the structure and function of the PPT1 protein.

Escherichia coli YigI is a Conserved Gammaproteobacterial Acyl-CoA Thioesterase Permitting Metabolism of Unusual Fatty Acid Substrates.

Thioesterases play a critical role in metabolism, membrane biosynthesis, and overall homeostasis for all domains of life. In this present study, we characterize a putative thioesterase from Escherichia coli MG1655 and define its role as a cytosolic enzyme. Building on structure-guided functional predictions, we show that YigI is a medium- to long-chain acyl-CoA thioesterase that is involved in the degradation of conjugated linoleic acid (CLA) in vivo, showing overlapping specificity with two previously defined E. coli thioesterases TesB and FadM. We then bioinformatically identify the regulatory relationships that induce YigI expression, which include: an acidic environment, high oxygen availability, and exposure to aminoglycosides. Our findings define a role for YigI and shed light on why the E. coli genome harbors numerous thioesterases with closely related functions. IMPORTANCE Previous research has shown that long chain acyl-CoA thioesterases are needed for E. coli to grow in the presence of carbon sources such as conjugated linoleic acid, but that E. coli must possess at least one such enzyme that had not previously been characterized. Building off structure-guided function predictions, we showed that the poorly annotated protein YigI is indeed the previously unidentified third acyl CoA thioesterase. We found that the three potentially overlapping acyl-CoA thioesterases appear to be induced by nonoverlapping conditions and use that information as a starting point for identifying the precise reactions catalyzed by each such thioesterase, which is an important prerequisite for their industrial application and for more accurate metabolic modeling of E. coli.