Crosstalk in extrahepatic and hepatic system in NAFLD/NASH.

Non-alcoholic fatty liver disease (NAFLD) has emerged as the most prevalent chronic liver disease globally. Non-alcoholic steatohepatitis (NASH) represents an extremely progressive form of NAFLD, which, without timely intervention, may progress to cirrhosis or hepatocellular carcinoma. Presently, a definitive comprehension of the pathogenesis of NAFLD/NASH eludes us, and pharmacological interventions targeting NASH specifically remain constrained. The aetiology of NAFLD encompasses a myriad of external factors including environmental influences, dietary habits and gender disparities. More significantly, inter-organ and cellular interactions within the human body play a role in the development or regression of the disease. In this review, we categorize the influences affecting NAFLD both intra- and extrahepatically, elaborating meticulously on the mechanisms governing the onset and progression of NAFLD/NASH. This exploration delves into progress in aetiology and promising therapeutic targets. As a metabolic disorder, the development of NAFLD involves complexities related to nutrient metabolism, liver-gut axis interactions and insulin resistance, among other regulatory functions of extraneous organs. It further encompasses intra-hepatic interactions among hepatic cells, Kupffer cells (KCs) and hepatic stellate cells (HSCs). A comprehensive understanding of the pathogenesis of NAFLD/NASH from a macroscopic standpoint is instrumental in the formulation of future therapies for NASH.

Polysaccharide extracted from Sarcandra glabra residue attenuate cognitive impairment by regulating gut microbiota in diabetic mice.

Diabetic encephalopathy (DE), characterized by cognitive impairment, currently lacks targeted treatment. Previous studies have shown that Sarcandra glabra extracted residue polysaccharide (SERP) exhibited hypoglycemic effects either in vitro or in streptozotocin-induced diabetes mice. However, the therapeutic effect of SERP on DE was not elucidated. This study investigated the therapeutic effect of SERP on DE and its underlying mechanism. Our results revealed that SERP regulates glucose and lipid metabolism, improves cognitive function, and exhibits diminished activity post-antibiotic intervention. Importantly, we discovered a novel mechanism by which SERP modulates the gut microbiota, specifically enriching Bacteroidales S24-7, resulting in elevated levels of butyric acid in the intestine. This regulation modulates the intestinal endocrine cell lipid metabolism level, restores damaged intestinal barriers and neural epithelial circuits, thus exhibiting cure effects. Our findings suggest that SERP could become a candidate for treating DE, potentially involving the regulation mechanism of the "microbiota-gut-brain axis". This study underscores the unique therapeutic efficacy of SERP in managing DE, offering fresh drug candidates and innovative treatment strategies for this challenging condition.

Enhanced bioactivity and stability of a long-acting FGF21: A novel variant for the treatment of NASH.

Fibroblast growth factor 21 (FGF21) is pivotal in regulating energy metabolism, highlighting substantial therapeutic potential for non-alcoholic steatohepatitis (NASH). Previously, we reported a long-acting FGF21 fusion protein, PsTag-FGF21, which was prepared by genetically fusing human FGF21 with a 648-residue polypeptide (PsTag). While this fusion protein demonstrated therapeutic efficacy against NASH, our final product analysis revealed the presence of fixed impurities resistant to effective removal, indicating potential degradation of PsTag-FGF21. Here, we enriched and analyzed the impurities, confirming our hypothesis regarding the C-terminal degradation of PsTag-FGF21. We now describe a new variant developed to eliminate the C-terminal degradation. By introducing one mutation located at the C-terminal of PsTag-FGF21(V169L), we demonstrated that the new molecule, PsTag-FGF21(V169L), exhibits many improved attributes. Compared with PsTag-FGF21, PsTag-FGF21(V169L) displayed elevated bioactivity and stability, along with a twofold enhanced binding affinity to the coreceptor ß-Klotho. In vivo, the circulating half-life of PsTag-FGF21(V169L) was further enhanced compared with that of PsTag-FGF21. In NASH mice, PsTag-FGF21(V169L) demonstrated efficacy with sustained improvements in multiple metabolic parameters. Besides, PsTag-FGF21(V169L) demonstrated the ability to alleviate NASH by decreasing hepatocyte apoptosis. The superior biophysical, pharmacokinetic, and pharmacodynamic properties, along with the positive metabolic effects, imply that further clinical development of PsTag-FGF21(V169L) as a metabolic therapy for NASH patients may be warranted.

A long-acting FGF21 attenuates metabolic dysfunction-associated steatohepatitis-related fibrosis by modulating NR4A1-mediated Ly6C phenotypic switch in macrophages.

BACKGROUND AND PURPOSE: Because of the absence of effective therapies for metabolic dysfunction-associated steatohepatitis (MASH), there is a rising interest in fibroblast growth factor 21 (FGF21) analogues due to their potential anti-fibrotic activities in MASH treatment. PsTag-FGF21, a long-acting FGF21 analogue, has demonstrated promising therapeutic effects in several MASH mouse models. However, its efficacy and mechanism against MASH-related fibrosis remain less well defined, compared with the specific mechanisms through which FGF21 improves glucose and lipid metabolism. EXPERIMENTAL APPROACH: The effectiveness of PsTag-FGF21 was evaluated in two MASH-fibrosis models. Co-culture systems involving macrophages and hepatic stellate cells (HSCs) were employed for further assessment. Hepatic macrophages were selectively depleted by administering liposome-encapsulated clodronate via tail vein injections. RNA sequencing and cytokine profiling were conducted to identify key factors involved in macrophage-HSC crosstalk. KEY RESULTS: We first demonstrated the significant attenuation of hepatic fibrosis by PsTag-FGF21 in two MASH-fibrosis models. Furthermore, we highlighted the crucial role of macrophage phenotypic switch in PsTag-FGF21-induced HSC deactivation. FGF21 was demonstrated to regulate macrophages in a PsTag-FGF21-like manner. NR4A1, a nuclear factor which is notably down-regulated in human livers with MASH, was identified as a mediator responsible for PsTag-FGF21-induced phenotypic switch. Transcriptional control over insulin-like growth factor 1, a crucial factor in macrophage-HSC crosstalk, was exerted by the intrinsically disordered region domain of NR4A1. CONCLUSION AND IMPLICATIONS: Our results have elucidated the previously unclear mechanisms through which PsTag-FGF21 treats MASH-related fibrosis and identified NR4A1 as a potential therapeutic target for fibrosis.

A comprehensive comparison of molecular and phenotypic profiles between hepatitis B virus (HBV)-infected and non-HBV-infected hepatocellular carcinoma by multi-omics analysis.

Hepatitis B virus (HBV) infection is a major etiology of hepatocellular carcinoma (HCC). An interesting question is how different are the molecular and phenotypic profiles between HBV-infected (HBV+) and non-HBV-infected (HBV-) HCCs? Based on the publicly available multi-omics data for HCC, including bulk and single-cell data, and the data we collected and sequenced, we performed a comprehensive comparison of molecular and phenotypic features between HBV+ and HBV- HCCs. Our analysis showed that compared to HBV- HCCs, HBV+ HCCs had significantly better clinical outcomes, higher degree of genomic instability, higher enrichment of DNA repair and immune-related pathways, lower enrichment of stromal and oncogenic signaling pathways, and better response to immunotherapy. Furthermore, in vitro experiments confirmed that HBV+ HCCs had higher immunity, PD-L1 expression and activation of DNA damage response pathways. This study may provide insights into the profiles of HBV+ and HBV- HCCs, and guide rational therapeutic interventions for HCC patients.

Targeting aberrant glycosylation to modulate microglial response and improve cognition in models of Alzheimer's disease.

Altered glycosylation profiles have been correlated with potential drug targets in various diseases, including Alzheimer's disease (AD). In this area, the linkage between bisecting N-acetylglucosamine (GlcNAc), a product of N-acetylglucosaminyltransferase III (GnT-III), and AD has been recognized, however, our understanding of the cause and the causative role of this aberrant glycosylation in AD are far from completion. Moreover, the effects and mechanisms of glycosylation-targeting interventions on memory and cognition, and novel targeting strategies are worth further study. Here, we showed the characteristic amyloid pathology-induced and age-related changes of GnT-III, and identified transcription factor 7-like 2 as the key transcription factor responsible for the abnormal expression of GnT-III in AD. Upregulation of GnT-III aggravated cognitive dysfunction and Alzheimer-like pathologies. In contrast, loss of GnT-III could improve cognition and alleviate pathologies. Furthermore, we found that an increase in bisecting GlcNAc modified ICAM-1 resulted in impairment of microglial responses, and genetic inactivation of GnT-III protected against AD mechanistically by blocking the aberrant glycosylation of ICAM-1 and subsequently modulating microglial responses, including microglial motility, phagocytosis ability, homeostatic/reactive state and neuroinflammation. Moreover, by target-based screening of GnT-III inhibitors from FDA-approved drug library, we identified two compounds, regorafenib and dihydroergocristine mesylate, showing pharmacological potential leading to modulation of aberrant glycosylation and microglial responses, and rescue of memory and cognition deficits.

DHEC mesylate attenuates pathologies and aberrant bisecting N-glycosylation in Alzheimer's disease models.

Tremendous progress has been made to develop the therapy of Alzheimer's disease (AD). Existing several anti-AD remedies, with certain limitations, are far from adequate. Evidence suggests that dihydroergocristine (DHEC) mesylate, one of the main components of Ergoloid mesylates, can reduce the production of amyloid-ß in vitro. However, the therapeutic effect of DHEC mesylate in AD and its underlying mechanism are still largely unknown. Herein, we characterized the pharmacological effect of DHEC mesylate in AD and found that the spatial memory disorders and Alzheimer-type pathologies were alleviated by DHEC mesylate administration. Moreover, we demonstrated that DHEC mesylate improved aberrant bisecting N-glycosylation, which was identified as a potential biomarker of AD. We further explored the underlying mechanism and confirmed that DHEC mesylate protected against AD via AMPK and ERK signaling, in which, AMPK was the dominant down-stream molecule of DHEC mesylate. In summary, our findings provide foundations for development of DHEC mesylate as a therapeutic approach for AD.

Nitrated T cell epitope linked vaccine targeting CD47 elicits antitumor immune responses and acts synergistically with vaccine targeting PDL1.

Despite the clinical breakthrough made by immune checkpoint blockades (ICB) in cancer immunotherapy, immunosuppressed tumor microenvironment (TME) remains a major impediment in the efficacy of ICB immunotherapy. In this study, we constructed a Nitrated T cell epitope (NitraTh) linked vaccine targeting CD47, namely CD47-NitraTh. CD47-NitraTh could repress the progression of tumor by inducing tumor-specific immune response. Furthermore, combination vaccination with CD47-NitraTh and PDL1-NitraTh could reconstruct tumor associated macrophage, enhance macrophage-mediated phagocytosis for tumor cells, and promote the activation of tumor infiltrating T cells. Notably, by activating chemokine signaling pathway, NitraTh based vaccines reversed immunosuppressed TME, resulting in improved therapeutic outcome for tumor. With the advantage of reversing immunosuppressed TME, NitraTh based vaccine seems an optimal immunotherapy strategy for patients who are not sensitive to antibody based ICB.

von Hippel-Lindau-targeting microRNA-143-3p attenuates mitochondrial abnormality via AMPK/PGC-1α axis in Parkinson's disease.

Parkinson's disease (PD) is the second most common neurodegenerative disease characterized by selective loss of dopaminergic neurons. We previously found that inhibition of von Hippel-Lindau (VHL) can alleviate dopaminergic neuron degeneration in PD models via regulation of mitochondrial homeostasis, however, the disease-related alterations of VHL and the regulatory mechanisms of VHL level in PD need to be further investigated. In this study, we found that the levels of VHL were markedly increased in multiple cell models of PD and identified microRNA-143-3p (miR-143-3p) as a promising candidate for regulating VHL expression involved in PD. miR-143-3p directly bound to the 3'untranslated region of human VHL mRNA and inhibited its translation, and exerted neuroprotective effects by improving cell viability, apoptosis and tyrosine hydroxylase abnormality. Furthermore, we demonstrated that miR-143-3p exerted neuroprotection by attenuating mitochondrial abnormality via AMP-activated protein kinase (AMPK)/peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) axis, and AMPK inhibitor abolished the beneficial effects of miR-143-3p on the cell model of PD. Therefore, we identify the dysregulated VHL and miR-143-3p in PD, and propose the therapeutic potential of miR-143-3p to alleviate PD by improving mitochondrial homeostasis via AMPK/PGC-1α axis.

Structure-activity relationship of low molecular weight Astragalus membranaceus polysaccharides produced by Bacteroides.

Astragalus membranaceus polysaccharides (APS) possess significant biological activities, such as anti-tumor, antiviral, and immunomodulatory activities. However, there is still a lack of research on the structure-activity relationship of APS. In this paper, two carbohydrate-active enzymes from Bacteroides in living organisms were used to prepare degradation products. The degradation products were divided into APS-A1, APS-G1, APS-G2, and APS-G3 according to molecular weight. Structural analysis showed that all degradation products had an α-1,4-linked glucose backbone, but APS-A1 and APS-G3 also had branched chains of α-1,6-linked galactose or arabinogalacto-oligosaccharide. In vitro, immunomodulatory activity evaluation results indicated that APS-A1 and APS-G3 had better immunomodulatory activity, while the immunomodulatory activities of APS-G1 and APS-G2 were comparatively weaker. Molecular interaction detection showed that APS-A1 and APS-G3 could bind to toll-like receptors-4 (TLR-4) with a binding constant of 4.6 × 10-5 and 9.4 × 10-6, respectively, while APS-G1 and APS-G2 failed to bind to TLR-4. Therefore, the branched chains of galactose or arabinogalacto-oligosaccharide played a crucial role in the immunomodulatory activity of APS.

Structure Characterization and Anti-Rheumatoid Arthritis Activity of a Polysaccharide from Notopterygium incisum.

SCOPE: Notopterygium incisum is a traditional Chinese medicine that is commonly used to treat rheumatoid arthritis. Polysaccharides from N. incisum can be one of its main active components. However, there have been little investigations on N. incisum polysaccharides. METHODS AND RESULTS: A novel polysaccharide named NIP is extracted from N. incisum with a molecular weight of 2.34 × 106  Da. NIP, composed of arabinose, galactose, glucose, and galacturonic acid, is linked by methyl esterified 1,4-linked α-galacturonic acid, 1,6-linked ß-galactose, 1,5-linked α-arabinose, and 1,4,6-linked ß- glucose. In vitro, NIP can inhibit the NO production of LPS-stimulated RAW264.7 cells. In vivo, NIP relieves toe redness and swelling of AIA rats, reduces the release of inflammatory factors in the serum, and inhibits the activation of NF-κB and JAK/STAT3 signaling pathways. In addition, NIP can effectively decrease oxidative stress, reverse intestinal flora imbalance, and promote butyric acid-producing bacteria's proliferation to exert anti-RA activity. CONCLUSION: NIP may be recommended as a functional food that can alleviate the damage of rheumatoid arthritis.

Minimizing the Anticodon-Recognized Loop of Methanococcus jannaschii Tyrosyl-tRNA Synthetase to Improve the Efficiency of Incorporating Noncanonical Amino Acids.

In the field of genetic code expansion (GCE), improvements in the efficiency of noncanonical amino acid (ncAA) incorporation have received continuous attention. By analyzing the reported gene sequences of giant virus species, we noticed some sequence differences at the tRNA binding interface. On the basis of the structural and activity differences between Methanococcus jannaschii Tyrosyl-tRNA Synthetase (MjTyrRS) and mimivirus Tyrosyl-tRNA Synthetase (MVTyrRS), we found that the size of the anticodon-recognized loop of MjTyrRS influences its suppression activity regarding triplet and specific quadruplet codons. Therefore, three MjTyrRS mutants with loop minimization were designed. The suppression of wild-type MjTyrRS loop-minimized mutants increased by 1.8-4.3-fold, and the MjTyrRS variants enhanced the activity of the incorporation of ncAAs by 15-150% through loop minimization. In addition, for specific quadruplet codons, the loop minimization of MjTyrRS also improves the suppression efficiency. These results suggest that loop minimization of MjTyrRS may provide a general strategy for the efficient synthesis of ncAAs-containing proteins.

Structure and function characterization of the α-L-arabinofuranosidase from the white-rot fungus Trametes hirsuta.

α-L-Arabinofuranosidases (Abfs) play a crucial role in the degradation of hemicelluloses, especially arabinoxylans (AX). Most of the available characterized Abfs are from bacteria, while fungi, as natural decomposers, contain Abfs with little attention given. An arabinofuranosidase (ThAbf1), belonging to the glycoside hydrolase 51 (GH51) family, from the genome of the white-rot fungus Trametes hirsuta, was recombinantly expressed, characterized, and functionally determined. The general biochemical properties showed that the optimal conditions for ThAbf1 were pH 6.0 and 50°C. In substrate kinetics assays, ThAbf1 preferred small fragment arabinoxylo-oligosaccharides (AXOS) and could surprisingly hydrolyze di-substituted 23,33-di-L-arabinofuranosyl-xylotriose (A2,3XX). It also synergized with commercial xylanase (XYL) and increased the saccharification efficiency of arabinoxylan. The crystal structure of ThAbf1 indicated the presence of an adjacent cavity next to the catalytic pocket which led to the ability of ThAbf1 to degrade di-substituted AXOS. The narrow binding pocket prevents ThAbf1 from binding larger substrates. These findings have strengthened our understanding of the catalytic mechanism of GH51 family Abfs and provided a theoretical foundation for the development of more efficient and versatile Abfs to accelerate the degradation and biotransformation of hemicellulose in biomass. KEY POINTS: • ThAbf1 from Trametes hirsuta degraded di-substituted arabinoxylo-oligosaccharide. • ThAbf1 performed detailed biochemical characterization and kinetics. • ThAbf1 structure has been obtained to illustrate the substrate specificity.

Unstructured polypeptides as a versatile drug delivery technology.

Although polyethylene glycol (PEG), or "PEGylation" has become a widely applied approach for improving the efficiency of drug delivery, the immunogenicity and non-biodegradability of this synthetic polymer have prompted an evident need for alternatives. To overcome these caveats and to mimic PEG -or other natural or synthetic polymers- for the purpose of drug half-life extension, unstructured polypeptides are designed. Due to their tunable length, biodegradability, low immunogenicity and easy production, unstructured polypeptides have the potential to replace PEG as the preferred technology for therapeutic protein/peptide delivery. This review provides an overview of the evolution of unstructured polypeptides, starting from natural polypeptides to engineered polypeptides and discusses their characteristics. Then, it is described that unstructured polypeptides have been successfully applied to numerous drugs, including peptides, proteins, antibody fragments, and nanocarriers, for half-life extension. Innovative applications of unstructured peptides as releasable masks, multimolecular adaptors and intracellular delivery carriers are also discussed. Finally, challenges and future perspectives of this promising field are briefly presented. STATEMENT OF SIGNIFICANCE: Polypeptide fusion technology simulating PEGylation has become an important topic for the development of long-circulating peptide or protein drugs without reduced activity, complex processes, and kidney injury caused by PEG modification. Here we provide a detailed and in-depth review of the recent advances in unstructured polypeptides. In addition to the application of enhanced pharmacokinetic performance, emphasis is placed on polypeptides as scaffolders for the delivery of multiple drugs, and on the preparation of reasonably designed polypeptides to manipulate the performance of proteins and peptides. This review will provide insight into future application of polypeptides in peptide or protein drug development and the design of novel functional polypeptides.

Self-assembled nanoparticles based on DNA origami and a nitrated T helper cell epitope as a platform for the development of personalized cancer vaccines.

Neoantigen vaccines constitute an emerging and promising cancer immunotherapy. However, not all neoantigens have anti-tumor activity, as poor CD4+ epitope recognition can lead to the lack of greatly limit the persistence of the CD8+ T cell response. Therefore, we designed a self-assembled nanoplatform hereinafter referred to as DNA-coupled nitrated T helper cell epitope nanoparticle (DCNP) based on DNA origami containing a nitrated CD4 + T cell epitope, which can facilitate the effective activation of neoantigen-specific CD8+ T cells. Moreover, we embedded the cytidine-phosphate-guanosine oligonucleotide (CpG ODN) motif sequence in the DNA skeleton to function as a built-in adjuvant to activate Toll-like receptor 9. DCNP can markedly improve adjuvant and neoantigen co-delivery to lymphoid organs and promote neoantigen presentation on dendritic cells. Moreover, DCNP induced robust, and long-lived neoantigen-specific CD8+ T cell responses that significantly delayed tumor growth. Further, these effects were largely dependent on the nitrated T cell epitope. Collectively, our findings indicate that DCNP is a promising platform that could improve the development of personalized therapeutic neoantigen vaccines for cancer immunotherapy.

A peptide rich in glycine-serine-alanine repeats ameliorates Alzheimer-type neurodegeneration.

BACKGROUND AND PURPOSE: Repeated amino acid sequences in proteins are widely found, and the glycine-serine-alanine repeat is an element with a general propensity to form ß-sheet aggregates as found in key pathological factors, in several neurodegenerative diseases. Such properties of this repeat may guide development of disease-modifying therapies for neurodegenerative disease. However, details of its role and underlying mechanism(s) remain largely unknown. EXPERIMENTAL APPROACH: Actions of specific glycine-serine-alanine repeat peptides (SNPs), especially SNP-9, on Alzheimer's disease (AD)-like abnormalities were evaluated in transgenic mice and Caenorhabditis elegans, and in rat and cell models. Entry of SNPs into the brain, SNP activity in neuronal cells and peptide entry into cells were analysed in vivo and in vitro. Cell-free systems and the yeast two-hybrid system were also used to explore possible targets of SNP-9, and interactions of potential targets with SNP-9 were confirmed in cell-based systems. KEY RESULTS: We first identified SNP-9 as a potent neuroprotective peptide with the activity to decrease oligomeric amyloid ß (Aß) via co-assembling with the toxic Aß oligomer to form hetero-oligomers. Also, calcyclin-binding protein was found to act as a SNP-9-binding protein, by screening of a human brain cDNA library. Such binding showed that SNP-9 could regulate the abnormal hyperphosphorylation of tau via calcyclin-binding protein. CONCLUSION AND IMPLICATIONS: Our study provides a foundation for development of SNPs, especially SNP-9, as potential therapeutic interventions for AD. We propose SNP-9 as a potential therapeutic agent for the treatment of AD.

Microenvironment-responsive anti-PD-L1 × CD3 bispecific T-cell engager for solid tumor immunotherapy.

Bispecific T-cell Engager (BiTE) antibodies can redirect T-cells to tumor cells, and turn on the targeted lysis of tumor cells. However, BiTE has been challenging in solid tumors due to short plasma half-life, "off-target" effect, and immunosuppression via PD-1/PD-L1 axis. This study designed a safe, long-acting, and highly effective Protease-Activated PSTAGylated BiTE, named PAPB, which includes a shielding polypeptide domain (PSTAG), a protease-activated linker, and a BiTE core. The BiTE core consists of two scFvs targeting PD-L1 and CD3. BiTE core bound PD-L1 and CD3 in a dose-dependent manner, and PAPB can release BiTE core in response to MMP2 in the tumor microenvironment to exert antitumor activity. The plasma half-life of PAPB in mice was significantly prolonged from 2.46 h to 6.34 h of the BiTE core. In mice bearing melanoma (A375) xenografts, PAPB significantly increased infiltration of T lymphocytes in tumor tissue, and inhibited tumor proliferation without activating T-cells in the peripheral blood. Overall, the engineering protein PAPB could be a promising drug candidate for solid tumor immunotherapy.

Roles of the transcriptional regulators Fts1, YlNrg1, YlTup1, and YlSsn6 in the repression of the yeast-to-filament transition in the dimorphic yeast Yarrowia lipolytica.

In dimorphic fungi, the yeast-to-filament transition critical for cell survival under nutrient starvation is controlled by both activators and repressors. However, very few filamentation repressors are known. Here we report that, in the dimorphic yeast Yarrowia lipolytica, the conserved transcription factor YlNrg1 plays a minor role whereas Fts1, a newly identified Zn(II)2 Cys6 zinc cluster transcription factor, plays a key role in filamentation repression. FTS1 deletion caused hyperfilamentation whereas Fts1 overexpression drastically reduced filamentation. The expression of FTS1 is downregulated substantially during the yeast-to-filament transition. Transcriptome sequencing revealed that Fts1 represses 401 genes, including the filamentation-activating transcription factor genes MHY1, YlAZF1, and YlWOR4 and key cell wall protein genes. Tup1-Ssn6, a general transcriptional corepressor, is involved in the repression of many cellular functions in fungi. We show that both YlTup1 and YlSsn6 strongly repress filamentation in Y. lipolytica. YlTup1 and YlSsn6 together repress 1383 genes, including a large number of transcription factor and cell wall protein genes, which overlap substantially with Fts1-repressed genes. Fts1 interacts with both YlTup1 and YlSsn6, and LexA-Fts1 fusion represses a lexAop-promoter-lacZ reporter in a Tup1-Ssn6-dependent manner. Our findings suggest that Fts1 functions as a transcriptional repressor, directing the repression of target genes through the Tup1-Ssn6 corepressor.

Inhibiting von Hippel‒Lindau protein-mediated Dishevelled ubiquitination protects against experimental parkinsonism.

Dopaminergic neuron degeneration is a hallmark of Parkinson's disease (PD). We previously reported that the inactivation of von Hippel‒Lindau (VHL) alleviated dopaminergic neuron degeneration in a C. elegans model. In this study, we investigated the specific effects of VHL loss and the underlying mechanisms in mammalian PD models. For in vivo genetic inhibition of VHL, AAV-Vhl-shRNA was injected into mouse lateral ventricles. Thirty days later, the mice received MPTP for 5 days to induce PD. Behavioral experiments were conducted on D1, D3, D7, D14 and D21 after the last injection, and the mice were sacrificed on D22. We showed that knockdown of VHL in mice significantly alleviated PD-like syndromes detected in behavioral and biochemical assays. Inhibiting VHL exerted similar protective effects in MPP+-treated differentiated SH-SY5Y cells and the MPP+-induced C. elegans PD model. We further demonstrated that VHL loss-induced protection against experimental parkinsonism was independent of hypoxia-inducible factor and identified the Dishevelled-2 (DVL-2)/ß-catenin axis as the target of VHL, which was evolutionarily conserved in both C. elegans and mammals. Inhibiting the function of VHL promoted the stability of ß-catenin by reducing the ubiquitination and degradation of DVL-2. Thus, in vivo overexpression of DVL-2, mimicking VHL inactivation, protected against PD. We designed a competing peptide, Tat-DDF-2, to inhibit the interaction between VHL and DVL-2, which exhibited pharmacological potential for protection against PD in vitro and in vivo. We propose the therapeutic potential of targeting the interaction between VHL and DVL-2, which may represent a strategy to alleviate neurodegeneration associated with PD.

The Zinc Finger Transcription Factor Fts2 Represses the Yeast-to-Filament Transition in the Dimorphic Yeast Yarrowia lipolytica.

The yeast-to-filament transition is an important cellular response to environmental stimulations in dimorphic fungi. In addition to activators, there are repressors in the cells to prevent filament formation, which is important to keep the cells in the yeast form when filamentation is not necessary. However, very few repressors of filamentation are known so far. Here, we identify a novel repressor of filamentation in the dimorphic yeast Yarrowia lipolytica, Fts2, which is a C2H2-type zinc finger transcription factor. We show that fts2Δ cells exhibited increased filamentation under mild filament-inducing conditions and formed filaments under non-filament-inducing conditions. We also show that Fts2 interacts with YlSsn6, component of the Tup1-Ssn6 transcriptional corepressor, and Fts2-LexA represses a lexAop-PYlACT1-lacZ reporter in a Tup1-Ssn6-dependent manner, suggesting that Fts2 has transcriptional repressor activity and represses gene expression via Tup1-Ssn6. In addition, we show that Fts2 represses a large number of cell wall protein genes and transcription factor genes, some of which are implicated in the filamentation response. Interestingly, about two-thirds of Fts2-repressed genes are also repressed by Tup1-Ssn6, suggesting that Fts2 may repress the bulk of its target genes via Tup1-Ssn6. Lastly, we show that Fts2 expression is downregulated in response to alkaline pH and the relief of negative control by Fts2 facilitates the induction of filamentation by alkaline pH. IMPORTANCE The repressors of filamentation are important negative regulators of the yeast-to-filament transition. However, except in Candida albicans, very few repressors of filamentation are known in dimorphic fungi. More importantly, how they repress filamentation is often not clear. In this paper, we report a novel repressor of filamentation in Y. lipolytica. Fts2 is not closely related in amino acid sequence to CaNrg1 and Rfg1, two major repressors of filamentation in C. albicans, yet it represses gene expression via the transcriptional corepressor Tup1-Ssn6, similar to CaNrg1 and Rfg1. Using transcriptome sequencing, we determined the whole set of genes regulated by Fts2 and identified the major targets of Fts2 repression, which provide clues to the mechanism by which Fts2 represses filamentation. Our results have important implications for understanding the negative control of the yeast-to-filament transition in dimorphic fungi.