Heparan sulfate-dependent phase separation of CCL5 and its chemotactic activity.

Secreted chemokines form concentration gradients in target tissues to control migratory directions and patterns of immune cells in response to inflammatory stimulation; however, how the gradients are formed is much debated. Heparan sulfate (HS) binds to chemokines and modulates their activities. In this study, we investigated the roles of HS in the gradient formation and chemoattractant activity of CCL5 that is known to bind to HS. CCL5 and heparin underwent liquid-liquid phase separation and formed gradient, which was confirmed using CCL5 immobilized on heparin-beads. The biological implication of HS in CCL5 gradient formation was established in CHO-K1 (wild-type) and CHO-677 (lacking HS) cells by Transwell assay. The effect of HS on CCL5 chemoattractant activity was further proved by Transwell assay of human peripheral blood cells. Finally, peritoneal injection of the chemokines into mice showed reduced recruitment of inflammatory cells either by mutant CCL5 (lacking heparin-binding sequence) or by addition of heparin to wild-type CCL5. Our experimental data propose that co-phase separation of CCL5 with HS establishes a specific chemokine concentration gradient to trigger directional cell migration. The results warrant further investigation on other heparin-binding chemokines and allows for a more elaborate insight into disease process and new treatment strategies.

[Clinical observation on electroacupuncture at "four points of sacral region" on moderate to severe stress urinary incontinence after radical prostatectomy].

OBJECTIVE: To compare the clinical therapeutic effect between electroacupuncture at "four points of sacral region" and transurethral Erbium laser in treatment of moderate to severe stress urinary incontinence after radical prostatectomy. METHODS: A total of 68 patients of moderate to severe stress urinary incontinence after radical prostatectomy were divided into an electroacupuncture group (34 cases) and an Erbium laser group (34 cases, 3 cases dropped off) according to the settings. In the electroacupuncture group, electroacupuncture was applied at "four points of sacral region", i.e. points of 0.5 cun beside bilateral sacrococcygeal joints and bilateral Huiyang (BL 35), with continuous wave, 2 Hz in frequency, 60 min each time, once every other day, 3 times a week, 12 times as one course of treatment. In the Erbium laser group, transurethral Erbium laser technology was given, once every 4 weeks as one course of treatment. Both groups were treated for 5 courses. The scores of the International Consultation on Incontinence questionnaire-short form (ICI-Q-SF) and the incontinence quality of life questionnaire (I-QOL) were observed before treatment, after each course of treatment and in follow-up after 1 and 2 months of treatment completion, respectively, and the clinical efficacy was evaluated after treatment in the two groups. RESULTS: Compared with those before treatment, the ICI-Q-SF scores were decreased while the I-QOL scores were increased after 5 courses of treatment and in follow-up after 1, 2 months of treatment completion in the two groups (P<0.01). The ICI-Q-SF score in follow-up after 2 months of treatment completion were higher than that after 5 courses of treatment in the Erbium laser group (P<0.05). After 3, 4, 5 courses of treatment and in follow-up after 1 and 2 months of treatment completion, the ICI-Q-SF scores in the electroacupuncture group were lower than those in the Erbium laser group (P<0.05, P<0.01); after 2, 3, 4, 5 courses of treatment and in follow-up after 1 and 2 months of treatment completion, the I-QOL scores in the electroacupuncture group were higher than those in the Erbium laser group (P<0.01). The change ranges of ICI-Q-SF score and I-QOL score between before treatment and after each course of treatment in the electroacupuncture group were lager than those in the Erbium laser group (P<0.01, P<0.05). The total effective rate was 61.8% (21/34) in the electroacupuncture group, which was superior to 19.4 (6/31) in the Erbium laser group (P<0.01). CONCLUSION: Both electroacupuncture at "four points of sacral region" and transurethral Erbium laser can improve the clinical symptoms and the quality of life in patients of moderate to severe stress urinary incontinence after radical prostatectomy. The short-term efficacy and long-term efficacy of electroacupuncture are superior to the Erbium laser technology.

Interruption of post-Golgi STING trafficking activates tonic interferon signaling.

Activation of the cGAS-STING pathway is traditionally considered a "trigger-release" mechanism where detection of microbial DNA or cyclic di-nucleotides sets off the type I interferon response. Whether this pathway can be activated without pathogenic ligand exposure is less well understood. Here we show that loss of Golgi-to-lysosome STING cofactors, but not ER-to-Golgi cofactors, selectively activates tonic interferon signalling. Impairment of post-Golgi trafficking extends STING Golgi-dwell time, resulting in elevated immune signalling and protection against infection. Mechanistically, trans-Golgi coiled coil protein GCC2 and several RAB GTPases act as key regulators of STING post-Golgi trafficking. Genomic deletion of these factors potently activates cGAS-STING signalling without instigating any pathogenic trigger for cGAS. Gcc2-/- mice develop STING-dependent serologic autoimmunity. Gcc2-deleted or Rab14-deleted cancer cells induce T-cell and IFN-dependent anti-tumour immunity and inhibit tumour growth in mice. In summary, we present a "basal flux" mechanism for tonic cGAS-STING signalling, regulated at the level of post-Golgi STING trafficking, which could be exploited for cancer immunotherapy.

Tonic prime-boost of STING signalling mediates Niemann-Pick disease type C.

The classic mode of STING activation is through binding the cyclic dinucleotide 2'3'-cyclic GMP-AMP (cGAMP), produced by the DNA sensor cyclic GMP-AMP synthase (cGAS), which is important for the innate immune response to microbial infection and autoimmune disease. Modes of STING activation that are independent of cGAS are much less well understood. Here, through a spatiotemporally resolved proximity labelling screen followed by quantitative proteomics, we identify the lysosomal membrane protein Niemann-Pick type C1 (NPC1) as a cofactor in the trafficking of STING. NPC1 interacts with STING and recruits it to the lysosome for degradation in both human and mouse cells. Notably, we find that knockout of Npc1 'primes' STING signalling by physically linking or 'tethering' STING to SREBP2 trafficking. Loss of NPC1 protein also 'boosts' STING signalling by blocking lysosomal degradation. Both priming and boosting of STING signalling are required for severe neurological disease in the Npc1-/- mouse. Genetic deletion of Sting1 (the gene that encodes STING) or Irf3, but not that of Cgas, significantly reduced the activation of microglia and relieved the loss of Purkinje neurons in the cerebellum of Npc1-/- mice, leading to improved motor function. Our study identifies a cGAS- and cGAMP-independent mode of STING activation that affects neuropathology and provides a therapeutic target for the treatment of Niemann-Pick disease type C.

Uncovering the pathological functions of Ser404 phosphorylation by semisynthesis of a phosphorylated TDP-43 prion-like domain.

Herein, we have successfully semi-synthesized a TDP-43 prion-like domain with Ser404 phosphorylation. We have demonstrated that Ser404 phosphorylation could accelerate the amyloid aggregation of the TDP-43 prion-like domain and aggravate its cytotoxicity.

TDP-43 specific reduction induced by Di-hydrophobic tags conjugated peptides.

TAR DNA binding protein 43 (TDP-43) is a key target in amyotrophic lateral sclerosis (ALS) treatment. Here, based on hydrophobic tagging strategy, we designed and synthesized a series of single or double hydrophobic tags conjugated peptides D1-D8. Among them, it was found that D4 displayed strongest ability to induce TDP-43 degradation in cells. D4 could reduce TDP-43 induced cytotoxicity. Besides, D4 could reduce TDP-43 levels in a transgenic drosophila model.

Semi-synthesis of murine prion protein by native chemical ligation and chemical activation for preparation of polypeptide-α-thioester.

Prions are suspected as pathogen of the fatal transmissible spongiform encephalopathies. Strategies to access homogenous prion protein (PrP) are required to fully comprehend the molecular mechanism of prion diseases. However, the polypeptide fragments from PrP show a high tendency to form aggregates, which is a gigantic obstacle of protein synthesis and purification. In this study, murine prion sequence 90 to 230 that is the core three-dimensional structure domain was constructed from three segments murine PrP (mPrP)(90-177), mPrP(178-212), and mPrP(213-230) by combining protein expression, chemical synthesis and chemical ligation. The protein sequence 90 to 177 was obtained from expression and finally converted into the polypeptide hydrazide by chemical activation of a cysteine in the tail. The other two polypeptide fragments of the C-terminal were obtained by chemical synthesis, which utilized the strategies of isopeptide and pseudoproline building blocks to complete the synthesis of such difficult sequences. The three segments were finally assembled by sequentially using native chemical ligation. This strategy will allow more straightforward access to homogeneously modified PrP variants. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Specific Knockdown of Endogenous Tau Protein by Peptide-Directed Ubiquitin-Proteasome Degradation.

Tau, an important pathological protein of Alzheimer's disease (AD), can mediate the toxicity of amyloid ß (Aß). Thus, reduction of Tau with chemical molecules may offer a novel strategy for treating AD. Here, we designed and synthesized a series of multifunctional molecules that contained Tau-recognition moieties and E3 ligase-binding moieties to enhance Tau degradation. Among these molecules, TH006 had the highest activity of inducing Tau degradation by increasing its poly-ubiquitination. The decrement in Tau induced by TH006 could decrease the cytotoxicity caused by Aß. Furthermore, TH006 could regulate the Tau level in the brain of an AD mouse model. Therefore, partial reduction of Tau with such multifunctional peptides may open up a novel therapeutic strategy for AD treatment.

Clearance of the intracellular high level of the tau protein directed by an artificial synthetic hydrolase.

Promoting clearance of intracellular excessive tau is a potential therapeutic strategy for treating Alzheimer's disease. In this work, we designed and synthesized a cyclen-hybrid artificial 'hydrolase' I1-Cu(II) to cleave tau in vitro. Furthermore, a cell-permeable 'hydrolase' I2-Cu(II), derived from I1-Cu(II), was also synthesized to cleave intracellular tau proteins.

Endogenous protection derived from activin A/Smads transduction loop stimulated via ischemic injury in PC12 cells.

Activin A (ActA), a member of transforming growth factor-beta (TGF-b) super- family, affects many cellular processes, including ischemic stroke. Though the neuroprotective effects of exogenous ActA on oxygen-glucose deprivation (OGD) injury have already been reported by us, the endogenous role of ActA remains poorly understood. To further define the role and mechanism of endogenous ActA and its signaling in response to acute ischemic damage, we used an OGD model in PC12 cells to simulate ischemic injury on neurons in vitro. Cells were pre-treated by monoclonal antibody against activin receptor type IIA (ActRII-Ab). We found that ActRII-Ab augments ischemic injury in PC12 cells. Further, the extracellular secretion of ActA as well as phosphorylation of smad3 in PC12 cells was also up-regulated by OGD, but suppressed by ActRII-Ab. Taken together, our results show that ActRII-Ab may augment ischemic injury via blocking of transmembrane signal transduction of ActA, which confirmed the existence of endogenous neuroprotective effects derived from the ActA/Smads pathway. ActRIIA plays an important role in transferring neuronal protective signals inside. It is highly possible that ActA transmembrance signaling is a part of the positive feed-back loop for extracellular ActA secretion.