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To successfully feed, ticks inject pharmacoactive molecules into the vertebrate host including cystatin cysteine protease inhibitors. However, the molecular and cellular events modulated by tick saliva remain largely unknown. Here, we describe and characterize a novel immunomodulatory cystatin, Iristatin, which is upregulated in the salivary glands of feeding Ixodes ricinus ticks. We present the crystal structure of Iristatin at 1.76 Å resolution. Purified recombinant Iristatin inhibited the proteolytic activity of cathepsins L and C and diminished IL-2, IL-4, IL-9, and IFN-γ production by different T-cell populations, IL-6 and IL-9 production by mast cells, and nitric oxide production by macrophages. Furthermore, Iristatin inhibited OVA antigen-induced CD4 + T-cell proliferation and leukocyte recruitment in vivo and in vitro. Our results indicate that Iristatin affects wide range of anti-tick immune responses in the vertebrate host and may be exploitable as an immunotherapeutic.

The Neotropical tick is the primary vector of , the causative agent of Brazilian spotted fever, a disease associated with high fatality rates. Tick saliva, a complex mixture of bioactive molecules essential for successful blood feeding, facilitates pathogen transmission and modulates host immune responses. A comprehensive evaluation of the salivary gland transcriptome database reveals that protease inhibitors are abundantly expressed molecules in tick saliva during feeding. Thus, this study aims to describe and characterize the most expressed member of the cystatin family identified in salivary transcriptome, named Amblyostatin-1. Bioinformatic tools were employed for analysis of the Amblyostatin-1 sequence and structure. A recombinant version of Amblyostatin-1 was expressed in an system, evaluated against a panel of cysteine proteases in biochemical assays, and used to generate antibodies in immunized mice. The biological activities of Amblyostatin-1 were assessed by its effects on dendritic cell maturation and in a carrageenan-induced inflammation model . Based on its sequence and predicted three-dimensional structure, Amblyostatin-1 is classified as an I25B cystatin, and its recombinant form selectively inhibits cathepsins L, C, and S at different rates, with a low nanomolar value of 0.697 ± 0.22 nM against cathepsin L. Regarding its biological activities, recombinant Amblyostatin-1 partially affects LPS-induced dendritic cell maturation by downmodulating the costimulatory molecules CD80 and CD86 at higher micromolar concentrations (3 µM) while promoting IL-10 production at nanomolar concentrations (100 nM). The apparent lack of Amblyostatin-1-specific antibody responses in immunized mice suggests an impairment of antigen processing and presentation . Furthermore, in a carrageenan-induced inflammation model, Amblyostatin-1 decreased edema formation and neutrophil infiltration into the skin without affecting other myeloid cells. These findings establish Amblyostatin-1 as a novel salivary cystatin with immunomodulatory and anti-inflammatory properties, highlighting its potential as an immunobiological agent.

Hereditary cystatin C amyloid angiopathy is a dominantly inherited disease caused by a leucine to glutamine variant of human cystatin C (hCC). L68Q-hCC forms amyloid deposits in brain arteries associated with micro-infarcts, leading ultimately to paralysis, dementia and death in young adults. To evaluate the ability of molecules to interfere with aggregation of hCC while informing about cellular toxicity, we generated cells that produce and secrete WT and L68Q-hCC and have detected high-molecular weight complexes formed from the mutant protein. Incubations of either lysate or supernatant containing L68Q-hCC with reducing agents glutathione or N-acetyl-cysteine (NAC) breaks oligomers into monomers. Six L68Q-hCC carriers taking NAC had skin biopsies obtained to determine if hCC deposits were reduced following NAC treatment. Remarkably, ~50–90% reduction of L68Q-hCC staining was observed in five of the treated carriers suggesting that L68Q-hCC is a clinical target for reducing agents. HCCAA is a dominantly inherited disease which causes brain hemorrhages as a result of mutant cystatin C aggregation in carriers. Here, the authors show that n- acetyl cysteine can prevent aggregation of mutant protein in a cell model system and reverse protein deposition in the skin of mutation-carrying subjects.

• Arginine rich, mutated in early stage of tumours (Armet), is a well-characterized bifunctional protein as an unfolded protein response component intracellularly and a neurotrophic factor extracellularly in mammals. Recently, a new role of Armet as an effector protein mediating insect–plant interactions has been reported; however, its molecular mechanisms underlying the regulation of plant defences remain unclear. • We investigated the molecular mechanisms underlying whitefly-secreted Armet-mediated regulation of insect–plant interaction by agrobacterium-mediated transient expression, RNA interference, electrical penetration graph, protein–protein interaction studies, virus-induced gene silencing assay, phytohormone analysis and whitefly bioassays. • Armet, secreted by Bemisia tabaci whitefly, is highly expressed in the primary salivary gland and is delivered into tobacco plants during feeding. Overexpression of the BtArmet gene in tobacco enhanced whitefly performance, while silencing the BtArmet gene in whitefly interrupted whitefly feeding and suppressed whitefly performance on tobacco plants. BtArmet was shown to interact with NtCYS6, a cystatin protein essential for tobacco anti-whitefly resistance, and counteract the negative effects of NtCYS6 on whitefly. • These results indicate that BtArmet is a salivary effector and acts to promote whitefly performance on tobacco plants through binding to the tobacco cystatin NtCYS6. Our findings provide novel insight into whitefly–plant interactions.

Preeclampsia results from placental insufficiency and causes maternal endothelial dysfunction and multi-organ damage. Our in-silico analysis identified Cystatin 6 (CST6), a cysteine protease inhibitor, as located on the placental surface where it might be released into maternal circulation. This study aimed to characterise CST6 and one of its high affinity targets, Legumain (LGMN), in preeclampsia and assess its biomarker potential by measuring levels in maternal circulation. Placental CST6 mRNA expression was significantly increased in 78 pregnancies complicated by early-onset preeclampsia (delivering at < 34 weeks’ gestation) relative to 30 gestation matched controls (P  < 0.0001). LGMN mRNA expression was significantly decreased ( P  = 0.0309). Circulating CST6 was increased in 35 pregnancies complicated by early-onset preeclampsia (< 34 weeks’ gestation) relative to 27 gestation matched controls ( P  = 0.0261), and LGMN levels remained unchanged. At 36 weeks’ gestation, circulating CST6 was significantly increased (P =  0.001), while LGMN was significantly decreased ( P  = 0.0135) in 21 pregnancies preceding diagnosis of preeclampsia at term, compared to 184 pregnancies that did not develop preeclampsia. Human trophoblast stem cells (hTSC) were differentiated into syncytiotrophoblast or extravillous trophoblast (EVT) to evaluate CST6 and LGMN expression in these trophoblast lineages. CST6 and LGMN mRNA expression were significantly increased across 96 h after syncytiotrophoblast ( P  = 0.0066 and P  = 0.0010 respectively) and EVT differentiation (P =  0.0618 and P  = 0.0016 respectively), with the highest expression in syncytiotrophoblast. Computational analysis of two publicly available single-cell and single-nuclei RNA sequencing datasets correlated with the expression pattern observed in vitro. When syncytiotrophoblast cells were exposed to hypoxia (1% O 2 vs. 8% O 2 ), CST6 expression significantly increased ( P  = 0.0079), whilst LGMN expression was unchanged. The vascular endothelium may serve as an additional source of circulating CST6 and LGMN in preeclampsia.  Induction of dysfunction in endothelial cells by TNFα, caused reduced CST6 expression ( P  = 0.0036), whilst LGMN expression remained unchanged. Administering recombinant CST6 to endothelial cells enhanced markers of endothelial dysfunction and LGMN expression in the presence of TNFα. These findings indicate an inverse relationship between CST6 and LGMN in the placenta and maternal circulation in preeclampsia. We suggest elevated circulating levels of CST6 may be induced by placental hypoxia. This study provides novel insight into the dysregulation of CST6 and LGMN in preeclampsia and introduces their potential roles in human pregnancy and associated pathology.

Background Cystatins are cysteine protease inhibitor in plant and animal that not only regulates the intracellular transport of endogenous proteins in plants but also plays an important role in plant defense against biotic and abiotic stresses. However, the cystatin gene family in alfalfa remains poorly characterized. Result In this study, 55 cystatin genes distributed across 18 chromosomes in alfalfa were identified. GO enrichment analysis indicated that the 55 MsCYS genes were primarily associated with cysteine-type endopeptidase inhibitor activity. These genes were classified into six distinct subgroups, with each containing one or two typical cystatin domains. Analysis of cis -acting regulatory elements suggested the potential involvement of MsCYS genes in stress responses. Transcriptome analysis revealed that nearly half of the MsCYS genes were not expressed across the examined tissues and stress conditions and those that are expressed showed higher expression levels in stems and leaves than in roots. Expression levels of MsCYS genes were higher in stems and leaves than in roots. Under stress caused by thrips, MsCYS49 and MsCYS54 were significantly up-regulated. Real-time RT-PCR results corroborated the transcriptome data. The expression of MsCYS49 and MsCYS54 increased markedly, reaching 5.5-fold and 8.3-fold that of the control levels. After 14 days of thrips infestation. The cysteine content in alfalfa leaves increased significantly, which was associated with the up-regulation of MsCYS genes. Additionally, exogenous application of jasmonic acid enhanced the expression of certain MsCYS genes, thereby conferring resistance to thrips in alfalfa. Exogenous salicylic acid also up-regulated the expression of certain MsCYS genes and modulated insect defense. Conclusion Our study identified 55 members of the cystatin gene family in alfalfa and analyzed their phylogenetic relationships, structural characteristics, and putative functions in resistance to biotic and abiotic stress. This work establishes a robust foundation for further functional characterization of each cystatin gene member, which provides novel insights for improving the stress tolerance of alfalfa.

Metastasis is an important factor contributing to poor prognosis in patients with gastric cancer; yet, the molecular mechanism leading to this cell behavior is still not well understood. In this study, we explored the role of cysteine protease inhibitor SN (Cystatin SN, CST1) in promoting gastric cancer metastasis. We hypothesized that CST1 could regulate gastric cancer progression by regulating GPX4 and ferroptosis. Whole transcriptome sequencing suggested that the expression of CST1 was significantly increased in metastatic cancer, and high CST1 expression was correlated with a worse prognosis. Our data further confirmed that the overexpression of CST1 may significantly promote the migration and invasion of gastric cancer cells in vitro and enhance liver, lung, and peritoneal metastasis of gastric cancer in nude mice. Meanwhile, high expression of CST1 promoted the epithelial-mesenchymal transition (EMT) of gastric cancer cells. Mechanistically, a co-immunoprecipitation experiment combined with mass spectrometry analysis confirmed that CST1 could interact with GPX4, a key protein regulating ferroptosis. CST1 relieves GPX4 ubiquitination modification by recruiting OTUB1, improving GPX4 protein stability and reducing intracellular reactive oxygen species (ROS), thereby inhibiting ferroptosis and, in turn, promoting gastric cancer metastasis. Moreover, clinical data suggested that CST1 is significantly increased in peripheral blood and ascites of gastric cancer patients with metastasis; multivariate Cox regression model analysis showed that CST1 was an independent risk factor for the prognosis of gastric cancer patients. Overall, our results elucidated a critical pathway through which high CST1 expression protects gastric cancer cells from undergoing ferroptosis, thus promoting its progression and metastasis. CST1 may be used as a new oncological marker and potential therapeutic target for gastric cancer metastasis.

Alveolar echinococcosis (AE), caused by the metacestode larval of Echinococcus multilocularis , is one of the most lethal helminthic diseases in humans. Current treatment options, such as albendazole, are limited in their efficacy, highlighting the need for a deeper understanding of the parasite-host interaction to identify new therapeutic targets. One promising area of research involves helminth-derived cystatins, which are known to modulate host immune responses to facilitate parasite survival. A cystatin homologue from E. multilocularis ( Em Cystatin-B) was identified and analyzed. Em Cystatin-B was cloned, expressed and purified. Its expression patterns were evaluated by western blot, qPCR and Immunohistochemistry The Em Cystatin-B structure was solved by X-ray crystallography. Em Cystatin-B was expressed in the mature protoscoleces as well as in the cytosol and nucleus of the metacestode vesicles. Moverover, Em Cystatin-B adopts a conserved typical cystatin fold, but also exhibits unique structural features. Notably, a novel feature characterized by two intermolecular disulfide bridges between Cys4 in a Em Cystatin-B molecular and Cys76 in adjacent molecule was discovered. Further investigation demonstrated this distinctive feature appears to be involved in the oligomerization of Em Cystatin-B, facilitating a monomer-dimer-tetramer assembly pathway. The crystal structure of Em Cystatin-B reveals a novel feature in classical stefins, and provides species-specific insights into the sequence, structure, and functional characteristics of Em Cystatin-B.

Genes underneath signals from genome-wide association studies (GWAS) for kidney function are promising targets for functional studies, but prioritizing variants and genes is challenging. By GWAS meta-analysis for creatinine-based estimated glomerular filtration rate (eGFR) from the Chronic Kidney Disease Genetics Consortium and UK Biobank (n = 1,201,909), we expand the number of eGFRcrea loci (424 loci, 201 novel; 9.8% eGFRcrea variance explained by 634 independent signal variants). Our increased sample size in fine-mapping (n = 1,004,040, European) more than doubles the number of signals with resolved fine-mapping (99% credible sets down to 1 variant for 44 signals, ≤5 variants for 138 signals). Cystatin-based eGFR and/or blood urea nitrogen association support 348 loci (n = 460,826 and 852,678, respectively). Our customizable tool for Gene PrioritiSation reveals 23 compelling genes including mechanistic insights and enables navigation through genes and variants likely relevant for kidney function in human to help select targets for experimental follow-up. Identifying causal variants and genes in genome-wide association studies remains a challenge, an issue that is ameliorated with larger sample sizes. Here the authors meta-analyze kidney function genome-wide association studies to identify new loci and fine-map loci to home in on variants and genes involved in kidney function.

Background Sepsis is a life-threatening complication of an infection and remains one of the leading causes of mortality in surgical patients. Bacteremia induces excessive inflammatory responses that result in multiple organ damage. Chronic helminth infection and helminth-derived materials have been found to immunomodulate host immune system to reduce inflammation against some allergic or inflammatory diseases. Schistosoma japonicum cystatin ( Sj -Cys) is a cysteine protease inhibitor that induces regulatory T-cells and a potential immunomodulatory. The effect of Sj -Cys on reducing sepsis inflammation and mortality was investigated. Methods Sepsis was induced in BALB/c mice using cecal ligation and puncture (CLP), followed by intraperitoneal injection of different doses (10, 25 or 50 μg) of recombinant Sj -Cys (r Sj -Cys). The therapeutic effect of r Sj -Cys on sepsis was evaluated by observing the survival rates of mice for 96 h after CLP and the pathological injury of liver, kidney and lung by measuring the levels of alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN) and creatinine (Cr) in sera and the tissue sections pathology, and the expression of MyD88 in liver, kidney and lung tissues. The immunological mechanism was investigated by examining pro-inflammatory cytokines (TNF-α, IL-6, IL-1β) and IL-10 and TGF-β1 in mice sera and in culture of macrophages stimulated by lipopolysaccharides (LPS). Results r Sj -Cys treatment provided significant therapeutic effects on CLP-induced sepsis in mice demonstrated with increased survival rates, alleviated overall disease severity and tissue injury of liver, kidney and lung. The r Sj -Cys conferred therapeutic efficacy was associated with upregualted IL-10 and TGF-β1 cytokines and reduced pro-inflammatory cytokines TNF-α, IL-6, IL-1β. MyD88 expression in liver, kidney and lung tissues of r Sj -Cys-treated mice was reduced. In vitro assay with macrophages also showed that r Sj -Cys inhibited the release of pro-inflammatory cytokines and mediator nitric oxide (NO) after being stimulated by lipopolysaccharide (LPS). Conclusions The results suggest the anti-inflammatory potential of r Sj -Cys as a promising therapeutic agent on sepsis. The immunological mechanism underlying its therapeutic effect may involve the downregulation of pro-inflammatory cytokines and upregulation of IL-10 and TGF-β1 cytokines possibly via downregulation of the TLR adaptor-transducer MyD88 pathway. The findings suggest r Sj -Cys is a potential therapeutic agent for sepsis and other inflammatory diseases.