Cystatin Gene Research Articles

Identification and functional characterization of a novel cystatin in amphioxus, ancient origin of vertebrate type-2 cystatin homologues

Cystatins are well known as a vast superfamily of functional proteins participated in the reversible competitive inhibition of cysteine proteases. Currently, increasing evidences point to the extensive phylogenetic diversity and crucial immune roles of type-2 cystatins in the vertebrate species. However, no information is available regarding the homologue in cephalochordate amphioxus, the representative of most basal living chordates, whose immune regulation are still ambiguous. Here, we clearly identified the presence of type-2 cystatin gene in amphioxus Branchiostoma japonicum, termed Bjcystatin-2, which was structurally characterized by typical wedge-shaped cystatin feature. Evolutionary analyses revealed that Bjcystatin-2 is the putative ancestral type-2 cystatin for chordates, with gene diversity emerging through duplication events. The expression of Bjcystatin-2 showed tissue-specific profile and was inducible upon invasive pathogens. Significantly, the recombinant Bjcystatin-2 exhibited not merely cathepsin L inhibitory activity, but also the ability to bind with bacteria and their characteristic molecules. Furthermore, Bjcystatin-2 also showed the capacity to enhance the macrophage-driven bacterial phagocytosis and to attenuate the generation of pro-inflammatory cytokines within macrophages. In summary, these findings demonstrate that Bjcystatin-2 exhibits dual role acting as both a protease inhibitor and an immunoactive molecule, greatly enriching our understanding of immune defense mechanisms of type-2 cystatin within the amphioxus.

The evaluation of cystatin protein vaccines based on the stress response of ticks triggered by low-temperature and toxin stress in Haemaphysalis doenitzi.

Ticks, which are obligate blood-feeding parasites, transmit a wide range of pathogens during their hematophagic process. Certain enzymes and macromolecules play a crucial role in inhibition of several tick physiological processes, including digestion and reproduction. In the present study, genes encoding type 2 cystatin were cloned and characterized from Haemaphysalis doenitzi, and the potential role of cystatin in tick control was further assessed. Two cystatin genes, HDcyst-1 and HDcyst-2, were successfully cloned from the tick H. doenitzi. Their open reading frames are 390 and 426 base pairs, and the number of coding amino acids are 129 and 141, respectively. In the midgut, salivary glands, Malpighian tubules and ovaries of ticks, the relative expression of HDcyst-1 was higher in the midgut and Malpighian tubules, and HDcyst-2 was higher in the salivary glands of H. doenitzi, respectively. Lipopolysaccharide (LPS) injection and low-temperature stress elevated cystatin expression in ticks. Enzyme-linked immunosorbent assay showed that both rHDcyst-1 and rHDcyst-2 protein vaccines increased antibody levels in immunized rabbits. A vaccination trial in rabbits infected with H. doenitzi showed that both recombinant cystatin proteins significantly reduced tick engorgement weights and egg mass weight, in particular, rHDcyst-1 significantly prolonged tick engorgement time by 1 day and reduced egg hatching rates by 16.9%. In total, rHDcyst-1 and rHDcyst-2 protein vaccinations provided 64.1% and 51.8% protection to adult female ticks, respectively. This is the first report on the immunological characterization of the cystatin protein and sequencing of the cystatin gene in H. doenitzi. Cystatin proteins are promising antigens that have the potential to be used as vaccines for infestation of H. doenitzi control. © 2024 Society of Chemical Industry.

Abstract 6923: Deconvolution of breast cancer dissemination and therapy resistance via single-cell transcriptional lineage tracing

Abstract Chemotherapy remains the most used systemic treatment option for triple-negative breast cancer (TNBC). Lineage tracing studies demonstrated that treatment does not significantly alter clonal composition of primary tumors (PTs). The specific transcriptional phenotype(s) conferring chemoresistance in PTs and metastasis, however, are still largely undetermined, and have not been analyzed at single-clone resolution. We first modelled clinically relevant contexts of chemoresistance in our recently published single-cell lineage tracing approach (DOI: 10.1158/0008-5472.CAN-22-2717), using the standard-of-care combination of adriamycin and cyclophosphamide (A+C) in TNBC xenografts. Treatment was administered in neoadjuvant or adjuvant settings, or both together. In all cases, surgical PT resection was followed by metastatic disease. The neoadjuvant treatment resulted into a stable disease, thus resembling a significant fraction of the treated patients. To investigate clonal and transcriptional dynamics we performed scRNAseq analysis of 12 replicates (3 matched PT-lung replicates for each condition, for a total of 24 samples). Clustering analyses revealed a separation of treatments and tissue of origin. We retrieved a large number of longitudinal clones, including rare clones with high metastatic potential, in all the experimental conditions. A+C did not significantly alter clonal abundances, neither in PTs nor in metastases. Pseudobulk transcriptional analyses revealed key transcriptional traits associated with chemoresistance. Differential expression on longitudinal clones gave insights about transcriptional features of chemoresistant and pro-metastatic clones under chemotherapy. Integration at single-clone level of cluster analysis, pseudobulk and differential expression revealed cystatin (CST) family genes as highly-enriched in the chemoresistant and pro-metastatic phenotype. We are currently validating the role of CST genes in tumor growth, dissemination and chemoresistance in vivo using a panel of specific shRNAs. In parallel, using the same experimental model, we successfully deconvoluted the metastatic cascade through a single-cell lineage tracing approach on matched PT-CTC (circulating tumor cells)-lung replicates. scRNAseq analyses of pro-metastatic, circulating and metastatic clones is ongoing. Our preliminary data showed that all the dominant clones in the metastatic lungs are also found as CTCs in the bloodstream, thus suggesting that all pro-metastatic cells are also capable of surviving in the bloodstream and reinforcing our concept of pro-metastatic clone. This approach led us to gain a complete overview around clonal and transcriptional evolution of breast cancer dissemination. Citation Format: Alberto Dalmasso, Andrea Cossa, Giulia Bertolini, Niccolò Roda, Ilaria Servidio, Francesco Antonio Tucci, Salvatore Pece, Gabriella Sozzi, Pier Giuseppe Pelicci. Deconvolution of breast cancer dissemination and therapy resistance via single-cell transcriptional lineage tracing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6923.

Investigating the prognostic and predictive value of the type II cystatin genes in gastric cancer

BackgroundAccumulating evidence indicates that type II cystatin (CST) genes play a pivotal role in several tumor pathological processes, thereby affecting all stages of tumorigenesis and tumor development. However, the prognostic and predictive value of type II CST genes in GC has not yet been investigated.MethodsThe present study evaluated the expression and prognostic value of type II CST genes in GC by using The Cancer Genome Atlas (TCGA) database and the Kaplan–Meier plotter (KM plotter) online database. The type II CST genes related to the prognosis of GC were then screened out. We then validated the expression and prognostic value of these genes by immunohistochemistry. We also used Database for Annotation, Visualization, and Integrated Discovery (DAVID), Gene Multiple Association Network Integration Algorithm (GeneMANIA), Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), nomogram, genome-wide co-expression analysis, and other bioinformatics tools to analyze the value of type II CST genes in GC and the underlying mechanism.ResultsThe data from the TCGA database and the KM plotter online database showed that high expression of CST2 and CST4 was associated with the overall survival (OS) of patients with GC. The immunohistochemical expression analysis showed that patients with high expression of CST4 in GC tissues have a shorter OS than those with low expression of CST4 (HR = 1.85,95%CI: 1.13–3.03, P = 0.015). Multivariate Cox regression analysis confirmed that the high expression level of CST4 was an independent prognostic risk factor for OS.ConclusionsOur findings suggest that CST4 could serve as a tumor marker that affects the prognosis of GC and could be considered as a potential therapeutic target for GC.

Industrial wastewater irrigation increased higher heavy metals uptake and expansins, metacaspases, and cystatin genes expression in Parthenium and maize.

Industrial wastewater irrigation of agricultural crops can cause a lot of environmental and health problems in developing countries due to heavy metals deposition in agricultural soils as well as edible plant consumption by human beings. Therefore, this study was conducted to find out the heavy metals concentration in industrial wastewater and soil irrigated with that wastewater. In addition, the aim was to determine the impact of industrial wastewater irrigation on Parthenium hysterophorus and Zea mays genes involved in growth improvement and inhibition. For this purpose, plant samples from agriculture fields irrigated with wastewater from Hattar Industrial Estate (HIE) of Haripur, Pakistan, and control plants from non-contaminated soil irrigated with tape water were collected after 15 and 45 days of germination. Heavy metals concentration in the collected plant samples, wastewater, and soil was determined. The results revealed that the soil of the sample collection site was predominantly contaminated with Cr, Pb, Ni, Cu, Co, Zn, and Cd up to the concentrations of 38.98, 21.14, 46.01, 155.73, 12.50, 68.50, and 7.01 mg/kg, respectively. The concentrations of these heavy metals were found to surpass the permissible limit in normal agricultural soil. Expansins, cystatins (plant growth enhancers), and metacaspases (plant growth inhibitor) gene expression were studied through reverse transcription polymerase chain reaction. The results showed that the expression of these genes was higher in samples collected from wastewater-irrigated soils as compared to control. The expression of these genes was observed in 45 days old samples, 15 days old samples, and control. Taken together, this study suggests the use of Parthenium and maize for phytoremediation and that they should not be used for eating purposes if irrigated with industrial wastewater.

Field test of Easter lilies transformed with a rice cystatin gene for root lesion nematode resistance.

Easter lilies, Lilium longiflorum cv. Nellie White are a staple of the floral industry. In the U.S. most of the Easter lilies are grown in Oregon and California along the coast where there is a micro climate that is favorable to growth of lilies. The main pest when growing lilies in the field is Pratylenchus penetrans, the root lesion nematode. Easter lilies are one of the most expensive crops to produce because of the cost of chemicals used to control P. penetrans and other pathogens that infect the lilies. Our previous study had shown that transgenic Easter lilies containing a rice cystatin gene (Oc-IΔD86 that has a deleted Asp86) were resistant to P. penetrans in vitro. This study examined growth characteristics of five independently transformed lines of the cystatin Easter lilies compared to non-transformed Nellie White for three seasons in the field in Brookings, Oregon. Liles grown in three soil chemical treatments 1) preplant fumigation, 2) preplant fumigation plus at plant organophosphate, and 3) at plant organophosphate were compared to those grown in nontreated soil. Growth characteristics evaluated included: time of shoot emergence, survival of plants, size of plants, visual ratings of plant health, basal roots and stem roots, weight of foliage and roots, and number and size of bulblets that developed on stems. Nematodes were counted following their extraction from the roots. While not totally resistant, when planted in the field, transformed lines demonstrated and maintained a degree of resistance to lesion nematode over two growing seasons and displayed desirable growth and quality characteristics similar to non-transformed lilies.

Loop replacement design: a new way to improve potency of plant cystatins.

Plant cystatins function as competitive inhibitors of cysteine proteases. Similar to other defence proteins, cystatins include hypervariable, positively selected amino acid sites presumably impacting their biological activity. Protein engineering approaches, such as point mutations, at these functionally relevant amino acid sites have already been found to be a powerful tool in improving the inhibitory properties of cystatins. Such engineered cystatins not only better protect against digestive proteases of herbivorous arthropods but also against cysteine proteases of several other plant pests as well as against cysteine proteases produced in plant during stress-induced senescence. Despite previous engineering successes, an urgent need still exists to further improve both plant cystatin potency and specificity. Tremblay and colleagues propose in this issue a new cystatin engineering strategy to substitute the function-related structural elements (SEs) of a cystatin by the corresponding elements of an alternative cystatin. This strategy, possibly combined with direct cystatin gene editing in a target plant, might provide an innovative way to control cysteine protease activity. Comment on https://doi.org/10.1111/febs.16288.

Genome-Wide Identification and Characterization of the Cystatin Gene Family in Bread Wheat (Triticum aestivum L.).

Cystatins, as reversible inhibitors of papain-like and legumain proteases, have been identified in several plant species. Although the cystatin family plays crucial roles in plant development and defense responses to various stresses, this family in wheat (Triticum aestivum L.) is still poorly understood. In this study, 55 wheat cystatins (TaCystatins) were identified. All TaCystatins were divided into three groups and both the conserved gene structures and peptide motifs were relatively conserved within each group. Homoeolog analysis suggested that both homoeolog retention percentage and gene duplications contributed to the abundance of the TaCystatin family. Analysis of duplication events confirmed that segmental duplications played an important role in the duplication patterns. The results of codon usage pattern analysis showed that TaCystatins had evident codon usage bias, which was mainly affected by mutation pressure. TaCystatins may be regulated by cis-acting elements, especially abscisic acid and methyl jasmonate responsive elements. In addition, the expression of all selected TaCystatins was significantly changed following viral infection and cold stress, suggesting potential roles in response to biotic and abiotic challenges. Overall, our work provides new insights into TaCystatins during wheat evolution and will help further research to decipher the roles of TaCystatins under diverse stress conditions.

Evolutionary Analysis of Cystatins of Early-Emerging Metazoans Reveals a Novel Subtype in Parasitic Cnidarians.

Simple SummaryCysteine protease inhibitors (cystatins) are molecules that play key protective roles in protein degradation and are involved in the immunomodulation of host responses to parasites. Little is known about the cystatin gene repertoire, evolution, and lineage-specific adaptations of early-emerging metazoans. Using bioinformatics searches, we identified orthologues of cystatins in basal animal lineages including free-living and parasite taxa. We aimed to explore whether their cystatin gene repertoire and evolution follow similar patterns recognized for derived metazoans and whether the modifications are linked to the organism’s life history. We revealed that cysteine protease inhibitors from early-emerging animal groups are highly diverse, with modifications in gene organization and protein architecture. A new subtype of cystatins was discovered in the parasitic cnidarians, the Myxozoa, which has so far been only reported for a group of derived animals: trematode flukes. We set out hypotheses to describe the driving forces for the origins of this unique cystatin subtype and propose evolutionary scenarios elucidating the current existence of cystatins in the Metazoa, especially in their early-emerging lineages. Our research identified molecules for which future functional studies may help to identify their roles in host–parasite interactions and for the parasite itself. The evolutionary aspects of cystatins are greatly underexplored in early-emerging metazoans. Thus, we surveyed the gene organization, protein architecture, and phylogeny of cystatin homologues mined from 110 genomes and the transcriptomes of 58 basal metazoan species, encompassing free-living and parasite taxa of Porifera, Placozoa, Cnidaria (including Myxozoa), and Ctenophora. We found that the cystatin gene repertoire significantly differs among phyla, with stefins present in most of the investigated lineages but with type 2 cystatins missing in several basal metazoan groups. Similar to liver and intestinal flukes, myxozoan parasites possess atypical stefins with chimeric structure that combine motifs of classical stefins and type 2 cystatins. Other early metazoan taxa regardless of lifestyle have only the classical representation of cystatins and lack multi-domain ones. Our comprehensive phylogenetic analyses revealed that stefins and type 2 cystatins clustered into taxonomically defined clades with multiple independent paralogous groups, which probably arose due to gene duplications. The stefin clade split between the subclades of classical stefins and the atypical stefins of myxozoans and flukes. Atypical stefins represent key evolutionary innovations of the two parasite groups for which their origin might have been linked with ancestral gene chimerization, obligate parasitism, life cycle complexity, genome reduction, and host immunity.

143 Therapeutic efficacy and safety of adipose tissue-derived mesenchymal stem cells in treating mastitis and metritis in dairy cattle

Mastitis and metritis are two inflammatory diseases with high economic impact on dairy industry and farmers are losing revenues. Antibiotic treatment for these disease conditions typically achieve suboptimal outcomes. Moreover, emerging antibiotic resistance in mastitis and metritis worldwide has a severe effect on productivity, fertility, and lifespan of animals. A paradigm shift in treatment strategies is much needed for these animals. Attracting properties of mesenchymal stem cells may act as an alternative strategy for treating these diseases. In addition to their ability to differentiate into multiple types of cells, mesenchymal stem cells can orchestrate immune responses and modulate tissue microenvironments. In the present study, we evaluated the safety and efficacy of adipose tissue–derived mesenchymal stem cells (AT-MSC) for mastitis and metritis. A well-characterised adipose-derived stem cell line with 3–5 passages cells were used for treatment for these diseases. In a safety trial, mastitis- and metritis-infected cattle were administered with 1×106 cells of AT-MSC through different routes (local, IV, IV+local). Animals were clinically evaluated during 15 days of the experimental period and blood samples were collected for hemogram determination. The efficacy was checked under both invivo and invitro conditions. Invitro efficiency of AT-MSC was determined using a transwell plate experiment, in which AT-MSC were co-cultured with bacteria (Escherichia coli and Staphylococcus). Invivo efficiency was evaluated by administration of in 2mL (1×106 cells) of AT-MSC dose; then, somatic cell count (SCC) in milk of mastitic cattle and polymorphonuclear (PMN) cell count in cervical vaginal fluid (CVF) of metritic cattle were determined. Gene expression profiling of antimicrobial (cathelicidin, lipocalin, cystatin) and anti-inflammatory (IL-4, IL-6, IL-10) peptides were quantified in all groups: IV, local, IV+local, and control ATB (antibiotic). Allogenic AT-MSC did not induce any immunological rejection response in treated animals. A significant reduction of bacteria in CVF invitro when co-cultured with AT-MSC was observed. The PMN count was significantly reduced in CVF of the IV+local group. The SCC in milk of mastitic animals was decreased in the local (intramammary) group. Maximum expression of IL-6, IL-10, cathelicidin, lipocalin, and cystatin and angiopoietin genes were observed on Day 3 followed by Day 7 in the local group in case of mastitis. In metritis, expression of IL-6, IL-10, cathelicidin, lipocalin, cystatin, and angiopoietin were observed at Day 3 in the IV+local group. All mastitic and metritic cattle were completely and permanently cured within 30 days after treatment with mesenchymal stem cells. In conclusion, we provide initial evidence for the use of allogenic AT-MSC and their safety and efficacy to treat mastitis and metritis in dairy cattle.

262 UV-endonuclease and photolyase DNA repair enzymes increase cystatin gene expression after UVB induced downregulation

262 UV-endonuclease and photolyase DNA repair enzymes increase cystatin gene expression after UVB induced downregulation

Cloning, expression and enzymatic characterization of a cystatin gene involved in herbivore defense in tea plant (Camellia sinensis)

Plant cystatins play crucial roles in the process of plant defense against herbivorous insects. A cDNA clone, designated CsCPI2, was isolated from Camellia sinensis cv. Longjing 43 using 5′-/3′-RACE extension. The full-length cDNA gene is 618 bp in size, encodes 205 amino acid residues, and has a deduced molecular weight of 23.07 kDa. CsCPI2 was different from the already published CsCPI1, as the two cDNA sequences share 69.31% and 60.4% pairwise identity at the nucleic acid and amino acid levels, respectively. The CsCPI2–MBP fusion protein was over-expressed in Escherichia coli. We found that the inhibitory effect of CsCPI2–MBP on ficin was 1.17, 3.46, and 3.73 times the effect on papain, chymopapain, and bromelain, respectively. The highest inhibitory activity of CsCPI2–MBP was found at pH 7 at 40 °C, and CsCPI2–MBP was stable at temperature below 60 °C. Moreover, CsCPI2–MBP was found to be more responsive to acidic environment than to alkaline one. Higher cystatin inhibitory activity was found in the tea leaves infested by Myllocerinus aurolineatus than that in intact healthy leaves. Finally, we found that CsCPI2 was a competitive inhibitor of the cysteine proteinases in tea weevil’s gut, and leaves treated with CsCPI2–MBP incurred compensatory damage, and the weight gain of tea weevils fed on CsCPI2–MBP treated leaves was significantly higher than that of MBP treated leaves, but did not cause mortality. Taken together, our research highlights potential defensive proteins for future tea engineering approaches, but more in vivo experiments are needed to confirm their effectiveness.

Opposing Functions of BRD4 Isoforms in Breast Cancer

Bromodomain-containing protein 4 (BRD4) is a cancer therapeutic target in ongoing clinical trials disrupting primarily BRD4-regulated transcription programs. The role of BRD4 in cancer has been attributed mainly to the abundant long isoform (BRD4-L). Here we show, by isoform-specific knockdown and endogenous protein detection, along with transgene expression, the less abundant BRD4 short isoform (BRD4-S) is oncogenic while BRD4-L is tumor-suppressive in breast cancer cell proliferation and migration, as well as mammary tumor formation and metastasis. Through integrated RNA-seq, genome-wide ChIP-seq, and CUT&RUN association profiling, we identify the Engrailed-1 (EN1) homeobox transcription factor as a key BRD4-S coregulator, particularly in triple-negative breast cancer. BRD4-S and EN1 comodulate the extracellular matrix (ECM)-associated matrisome network, including type II cystatin gene cluster, mucin 5, and cathepsin loci, via enhancer regulation of cancer-associated genes and pathways. Our work highlights the importance of targeted therapies for the oncogenic, but not tumor-suppressive, activity of BRD4.

The first characterization of a cystatin and a cathepsin L-like peptidase from Aedes aegypti and their possible role in DENV infection by the modulation of apoptosis

Recently, a salivary gland transcriptome study demonstrated that the transcripts of a putative cystatin gene (SeqID AAEL013287; Aacystatins) from Aedes aegypti were increased in DENV2-infected mosquitoes and that silencing of the Aacystatin gene resulted in an increase in DENV titres. In this work, Aacystatin was biochemically characterized; the purified recombinant inhibitor was able to inhibit typical cysteine proteases with a Ki in the nM range. Pulldown assays using Aag2 cell extracts identified a cathepsin L-like peptidase (AaCatL) as a possible target of Aacystatin. Purified recombinant AaCatL had an optimal pH of 5.0 and displayed a preference for Leu, Val and Phe residues at P2, which is common for other cathepsin L-like peptidases. Transcription analysis of Aacystatin and AaCatL in the salivary glands and midgut of DENV2-infected mosquitoes revealed a negative correlation between DENV2 titres and levels of the inhibitor and peptidase, suggesting their involvement in DENV2-mosquito interactions. Considering that apoptosis may play an important role during viral infections, the possible involvement of Aacystatin in staurosporine-induced apoptosis in Aag2 cells was investigated; the results showed higher expression of the inhibitor in treated cells; moreover, pre incubation with rAacystatin was able to increase Aag2 cell viability.

Investigation of the clinical significance and prospective molecular mechanisms of cystatin genes in patients with hepatitis B virus‑related hepatocellular carcinoma.

The present study aimed to investigate the clinical significance and prospective molecular mechanism of cystatin (CST) genes in patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). The role of CST genes in the molecular mechanism of HCC was revealed through bioinformatics analysis. The clinical significance of CST genes was investigated using GSE14520-derived data from patients with HBV-related HCC. Gene set enrichment analysis (GSEA) was used to identify pathways in which the CST genes were enriched, as well as the association between these pathways and HCC. The expression levels of CST1, CST2, CST5, CSTA and CSTB genes were higher in HCC tissue compared with in normal tissue; conversely, CST3 and CST7 were reduced in HCC tissue. Subsequent receiver operating characteristic analysis of the CST genes demonstrated that CST7 and CSTB genes may function as potential diagnostic markers for HCC. Furthermore, the expression levels of CST6 and CST7 were strongly associated with recurrence-free survival and overall survival of patients with HBV-related HCC. GSEA of the CST genes revealed that CST7 was significantly enriched in tumor evasion and tolerogenicity, cancer progenitors, liver cancer late recurrence, liver cancer progression and several liver cancer subclasses. In addition, CST genes demonstrated homology in terms of protein structure and were revealed to be strongly co-expressed. The present findings suggested that CST7 and CSTB genes may serve as potential prognostic and diagnostic biomarkers for HCC.

SpCYS, a cystatin gene from wild potato (Solanum pinnatisectum), is involved in the resistance against Spodoptera litura

Cysteine proteinase inhibitor (cystatin) genes belong to one family of proteinase inhibitors and play an important role in the resistance to insect pests. Cystatin (CYS) genes have been studied in different plant–insect interactions, but there are few reports about the importance of potato cystatin genes. In this study, a cystatin gene named SpCYS was characterized from the wild diploid potato Solanum pinnatisectum Dun, which exhibits high resistance to insect pests. We also investigated the impact of SpCYS in the development of Spodoptera litura when feed from leaves of genetically modified potato. Bioinformatic analysis revealed that SpCYS encoded protein possessed conserved domains of the cystatin family. The SpCYS expression was observed in leaves, stems, roots and tubers with the expression level being almost the same in roots, stems and leaves but greater in tubers. After mechanical damage, SpCYS expression in leaves was induced reaching its peak at 9 h and then decreased to normal levels. Further, we constructed transgenic lines of cultivated potato (cultivar Desiree) expressing SpCYS and performed insect bioassays and feeding preference experiments. Both tests showed that the over-expression of SpCYS lowered the weight increase rate of both Spodoptera litura larvae and pupa. S. litura larvae preferred to feed on the leaves of non-transformed controls rather than the transgenic lines. Taken together, our data suggest that SpCYS is a significant asset for the defense mechanism of potato against S. litura.

Cystatin F as a key family 2 cystatin subunit and prognostic biomarker for early‑stage pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, and early diagnosis and assessment may enhance the quality of life and survival of patients. The prognostic value of key family 2 cystatins subunit in PDAC patients remains unknown. The potential molecular roles of family 2 cystatins and related pathways were investigated using bioinformatics analysis. The relationship of family 2 cystatin expression levels and clinical outcomes of 112 patients with early-stage PDAC were evaluated via univariate and combined survival analysis. A prognostic nomogram model was also constructed and gene set enrichment analysis was performed to investigate potential pathways in PDAC. The pathways, interaction networks, and Gene Ontology term analysis of the cystatin gene family were analyzed in the present study. Cystatin F (CST7) was identified as the key subunit of family 2 cystatins in survival analysis. PDAC patients who harbored a higher expression level of CST7 had a lower risk in overall survival (adjusted HROS=0.44, 95% CI=0.25-0.77, P=0.004) and a longer survival time in various subgroups. The prognostic nomogram indicated that the CST7 expression model effectively predicted the outcomes of patients with early-stage PDAC (predictive ability >0.75). In the gene set enrichment analysis, it was revealed that CST7 expression may be involved in immune regulation and be associated with cell adhesion. CST7 could be a useful biomarker for the prognostic prediction of early-stage PDAC after pancreaticoduodenectomy.

Characteristics and function of a novel cystatin gene in the pine wood nematode Bursaphelenchus xylophilus.

ABSTRACTBursaphelenchus xylophilus is the pathogen that causes pine wilt disease (PWD). The disease has caused significant economic losses and damage to forests. However, the pathogenic mechanism of B. xylophilus remains unclear. Cystatins are involved in various biological processes where they regulate normal proteolysis and also play a role in pathogenicity, but their functions in B. xylophilus are unknown. Therefore, we cloned the full-length cDNA of a cystatin gene of B. xylophilus (Bx-cpi-1) by rapid-amplification of cDNA ends and analyzed its characteristics with bioinformatic methods. In situ mRNA hybridization analyses showed that transcripts of Bx-cpi-1 were abundantly expressed in the reproductive organs of B. xylophilus. The expression of Bx-cpi-1 was investigated using qPCR. Bx-cpi-1 was expressed during each of the different developmental stages of B. xylophilus. The highest gene expression was at the egg stage. After infection of Pinus massoniana, the expression of Bx-cpi-1 increased. The functions of Bx-cpi-1 were verified by RNA interference. The feeding rate, reproduction and pathogenicity of B. xylophilus all decreased as a result of silencing of the Bx-cpi-1 gene. These results revealed that Bx-cpi-1 may be a variant of a type II cystatin gene which is involved in the development and pathogenic process of B. xylophilus.

Cloning and characterization of ApCystatin, a plant cystatin gene from Agapanthus praecox ssp. orientalis responds to abiotic stress

Plant cystatins are involved in the regulation of protein turnover and play important roles in defense mechanisms. We cloned the ApCystatin gene from Agapanthus praecox ssp. orientalis, a famous ornamental and medical plant. The complete cDNA sequence of ApCystatin is comprised of 1439 nucleotides with a 423 bp ORF encoding 140 amino acids. The mRNA level of ApCystatin was significantly up-regulated under various abiotic stress, such as salt, osmosis, oxidative and cold stresses, which suggested that ApCystatin participated in the plant's resistance to stress. The recombinant ApCystatin fusion protein expressed in E. coli transetta (DE3) cells was approximate 18 kDa. 25 μg of ApCystatin inhibited more than 95% activity of papain, suggesting ApCystatin as a papain-like protease inhibitor. As an exogenous substance, 1.60 μg/mL ApCystatin protein improved the regrowth percentage of Arabidopsis 60-h seedlings after cryopreservation from 30% to 47%. In addition, the relative survival rate of A. praecox embryogenic callus after cryopreservation also increased for 30% with addition of 1.20 μg/mL ApCystatin protein. This indicated that ApCystatin performed protective property against cryoinjury to Arabidopsis 60-h seedlings and A. praecox embryogenic callus during cryopreservation. Under various abiotic stress conditions, the recombinant ApCystatin protein showed significant advantage in growth rates at NaCl, mannitol, PEG6000, cold, acidic and alkaline conditions, compared to control. In conclusion, ApCystatin as a new member of plant cystatins exhibited protective property against cryoinjury in plant cryopreservation and abiotic stress in E. coli.

Silencing barley cystatins HvCPI-2 and HvCPI-4 specifically modifies leaf responses to drought stress.

Protein breakdown and mobilization are some of the major metabolic features associated with abiotic stresses, essential for nutrient recycling and plant survival. Genetic manipulation of protease and/or protease inhibitors may contribute to modulate proteolytic processes and plant responses. The expression analysis of the whole cystatin family, inhibitors of C1A cysteine proteases, after water deprivation in barley leaves highlighted the involvement of Icy-2 and Icy-4 cystatin genes. Artificial microRNA lines independently silencing the two drought-induced cystatins were generated to assess their function in planta. Phenotype alterations at the final stages of the plant life cycle are represented by the stay-green phenotype of silenced cystatin 2 lines. Besides, the enhanced tolerance to drought and differential responses to water deprivation at the initial growing stages are observed. The mutual compensating expression of Icy-2 and Icy-4 genes in the silencing lines pointed to their cooperative role. Proteolytic patterns by silencing these cystatins were concomitant with modifications in the expression of potential target proteases, in particular, HvPap-1, HvPap-12, and HvPap-16 C1A proteases. Metabolomics analysis lines also revealed specific modifications in the accumulation of several metabolites. These findings support the use of plants with altered proteolytic regulation in crop improvement in the face of climate change.