The cystatin superfamily comprises a large group of the cystatin domain containing proteins, present in a wide variety of organisms, including humans. Cystatin inhibitory activity is vital for the ...delicate regulation of normal physiological processes by limiting the potentially highly destructive activity of their target proteases such as the papain (C1) family, including cysteine cathepsins. Some of the cystatins also inhibit the legumain (C13) family of enzymes. Failures in biological mechanisms controlling protease activities result in many diseases such as neurodegeneration, cardiovascular diseases, osteoporosis, arthritis, and cancer. Cystatins have been classified into three types: the stefins, the cystatins and the kininogens, although other cystatin-related proteins, such as CRES proteins, are emerging. The stefins are mainly intracellular proteins, whereas the cystatins and the kininogens are extracellular. The cystatins are tight binding and reversible inhibitors. The basic mechanism of interaction between cystatins and their target proteases has been established, based mainly on the crystal structures of various cathepsins, stefins and cystatins and their enzyme-inhibitor complexes. Cystatins, as rather non-selective inhibitors, discriminate only slightly between endo- and exopeptidases. They are also prone to form amyloids. The levels of some stefins and cystatins in tissue and body fluids can serve as relatively reliable markers for a variety of diseases. In this review we summarize present knowledge about cystatins and their role in some diseases.
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
Plant cystatins function as competitive inhibitors of cysteine proteases. Modifying functionally relevant amino acid sites is a powerful tool in improving the inhibitory properties of cystatins against digestive proteases of herbivorous arthropods. We discuss the potential of a recent ‘loop replacement design’ proposed by Tremblay et al. for cystatins by which a function‐related element of a protein is changed for the corresponding element of a related protein to create a cystatin structural element (SE) hybrid, which better binds to a digestive cysteine protease.
Comment on: https://doi.org/10.1111/febs.16288
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.
Protein engineering approaches have been proposed to improve the inhibitory properties of plant cystatins against herbivorous arthropod digestive proteases, generally involving the site‐directed ...mutagenesis of functionally relevant amino acids or the selection of improved inhibitor variants by phage display approaches. Here, we propose a novel approach where the function‐related structural elements of a cystatin are substituted by the corresponding elements of an alternative cystatin. Inhibitory assays were first performed with 20 representative plant cystatins and model Cys proteases, including arthropod proteases, to appreciate the extent of functional variability among the plant cystatin family. The most, and less, potent of these cystatins were then used as ‘donors’ of structural elements to create hybrids of tomato cystatin SlCYS8 used as a model ‘recipient’ inhibitor. In brief, inhibitory activities against Cys proteases strongly differed from one plant cystatin to another, with Ki (papain) values diverging by more than 30‐fold and inhibitory rates against arthropod proteases varying by up to 50‐fold depending on the enzymes assessed. In line with theoretical assumptions from docking models generated for different Cys protease–cystatin combinations, structural element substitutions had a strong impact on the activity of recipient cystatin SlCYS8, positive or negative depending on the basic inhibitory potency of the donor cystatin. Our data confirm the wide variety of cystatin inhibitory profiles among plant taxa. They also demonstrate the usefulness of these proteins as a pool of discrete structural elements for the design of cystatin variants with improved potency against herbivorous pest digestive Cys proteases.
Protein engineering approaches have been proposed to improve the inhibitory properties of plant cystatins against herbivorous arthropod digestive proteases, involving in most cases the site‐directed mutagenesis of functionally relevant amino acids or the selection of potent inhibitor variants by phage display procedures. Here, we propose a novel approach for improvement, where the function‐related structural elements of a cystatin are substituted by the corresponding elements of an alternative cystatin.
Cystatins have recently emerged as important players in a multitude of physiological and patho-physiological settings that range from cell survival and proliferation, to differentiation, cell ...signaling and immunomodulation. This group of cysteine protease inhibitors forms a large super-family of proteins composed of one, two, three, and, in some species, more than three cystatin domains. Over the last 20 years or so, members of the cystatin super-family have been primarily explored with respect to their capacity to inhibit intracellular cysteine proteases. Yet, this classical mode of action does not fully explain their remarkably diverse biological functions. Due to the space limitations, the author will discuss here the most recent findings that suggest that some of the single-domain, cytoplasmic and cell-secreted cystatins may play important roles in the promotion or suppression of tumor growth, invasion and metastasis. Based on the present understanding of cystatin function, novel avenues for anti-cancer strategies are proposed.
Protease allergens disrupt epithelial barriers to exert their allergenicity. Cystatin SN (encoded by CST1) is an endogenous cysteine protease inhibitor upregulated in nasal epithelia in patients with ...allergic rhinitis (AR).
We sought to investigate the protective effect of human cystatin SN on AR symptoms using pollen-induced AR mouse models.
We performed an in vitro protease activity assay to evaluate the effect of recombinant human cystatin SN (rhCystatin SN) on Japanese cedar (JC) or ragweed proteases. A human nasal epithelial cell line, RPMI 2650, was used to examine tight junction (TJ) disruption in vitro. Mice were sensitized and nasally challenged with JC or ragweed pollens with or without rhCystatin SN to examine the effect of rhCystatin SN on AR symptoms and the epithelial barrier in vivo. Because mice lack CST1, we generated transgenic (Tg) mice expressing human CST1 under control of its genomic control region (hCST1-Tg mice) to examine the role of cystatin SN in physiologically expressed conditions.
rhCystatin SN inhibited JC but not ragweed protease activities and prevented JC-induced but not ragweed-induced TJ disruption in vitro. Exogenous administration of rhCystatin SN ameliorated JC-induced but not ragweed-induced sneezing and nasal TJ disruption in vivo. Furthermore, hCST1-Tg mice showed decreased JC-induced but not ragweed-induced sneezing symptoms and nasal TJ disruption compared with wild-type mice.
Human cystatin SN suppresses AR symptoms through inhibiting allergen protease activities and protecting the nasal TJ barrier in an allergen-specific manner. We propose that upregulation of nasal endogenous protease inhibitors, including cystatin SN, is a novel therapeutic strategy for protease allergen–induced AR.
Cystatin SN/SA (CST1/2) are cysteine protease inhibitors which protect against allergen, viral, and bacterial proteases. Cystatins are overexpressed in the setting of allergic rhinitis and chronic ...rhinosinusitis with nasal polyps (CRSwNP), however, their role in promoting type 2 inflammation remains poorly characterized.
The purpose of this study was to use integrated poly-omics and a murine exposure model to explore the link between Cystatin overexpression in CRSwNP and type 2 inflammation.
IRB and IACUC approved study comparing tissue, exosome, and mucus CST1/2 between CRSwNP and controls (n=10/group) using matched whole exome sequencing, transcriptomic, proteomic, post-translational modification, histologic, functional, and bioinformatic analyses. C57/BL6 mice were dosed with 3.9 μg/mL CST1 or PBS intranasally for 5-18 days in the presence or absence of epithelial ABCB1a knockdown. Inflammatory cytokines were quantified using Quansys multiplex assays or ELISAs.
Among 1,305 proteins quantified, CST1/2 were among the most overexpressed protease inhibitors in tissue, exosome and mucus samples and localized to the epithelial layer. Multiple PTMs were identified in the polyp tissue. Exosomal CST1/2 strongly and significantly correlated with eosinophils and Lund-Mackay scores. Murine type 2 cytokine secretion and Th2 cell infiltration increased in a time dependent manner following CST1 exposure and was abrogated by epithelial knockdown of ABCB1a, a regulator of epithelial cytokine secretion.
CST1 is a potent upstream initiator of epithelial derived type 2 inflammation in CRSwNP. Therapeutic strategies targeting CST activity and its associated PTMs deserve further interrogation.
Cystatin SN/SA (CST 1/2) are profoundly overexpressed within CRSwNP and correlate with disease severity. CST1 acts as a primary upstream driver of epithelial type 2 inflammation and may be abrogated by ABCB1a knockdown.
Summary
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.
Functional characterization of a cystatin A from the bat Myotis davidii Costa, Gabriel Cerqueira Alves; Torquato, Ricardo Jose Soares; de Morais Gomes, Vinícius ...
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology,
October-December 2024, Letnik:
274
Journal Article
Recenzirano
Myotis davidii cystatin A (MdCSTA), a stefin A-like from the Chinese native bat species M. davidii, was expressed as a recombinant protein and functionally characterized as a strong inhibitor of the ...cysteine proteases papain, human cathepsins L and B and the tick cathepsin L-like BmCL1. Despite the highly conserved amino acid sequences among stefins A from different vertebrates, MdCSTA presents a Methionine-2 residue at the N-terminal region and the second binding loop (pos 73–79) that differs from human stefin A (HsCSTA) and might be related to the lower inhibition constant (Ki) value presented by this inhibitor in comparison to human stefin A inhibition to cathepsin B. Therefore, to investigate the importance of these variable regions in cathepsin B inhibition, recombinant stefins A MdCSTA and HsCSTA containing mutations at the second amino acid residue and second binding loop were expressed and evaluated in kinetic assays. Enzymatic inhibition assays with cathepsin B revealed that switching the amino acid residues at position 2 and second binding loop region between bat and human CSTAs improved the HsCSTA's and reduced MdCSTA's inhibitory activity. Additionally, molecular docking analysis estimated lower energy values for the complex between MdCSTA-cathepsin B, in comparison to human CSTA-cathepsin B, while the mutants presented intermediate values, suggesting that other regions might contribute to the higher inhibitory activity against cathepsin B by MdCSTA. In conclusion, MdCSTA, the first bat's stefin A-like inhibitor to be functionally characterized, presented a higher inhibitory activity against cathepsin B in comparison to the human inhibitor, which is partially related to the glutamine-rich second binding loop and Met-2. Further structural analysis should be performed to elucidate potential inhibitor effects on cysteine proteinases.
Display omitted
•Myotis davidii's cystatin A is a strong inhibitor of papain, cathepsins L and B.•Cystatin A from Myotis davidii and Homo sapiens differ at the N-terminal and second binding loop.•Myotis davidii's cystatin A has a 20 times lower Ki for cathepsin B than human cystatin A.
Cystatins are natural inhibitors of cysteine proteases, enzymes that are widely distributed in animals, plants, and microorganisms. Human cystatin C (hCC) has been also recognized as an aggregating ...protein directly involved in the formation of pathological amyloid fibrils, and these amyloidogenic properties greatly increase in a naturally occurring L68Q hCC variant. For a long time only dimeric structure of wild-type hCC has been known. The dimer is created through 3D domain swapping process, in which two parts of the cystatin structure become separated from each other and next exchanged between two molecules. Important role in the domain swapping plays the L1 loop, which connects the exchanging segments and, upon dimerization, transforms from a β-turn into a part of a long β-strand. In the very recently published first monomeric structure of human cystatin C (hCC-stab1), dimerization was abrogated due to clasping of the β-strands from the swapping domains by an engineered disulfide bridge. We have designed and constructed another mutated cystatin C with the smallest possible structural intervention, that is a single-point mutation replacing hydrophobic V57 from the L1 loop by polar asparagine, known as a stabilizer of a β-turn motif. V57N hCC mutant occurred to be stable in its monomeric form and crystallized as a monomer, revealing typical cystatin fold with a five-stranded antiparallel β-sheet wrapped around an α-helix. Here we report a 2.04
Å resolution crystal structure of V57N hCC and discuss the architecture of the protein in comparison to chicken cystatin, hCC-stab1 and dimeric hCC.
PDF
