Papain-like Cysteine Proteases Research Articles

Papain Degrades Tight Junction Proteins of Human Keratinocytes In Vitro and Sensitizes C57BL/6 Mice via the Skin Independent of its Enzymatic Activity or TLR4 Activation

Papain is commonly used in food, pharmaceutical, textile, and cosmetic industries and is known to induce occupational allergic asthma. We have previously shown that the papain-like cysteine protease Dermatophagoides pteronyssinus 1 from house dust mite exhibits percutaneous sensitization potential. We aimed here to investigate the potential of papain itself in epicutaneous sensitization. The effects of papain on tight junction (TJ) proteins were tested in vitro in human primary keratinocytes. Using C57BL/6 wild-type and Toll-like receptor 4 (TLR4)-deficient mice, we analyzed the sensitization potential of papain, its effects on the skin barrier, and immune cell recruitment. Our results show that papain affects the skin barrier by increasing transepidermal water loss, degrading TJ proteins and inducing vasodilation. When topically applied, papain exhibited a high epicutaneous inflammatory potential by recruiting neutrophils, mast cells, and CD3-positive cells and by induction of a TH2-biased antibody response. However, its high potency for specific sensitization via the skin was TLR4 independent and, in spite of its capacity to degrade epidermal TJ proteins, does not rely on its enzymatic function. From our data, we conclude that papain has all features to act as a strong allergen via the skin.

Cysteine cathepsins: their role in tumor progression and recent trends in the development of imaging probes.

Papain-like cysteine proteases bear an enormous potential as drug discovery targets for both infectious and systemic human diseases. The considerable progress in this field over the last two decades has also raised interest in the visualization of these enzymes in their native context, especially with regard to tumor imaging. After a short introduction to structure and general functions of human cysteine cathepsins, we highlight their importance for drug discovery and development and provide a critical update on the current state of knowledge toward their involvement in tumor progression, with a special emphasis on their role in therapy response. In accordance with a radiopharmaceutical point of view, the main focus of this review article will be the discussion of recently developed fluorescence and radiotracer-based imaging agents together with related molecular probes.

Potential use of phytocystatins in crop improvement, with a particular focus on legumes.

Phytocystatins are a well-characterized class of naturally occurring protease inhibitors that function by preventing the catalysis of papain-like cysteine proteases. The action of cystatins in biotic stress resistance has been studied intensively, but relatively little is known about their functions in plant growth and defence responses to abiotic stresses, such as drought. Extreme weather events, such as drought and flooding, will have negative impacts on the yields of crop plants, particularly grain legumes. The concepts that changes in cellular protein content and composition are required for acclimation to different abiotic stresses, and that these adjustments are achieved through regulation of proteolysis, are widely accepted. However, the nature and regulation of the protein turnover machinery that underpins essential stress-induced cellular restructuring remain poorly characterized. Cysteine proteases are intrinsic to the genetic programmes that underpin plant development and senescence, but their functions in stress-induced senescence are not well defined. Transgenic plants including soybean that have been engineered to constitutively express phytocystatins show enhanced tolerance to a range of different abiotic stresses including drought, suggesting that manipulation of cysteine protease activities by altered phytocystatin expression in crop plants might be used to improve resilience and quality in the face of climate change.

Approaches for the generation of active papain-like cysteine proteases from inclusion bodies of Escherichia coli.

Papain-like cysteine proteases are widely expressed, fulfill specific functions in extracellular matrix turnover, antigen presentation and processing events, and may represent viable drug targets for major diseases. In depth and rigorous studies of the potential for these proteins to be targets for drug development require sufficient amounts of protease protein that can be used for both experimental and therapeutic purposes. Escherichia coli was widely used to express papain-like cysteine proteases, but most of those proteases are produced in insoluble inclusion bodies that need solubilizing, refolding, purifying and activating. Refolding is the most critical step in the process of generating active cysteine proteases and the current approaches to refolding include dialysis, dilution and chromatography. Purification is mainly achieved by various column chromatography. Finally, the attained refolded proteases are examined regarding their protease structures and activities.

The Tomato yellow leaf curl virus (TYLCV) V2 protein inhibits enzymatic activity of the host papain-like cysteine protease CYP1

The viral V2 protein is one of the key factors that Tomato yellow leaf curl geminivirus (TYLCV), a major tomato pathogen worldwide, utilizes to combat the host defense. Besides suppressing the plant RNA silencing defense by targeting the host SGS3 component of the silencing machinery, V2 also interacts with the host CYP1 protein, a papain-like cysteine protease likely involved in hypersensitive response reactions. The biological effects of the V2-CYP1 interaction, however, remain unknown. We addressed this question by demonstrating that V2 inhibits the enzymatic activity of CYP1, but does not interfere with post-translational maturation of this protein.

Cysteine Protease Profiles of the Medicinal Plant Calotropis procera R. Br. revealed by de novo transcriptome analysis.

Calotropis procera R. Br., a traditional medicinal plant in India, is a promising source of commercial proteases, because the cysteine proteases from the plant exhibit high thermo-stability, broad pH optima, and plasma-clotting activity. Though several proteases such as Procerain, Procerain B, CpCp-1, CpCp-2, and CpCp-3 have been isolated and characterized, the information of their transcripts is limited to cDNAs encoding their mature peptides. Due to this limitation, in this study, to determine the cDNA sequences encoding full open reading frame of these cysteine proteases, transcripts were sequenced with an Illumina Hiseq2000 sequencer. A total of 171,253,393 clean reads were assembled into 106,093 contigs with an average length of 1,614 bp and an N50 of 2,703 bp, and 70,797 contigs with an average length of 1,565 bp and N50 of 2,082 bp using Trinity and Velvet-Oases software, respectively. Among these contigs, we found 20 unigenes related to papain-like cysteine proteases by BLASTX analysis against a non-redundant NCBI protein database. Our expression analysis revealed that the cysteine protease contains an N-terminal pro-peptide domain (inhibitor region), which is necessary for correct folding and proteolytic activity. It was evident that expression yields using an inducible T7 expression system in Escherichia coli were considerably higher with the pro-peptide domain than without the domain, which could contribute to molecular cloning of the Calotropis procera protease as an active form with correct folding.

Functional characterization of a cystatin from the tick Rhipicephalus haemaphysaloides.

BackgroundTicks and tick-borne diseases affect animal and human health worldwide and cause significant economic losses in the animal industry. Functional molecular research is important to understand the biological characteristics of ticks at the molecular level. Enzymes and enzyme inhibitory molecules play very important roles in tick physiology, and the cystatins are tight-binding inhibitors of papain-like cysteine proteases. To this end, a novel cystatin, designated RHcyst-1, was isolated from the tick Rhipicephalus haemaphysaloides.MethodsThe full-length gene of RHcyst-1 was cloning by RACE. The recombinant protein of RHcyst-1 was expressed in a glutathione S-transferase (GST)-fused soluble form in Escherichia coli, and its inhibitory activity against cathepsin L, B, C, H, and S, as well as papain, was identified by fluorogenic substrate analysis. Expression analysis of RHcyst-1 at different tick stages was performed by quantitative reverse transcription - PCR (qRT-PCR). An RNAi experiment for RHcyst-1 was performed to determine its function for tick physiology.ResultsThe full-length cDNA of RHcyst-1 is 471 bp, including an intact open reading frame encoding an expected protein of 98 amino acids, without a signal peptide, having a predicted molecular weight of ~11 kDa and an isoelectric point of 5.66. A sequence analysis showed that it has significant homology with the known type 1 cystatins. The results of proteinase inhibition assays showed that rRHcyst-1 can effectively inhibit the six cysteine proteases’ enzyme activities. An investigation of the RHcyst-1 genes’ expression profile showed that it was more richly transcribed in the embryo (egg) stage. A disruption of the RHcyst-1 gene showed a significant decrease in the rate of tick hatching.ConclusionsOur results suggested that RHcyst-1 may be involved in the early embryonic development of ticks.

Dental Abnormalities Caused by Novel Compound Heterozygous CTSK Mutations

Cathepsin K (CTSK) is an important protease responsible for degrading type I collagen, osteopontin, and other bone matrix proteins. The mutations in the CTSK gene can cause pycnodysostosis (OMIM 265800), a rare autosomal recessive bone dysplasia. Patients with pycnodysostosis have been reported to present specific dental abnormalities; however, whether these dental abnormalities are related to dysfunctional CTSK has never been reported. Here we investigated the histologic changes of cementum and alveolar bone in a pycnodysostosis patient, caused by novel compound heterozygous mutations in the CTSK gene (c.87 G>A p.W29X and c.848 A>G p.Y283C). The most impressive manifestations in tooth were extensive periradicular high-density clumps with unclear periodontal space by orthopantomography examination and micro–computed tomography scanning analysis. Hematoxylin/eosin and toluidine blue staining and atomic force microscopy analysis showed that the cementum became significantly thickened, softened, and full of cementocytes. The disorganized bone structure was the main character of alveolar bone. The p.W29X mutation may represent the loss-of-function allele with an earlier termination codon in the precursor CTSK polypeptide. Residue Y283 is highly conserved among papain-like cysteine proteases. Three-dimensional structure modeling analysis found that the loss of the hydroxybenzene residue in the Y283C mutation would interrupt the hydrogen network and possibly affect the self-cleavage of the CTSK enzyme. Furthermore, p.Y283C mutation did not affect the mRNA and protein levels of overexpressed CTSK in COS-7 system but did reduce CTSK enzyme activity. In conclusion, the histologic and ultrastructural changes of cementum and alveolar bone might be affected by CTSK mutation via reduction of its enzyme activity (clinical trial registration: ChiCTR-TNC-10000876).

Ectopic phytocystatin expression increases nodule numbers and influences the responses of soybean (Glycine max) to nitrogen deficiency

Cysteine proteases and cystatins have many functions that remain poorly characterised, particularly in crop plants. We therefore investigated the responses of these proteins to nitrogen deficiency in wild-type soybeans and in two independent transgenic soybean lines (OCI-1 and OCI-2) that express the rice cystatin, oryzacystatin-I (OCI). Plants were grown for four weeks under either a high (5mM) nitrate (HN) regime or in the absence of added nitrate (LN) in the absence or presence of symbiotic rhizobial bacteria. Under the LN regime all lines showed similar classic symptoms of nitrogen deficiency including lower shoot biomass and leaf chlorophyll. However, the LN-induced decreases in leaf protein and increases in root protein tended to be smaller in the OCI-1 and OCI-2 lines than in the wild type. When LN-plants were grown with rhizobia, OCI-1 and OCI-2 roots had significantly more crown nodules than wild-type plants. The growth nitrogen regime had a significant effect on the abundance of transcripts encoding vacuolar processing enzymes (VPEs), LN-dependent increases in VPE2 and VPE3 transcripts in all lines. However, the LN-dependent increases of VPE2 and VPE3 transcripts were significantly lower in the leaves of OCI-1 and OCI-2 plants than in the wild type. These results show that nitrogen availability regulates the leaf and root cysteine protease, VPE and cystatin transcript profiles in a manner that is in some cases influenced by ectopic OCI expression. Moreover, the OCI-dependent inhibition of papain-like cysteine proteases favours increased nodulation and enhanced tolerance to nitrogen limitation, as shown by the smaller LN-dependent decreases in leaf protein observed in the OCI-1 and OCI-2 plants relative to the wild type.

The Arabidopsis thaliana papain-like cysteine protease RD21 interacts with a root-knot nematode effector protein

The root-knot nematode Meloidogyne chitwoodi secretes effector proteins into the cells of host plants to manipulate plant-derived processes in order to achieve successful parasitism. Mc1194 is a M. chitwoodi effector that is highly expressed in pre-parasitic second-stage juvenile nematodes. Yeast two-hybrid assays revealed Mc1194 specifically interacts with a papain-like cysteine protease (PLCP), RD21A in Arabidopsis thaliana. Mc1194 interacts with both the protease and granulin domains of RD21A. PLCPs are targeted by effectors secreted by bacterial, fungal and oomycete pathogens and the hypersusceptibility of rd21-1 mutants to M. chitwoodi indicates RD21A plays a role in plant-parasitic nematode infection.

Proteomics of nitrogen remobilization in poplar bark.

Seasonal nitrogen (N) cycling in temperate deciduous trees involves the accumulation of bark storage proteins (BSPs) in phloem parenchyma and xylem ray cells. BSPs are anabolized using recycled N during autumn leaf senescence and later become a source of N during spring shoot growth as they are catabolized. Little is known about the catabolic processes involved in remobilization and reutilization of N from BSPs in trees. In this study, we used multidimensional protein identification technology (MudPIT) and spectral counting to identify protein changes that occur in the bark during BSP catabolism. A total of 4,178 proteins were identified from bark prior to and during BSP catabolism. The majority (62%) of the proteins were found during BSP catabolism, indicating extensive remodeling of the proteome during renewed shoot growth and N remobilization. Among these proteins were 30 proteases, the relative abundances of which increased during BSP catabolism. These proteases spanned a range of families including members of the papain-like cysteine proteases, serine carboxypeptidases, and aspartyl proteases. These data identify, for the first time, candidate proteases that could potentially provide hydrolase activity required for N remobilization from BSPs and provide the foundation for research to advance our knowledge of poplar N cycling.

The diversity of rice phytocystatins.

Phytocystatins encompass a family of plant competitive cysteine proteinase inhibitors. They are encoded by part of a conserved monophyletic group of genes that are found in all eukaryotes. The primary targets of phytocystatins are papain-like cysteine proteinases. However, a group of larger phytocystatins is also able to inhibit proteinases such as legumains. Phytocystatins have been implicated in several physiological processes and act within an intricate proteolytic regulatory network. The present work characterizes the gene family of rice phytocystatins, which contains twelve genes with different features. Phylogenetic analyses cluster rice phytocystatins into three main groups. Group 1 is composed of OcI, OcIII and OcXII and is nearly ubiquitous and highly expressed in plants under normal and stressed conditions including salt, wounding, ABA or a fungal elicitor such as chitosan. Rice phytocystatins can contribute to plant senescence and may exhibit an inverse correlation between their gene expression and the activities of their target proteinases. This work contributes to clarifying the roles of individual phytocystatin genes in plant processes such as germination and response to environmental stresses.

PIRIN2 stabilizes cysteine protease XCP2 and increases susceptibility to the vascular pathogen Ralstonia solanacearum in Arabidopsis.

PIRIN (PRN) is a member of the functionally diverse cupin protein superfamily. There are four members of the Arabidopsis thaliana PRN family, but the roles of these proteins are largely unknown. Here we describe a function of the Arabidopsis PIRIN2 (PRN2) that is related to susceptibility to the bacterial plant pathogen Ralstonia solanacearum. Two prn2 mutant alleles displayed decreased disease development and bacterial growth in response to R. solanacearum infection. We elucidated the underlying molecular mechanism by analyzing PRN2 interactions with the papain-like cysteine proteases (PLCPs) XCP2, RD21A, and RD21B, all of which bound to PRN2 in yeast two-hybrid assays and in Arabidopsis protoplast co-immunoprecipitation assays. We show that XCP2 is stabilized by PRN2 through inhibition of its autolysis on the basis of PLCP activity profiling assays and enzymatic assays with recombinant protein. The stabilization of XCP2 by PRN2 was also confirmed in planta. Like prn2 mutants, an xcp2 single knockout mutant and xcp2 prn2 double knockout mutant displayed decreased susceptibility to R. solanacearum, suggesting that stabilization of XCP2 by PRN2 underlies susceptibility to R. solanacearum in Arabidopsis.

Characterisation of a novel hypovirus from Sclerotinia sclerotiorum potentially representing a new genus within the Hypoviridae

A novel mycovirus tentatively assigned the name Sclerotinia sclerotiorum hypovirus 2 (SsHV2/5472) was detected in the phytopathogenic fungus Sclerotinia sclerotiorum. The genome is 14581 nucleotides (nts) long, excluding the poly (A) tail. A papain-like cysteine protease (Pro), an RNA-dependent RNA polymerase (RdRp) and a helicase (Hel) domain were detected in the polyprotein. Phylogenetic analysis based on multiple alignments of the aa sequence of the polyprotein placed it in a distinct clade from Alphahypovirus and Betahypovirus. The distinct aa sequence plus the fact that SsHV2/5472 possesses the longest reported genome for a hypovirus, suggests that SsHV2/5472 may represent a new genus in the family Hypoviridae. Eliminating SsHV2/5472 from S. sclerotiorum significantly increased the virulence of the protoplast virus-free derivative 5472-P5, although SsHV/5472-containing isolates showed significant variation in their virulence. In addition, membrane-bound vesicles (25–50nm) were observed in ultrathin mycelial sections of SsHV2/5472 containing isolates but not in SsHV2/5472-free isolate.

Hepatitis E virus inhibits type I interferon induction by ORF1 products.

Hepatitis E virus (HEV) causes both endemic and epidemic human hepatitis by fecal-oral transmission in many parts of the world. Zoonotic transmission of HEV from animals to humans has been reported. Due to the lack of an efficient cell culture system, the molecular mechanisms of HEV infection remain largely unknown. In this study, we found that HEV replication in hepatoma cells inhibited poly(I·C)-induced beta interferon (IFN-β) expression and that the HEV open reading frame 1 (ORF1) product was responsible for this inhibition. Two domains, X and the papain-like cysteine protease domain (PCP), of HEV ORF1 were identified as the putative IFN antagonists. When overexpressed in HEK293T cells, the X domain (or macro domain) inhibited poly(I·C)-induced phosphorylation of interferon regulatory factor 3 (IRF-3), which is the key transcription factor for IFN induction. The PCP domain was shown to have deubiquitinase activity for both RIG-I and TBK-1, whose ubiquitination is a key step in their activation in poly(I·C)-induced IFN induction. Furthermore, replication of a HEV replicon containing green fluorescent protein (GFP) (E2-GFP) in hepatoma cells led to impaired phosphorylation of IRF-3 and reduced ubiquitination of RIG-I and TBK-1, which confirmed our observations of X and PCP inhibitory effects in HEK293T cells. Altogether, our study identified the IFN antagonists within the HEV ORF1 polyprotein and expanded our understanding of the functions of several of the HEV ORF1 products, as well as the mechanisms of HEV pathogenesis. Importance: Type I interferons (IFNs) are important components of innate immunity and play a crucial role against viral infection. They also serve as key regulators to evoke an adaptive immune response. Virus infection can induce the synthesis of interferons; however, viruses have evolved many strategies to antagonize the induction of interferons. There is little knowledge about how hepatitis E virus (HEV) inhibits induction of host IFNs, though the viral genome was sequenced more than 2 decades ago. This is the first report of identification of the potential IFN antagonists encoded by HEV. By screening all the domains in the open reading frame 1 (ORF1) polyprotein, we identified two IFN antagonists and performed further research to determine how and at which step in the IFN induction pathway they antagonize host IFN induction. Our work provides valuable information about HEV-cell interaction and pathogenesis.

Hepatitis E virus (HEV) protease: a chymotrypsin-like enzyme that processes both non-structural (pORF1) and capsid (pORF2) protein.

Hepatitis E virus (HEV), a major cause of acute viral hepatitis across the world, is a non-enveloped, plus-strand RNA virus. Its genome codes three proteins, pORF1 (multifunctional polyprotein), pORF2 (capsid protein) and pORF3 (multi-regulatory protein). pORF1 encodes methyltransferase, putative papain-like cysteine protease, helicase and replicase enzymes. Of these, the protease domain has not been characterized. On the basis of sequence analysis, we cloned and expressed a protein covering aa 440-610 of pORF1, expression of which led to cell death in Escherichia coli BL-21 and Huh7 hepatoma cells. Finally, we expressed and purified this protein from E. coli C43 cells (resistant to toxic proteins). The refolded form of this protein showed protease activity in gelatin zymography. Digestion assays showed cleavage of both pORF1 and pORF2 as observed previously. MS revealed digestion of capsid protein at both the N and C termini. N-terminal sequencing of the ~35 kDa methyltransferase, ~35 kDa replicase and ~56 kDa pORF2 proteins released by protease digestion revealed that the cleavage sites were alanine15/isoleucine16, alanine1364/valine1365 in pORF1 and leucine197/valine198 in pORF2. Specificity of these cleavage sites was validated by site-directed mutagenesis. Further characterization of the HEV protease, carried out using twelve inhibitors, showed chymostatin and PMSF to be the most efficient inhibitors, indicating this protein as a chymotrypsin-like protease. The specificity was further confirmed by cleavage of the chymotrypsin-specific fluorogenic peptide N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin. Mutational analysis of the conserved serine/cysteine/histidine residues suggested that H443 and C472/C481/C483 are possibly the active site residues. To our knowledge, this is the first direct demonstration of HEV protease and its function.

The cysteine protease CEP1, a key executor involved in tapetal programmed cell death, regulates pollen development in Arabidopsis.

Tapetal programmed cell death (PCD) is a prerequisite for pollen grain development in angiosperms, and cysteine proteases are the most ubiquitous hydrolases involved in plant PCD. We identified a papain-like cysteine protease, CEP1, which is involved in tapetal PCD and pollen development in Arabidopsis thaliana. CEP1 is expressed specifically in the tapetum from stages 5 to 11 of anther development. The CEP1 protein first appears as a proenzyme in precursor protease vesicles and is then transported to the vacuole and transformed into the mature enzyme before rupture of the vacuole. cep1 mutants exhibited aborted tapetal PCD and decreased pollen fertility with abnormal pollen exine. A transcriptomic analysis revealed that 872 genes showed significantly altered expression in the cep1 mutants, and most of them are important for tapetal cell wall organization, tapetal secretory structure formation, and pollen development. CEP1 overexpression caused premature tapetal PCD and pollen infertility. ELISA and quantitative RT-PCR analyses confirmed that the CEP1 expression level showed a strong relationship to the degree of tapetal PCD and pollen fertility. Our results reveal that CEP1 is a crucial executor during tapetal PCD and that proper CEP1 expression is necessary for timely degeneration of tapetal cells and functional pollen formation.

Cysteine Protease 51 (CP51), an anther-specific cysteine protease gene, is essential for pollen exine formation in Arabidopsis

Cysteine proteases play important roles in intracellular protein degradation, programmed cell death and responses to environmental stimuli in plant. Although subclassification and biochemical analysis of major plant papain-like cysteine proteases (PLCPs) have been studied, the biological function of many PLCPs remained unknown. In this study, we identified a PLCP gene Cysteine Protease 51 (CP51) which participates in exine formation and anther development in Arabidopsis thaliana. Promoter-GUS fusion detection showed its specific expression in anthers at stages 9–12. RNA interference (RNAi) transgenic plants with reduced CP51 transcriptional levels exhibited a male sterile phenotype with aborted microspores, shortened siliques and fewer or no seeds. Cytological analysis indicated that pollen abortion occurred due to defective pollen exine and the tapetum degraded earlier during the transition from the uninucleated stage to the binucleated stage. Scanning electron microscopy demonstrated that aborted microspores lacked complete or normal reticulate exine, and the intine membrane was extruded in the pollens of CP51-RNAi plants. Transmission electron microscopy further revealed that the tapetum degeneration was initiated early and that normal tectum connections to the bacula were missing in anthers of CP51-RNAi plants. Taken together, these results suggested that CP51 critically mediates tapetum stability and pollen exine formation.

Overexpression of the protease inhibitor BoCPI-1 in broccoli delays chlorophyll loss after harvest and causes down-regulation of cysteine protease gene expression

Papain-like cysteine proteases are involved in many physiological processes in vascular plants, including senescence and programmed cell death. Here we report the isolation of a cysteine protease inhibitor (BoCPI-1) from broccoli (Brassica oleracea var. italica), and characterise its role in regulating protease activity. Biochemical analysis showed BoCPI-1 had inhibitory activity against papain. Broccoli was genetically modified to overexpress BoCPI-1, and both azocasein assays (which provide a relatively crude measure of total protease activity), together with DCG-04 assays (which allow more targeted analysis of cysteine protease activity), were used to examine the function of the inhibitor during postharvest senescence. In broccoli heads, overexpression of BoCPI-1 reduced total protease activity, retained cellular soluble protein content and delayed the onset of postharvest senescence as measured by chlorophyll loss. Up-regulating the expression of BoCPI-1 resulted in a lower mRNA accumulation of five different senescence-associated cysteine protease genes (BoCP1, BoCP2, BoCP3, BoCP4, BoCP5). The link between the transcription of a cysteine protease inhibitor (a phytocystatin) and the transcription of the structurally unrelated cysteine proteases suggests that the changed cysteine protease mRNA accumulation patterns are the result of a feedback loop that is regulated by cellular protease activity.

Computational study on substrate specificity of a novel cysteine protease 1 precursor from Zea mays.

Cysteine protease 1 precursor from Zea mays (zmCP1) is classified as a member of the C1A family of peptidases (papain-like cysteine protease) in MEROPS (the Peptidase Database). The 3D structure and substrate specificity of the zmCP1 is still unknown. This study is the first one to build the 3D structure of zmCP1 by computer-assisted homology modeling. In order to determine the substrate specificity of zmCP1, docking study is used for rapid and convenient analysis of large populations of ligand–enzyme complexes. Docking results show that zmCP1 has preference for P1 position and P2 position for Arg and a large hydrophobic residue (such as Phe). Gly147, Gly191, Cys189, and Asp190 are predicted to function as active residues at the S1 subsite, and the S2 subsite contains Leu283, Leu193, Ala259, Met194, and Ala286. SIFt results indicate that Gly144, Arg268, Trp308, and Ser311 play important roles in substrate binding. Then Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) method was used to explain the substrate specificity for P1 position of zmCp1. This study provides insights into the molecular basis of zmCP1 activity and substrate specificity.