A Novel Cysteine Protease Inhibitor of Naegleria fowleri That Is Specifically Expressed during Encystation and at Mature Cysts

Naegleria fowleri is a free-living amoeba that is ubiquitous in diverse natural environments. It causes a fatal brain infection in humans known as primary amoebic meningoencephalitis. Despite the medical importance of the parasitic disease, there is a great lack of knowledge about the biology and pathogenicity of N. fowleri. In this study, we identified and characterized a novel cysteine protease inhibitor of N. fowleri (NfCPI). NfCPI is a typical cysteine protease inhibitor belonging to the cystatin family with a Gln-Val-Val-Ala-Gly (QVVAG) motif, a characteristic motif conserved in the cystatin family of proteins. Bacterially expressed recombinant NfCPI has a dimeric structure and exhibits inhibitory activity against several cysteine proteases including cathespin Bs of N. fowleri at a broad range of pH values. Expression profiles of nfcpi revealed that the gene was highly expressed during encystation and cyst of the amoeba. Western blot and immunofluorescence assays also support its high level of expression in cysts. These findings collectively suggest that NfCPI may play a critical role in encystation or cyst formation of N. fowleri by regulating cysteine proteases that may mediate encystation or mature cyst formation of the amoeba. More comprehensive studies to investigate the roles of NfCPI in encystation and its target proteases are necessary to elucidate the regulatory mechanism and the biological significance of NfCPI.

Genome-wide identification and analysis of cystatin family genes in Sorghum (Sorghum bicolor (L.) Moench)

To set a systematic study of the Sorghum cystatins (SbCys) gene family, a genome-wide analysis of the SbCys family genes was performed by bioinformatics-based methods. In total, 18 SbCys genes were identified in Sorghum, which were distributed unevenly on chromosomes, and two genes were involved in a tandem duplication event. All SbCys genes had similar exon/intron structure and motifs, indicating their high evolutionary conservation. Transcriptome analysis showed that 16 SbCys genes were expressed in different tissues, and most genes displayed higher expression levels in reproductive tissues than in vegetative tissues, indicating that the SbCys genes participated in the regulation of seed formation. Furthermore, the expression profiles of the SbCys genes revealed that seven cystatin family genes were induced during Bipolaris sorghicola infection and only two genes were responsive to aphid infestation. In addition, quantitative real-time polymerase chain reaction (qRT-PCR) confirmed that 17 SbCys genes were induced by one or two abiotic stresses (dehydration, salt, and ABA stresses). The interaction network indicated that SbCys proteins were associated with several biological processes, including seed development and stress responses. Notably, the expression of SbCys4 was up-regulated under biotic and abiotic stresses, suggesting its potential roles in mediating the responses of Sorghum to adverse environmental impact. Our results provide new insights into the structural and functional characteristics of the SbCys gene family, which lay the foundation for better understanding the roles and regulatory mechanism of Sorghum cystatins in seed development and responses to different stress conditions.

Sugarcane phytocystatins: Identification, classification and expression pattern analysis

The cystatins are tightly binding, but reversible, inhibitors of cysteine proteinases, which constitute a superfamily of evolutionary related proteins. They have been subdivided into three families: the cystatin family which contain two disulfide bonds, the stefin family which lack disulfide bonds, and the kininogen family composed of large glycoproteins containing three repeats similar to those found in the cystatin family. Members of the cystatin superfamily occurring in plants are currently known as phytocystatins, defined as proteins lacking disulfide bonds but possessing a conserved N-terminal amino acid sequence (L-A-R-[FY]-A-[VI]-X(3)-N). We have used the protein sequence deduced from seven phytocystatins (from the Arabidopsis thaliana genome project) and from the SUgarCane EST project (SUCEST) database to identify 25 possible sugarcane phytocystatins. Phylogenetic analysis has allowed us to cluster these phytocystatins into four distinct groups: (i) those with a characteristic N-terminal consensus, (ii) those with the same consensus plus a long C-terminal extension; (iii) those that lack the consensus but contain the highly conserved QxVxG motif found in all members of the superfamily and (iv) those that lack both the consensus and the QxVxG motif.

Structure, alternative splicing and chromosomal localization of the cystatin-related epididymal spermatogenic gene

The cystatin superfamily of cysteine protease inhibitors consists of three major families, including the stefins, cystatins and kininogens. However, the recent identification of several genes that possess sequence similarity with the cystatins but have different gene or protein structures indicates that several new cystatin families or subgroups of families might exist. We previously identified the cystatin-related epididymal spermatogenic (Cres) gene, which is related to the family 2 cystatins but exhibits highly tissue-specific expression in the reproductive tract. In the studies presented here, an analysis of gene structure as well as chromosomal mapping studies suggest that the Cres gene might represent a new subgroup within the family 2 cystatins. Although the Cres gene possesses an additional exon encoding 5ʹ untranslated sequences, its coding exons are similar in size to the three coding exons of the cystatin family 2 genes, and the Cres exon/intron splice junctions occur in identical locations as in the cystatin C gene. Furthermore, chromosomal mapping studies show that the Cres gene co-segregates with the cystatin C gene on mouse chromosome 2. Similar to the cystatin family 2 proteins, the Cres protein possesses the type A and B disulphide loops that are necessary for cystatin folding. Interestingly, Cres protein also possesses half of a type C disulphide loop. Although probably related to the cystatin genes, the Cres gene is distinct in that its promoter contains consensus motifs typical of regulated genes. Finally, reverse transcriptase-mediated PCR studies and the identification of new Cres cDNA clones indicate that the Cres mRNA is alternatively spliced, resulting in two Cres