Comparative analysis of the alkali-liberated components of the Hyphantria cunea and the Diacrisia virginica granulosis viruses

The biochemical and biophysical characteristics of the closely related Diacrisia virginica and Hyphantria cunea granulosis virus isolates were examined. Sucrose gradient sedimentation patterns of alkali-solubilized DGV and HcGV capsules were identical. The top, middle, and bottom fractions from either viral isolate were infectious when injected into susceptible host larvae. Electrophoretic analysis of alkaline-solubilized granulin extracts demonstrated that both viruses contain alkaline proteolytic activity. The major granulin protein (～28,000 daltons) of both isolates comigrated in a SDS-PAGE. Electrophoretic separation of the virus proteins demonstrated some quantitative differences between the two granulosis viruses. The enveloped nucleocapsids and the nucleocapsids of the two viruses were morphologically indistinguishable.     

Structure dissection of human progranulin identifies well-folded granulin/epithelin modules with unique functional activities

Progranulin is a secreted protein with important functions in several physiological and pathological processes, such as embryonic development, host defense, and wound repair. Autosomal dominant mutations in the progranulin gene cause frontotemporal dementia, while overexpression of progranulin promotes the invasive progression of a range of tumors, including those of the breast and the brain. Structurally, progranulin consists of seven-and-a-half tandem repeats of the granulin/epithelin module (GEM), several of which have been isolated as discrete 6-kDa GEM peptides. We have expressed all seven human GEMs using recombinant DNA in Escherichia coli. High-resolution NMR showed that only the three GEMs, hGrnA, hGrnC, and hGrnF, contain relatively well-defined three-dimensional structures in solution, while others are mainly mixtures of poorly structured disulfide isomers. The three-dimensional structures of hGrnA, hGrnC, and hGrnF contain a stable stack of two beta-hairpins in their N-terminal subdomains, but showed a more flexible C-terminal subdomain. Interestingly, of the well-structured GEMs, hGrnA demonstrated potent growth inhibition of a breast cancer cell line, while hGrnF was stimulatory. Poorly folded peptides were either weakly inhibitory or without activity. The functionally active and structurally well-characterized human hGrnA offers a unique opportunity for detailed structure-function studies of these important GEM proteins as novel members of mammalian growth factors.     

棉铃虫核型多角体病毒多角体蛋白聚集体特性及与其它包涵体蛋白的血清学关系

纯化的多角体碱解释放多角体蛋白,经等电点沉淀和柱层析对多角体蛋白进行分离纯化,结合SDS-PAGE、免疫双向扩散、免疫电镜等方法,证明棉铃虫核型多角体病毒(HaNPV)的多角体蛋白以聚集体形式存在。用ELISA法检测包涵体蛋白之间的血清学关系,结果表明,与黄地老虎颗粒体病毒(AsGV)和粘虫颗粒体病毒(PsGV)颗粒体蛋白相比较,HaNPV多角体蛋白与葡萄天蛾核型多角体病毒(ArNPV)和黄地老虎核型多角体病毒(AsNPV)多角体蛋白之间的血清学关系更为密切。     

Solution structures of a 30-residue amino-terminal domain of the carp granulin-1 protein and its amino-terminally truncated 3-30 subfragment: implications for the conformational stability of the stack of two beta-hairpins

Carp granulins are members of an emerging class of proteins with a sequence motif encoding a parallel stack of two to four beta-hairpins. The carp granulin-1 protein forms a stack of four beta-hairpins, whereas its amino-terminal fragment appears to adopt a very stable stack of two beta-hairpins in solution. Here we determined a refined three-dimensional structure of this peptide fragment to examine potential conformational changes compared with the full-length protein. The structures were calculated with both a traditional method and a fast semiautomated method using ambiguous NMR distance restraints. The resulting sets of structures are very similar and show that a well-defined stack of two beta-hairpins is retained in the peptide. Conformational rearrangements compensating the loss of the carboxy-terminal subdomain of the native protein are restricted to the carboxy-terminal end of the peptide, the turn connecting the two beta-hairpins, and the Tyr(21) and Tyr(25) aromatic side chains. Further removal of the Val(1) and Ile(2) residues, which are part of the first beta-hairpin and components of two major hydrophobic clusters in the two beta-hairpin structure, results in the loss of the first beta-hairpin. The second beta-hairpin, which is closely associated with the first, retains a similar but somewhat less stable conformation. The invariable presence of the second beta-hairpin and the dependence of its stability on the first beta-hairpin suggest that the stack of two beta-hairpins may be an evolutionary conserved and autonomous folding unit. In addition, the high conformational stability makes the stack of two beta-hairpins an attractive scaffold for the development of peptide-based drug candidates.     

Disulfide bonds and disorder in granulin‐3: An unusual handshake between structural stability and plasticity

Granulins (GRNs) are a family of small (～6 kDa) proteins generated by the proteolytic processing of their precursor, progranulin (PGRN), in many cell types. Both PGRN and GRNs are implicated in a plethora of biological functions, often in opposing roles to each other. Lately, GRNs have generated significant attention due to their implicated roles in neurodegenerative disorders. Despite their physiological and pathological significance, the structure‐function relationships of GRNs are poorly defined. GRNs contain 12 conserved cysteines forming six intramolecular disulfide bonds, making them rather exceptional, even among a few proteins with high disulfide bond density. Solution NMR investigations in the past have revealed a unique structure containing putative interdigitated disulfide bonds for several GRNs, but GRN‐3 was unsolvable due to its heterogeneity and disorder. In our previous report, we showed that abrogation of disulfide bonds in GRN‐3 renders the protein completely disordered (Ghag et al., Prot Eng Des Sel 2016). In this study, we report the cellular expression and biophysical analysis of fully oxidized, native GRN‐3. Our results indicate that both E. coli and human embryonic kidney (HEK) cells do not exclusively make GRN‐3 with homogenous disulfide bonds, likely due to the high cysteine density within the protein. Biophysical analysis suggests that GRN‐3 structure is dominated by irregular loops held together only by disulfide bonds, which induced remarkable thermal stability to the protein despite the lack of regular secondary structure. This unusual handshake between disulfide bonds and disorder within GRN‐3 could suggest a unique adaptation of intrinsically disordered proteins towards structural stability.     

Retracted: Upregulation of microRNA-140-3p inhibits epithelial-mesenchymal transition,invasion, and metastasis of hepatocellular carcinoma through inactivation of the MAPK signaling pathway by targeting GRN

Invasion and metastasis in hepatocellular carcinoma (HCC) results in poor prognosis. Human intervention in these pathological processes may benefit the treatment of HCC. The aim of the present study is to elucidate the mechanism of miR-140-3p affecting epithelial-mesenchymal transition (EMT), invasion, and metastasis in HCC. Microarray analysis was performed for differentially expressed genes screening. The target relationship between miR-140-3p and GRN was analyzed. Small interfering RNA (siRNA) against granulin (GRN) was synthesized. EMT markers were detected, and invasion and migration were evaluated in HCC cells introduced with a miR-140-3p inhibitor or mimic, or siRNA against GRN. A mechanistic investigation was conducted for the determination of mitogen-activated protein kinase (MAPK) signaling pathway-related genes and EMT markers (E-cadherin, N-cadherin, and Vimentin). GRN was highlighted as an upregulated gene in HCC. GRN was a target gene of miR-140-3p. Elevation of miR-140-3p or inhibition of GRN restrained the EMT process and suppressed the HCC cell migration and invasion. HCC cells treated with the miR-140-3p mimic or siRNA-GRN exhibited decreased GRN expression and downregulated the expressions of the MAPK signaling pathway-related genes, N-cadherin, and Vimentin but upregulated the expression of E-cadherin. GRN silencing can reverse the activation of the MAPK signaling pathway and induction of EMT mediated by miR-140-3p inhibition. Taken together, the results show that miR-140-3p confers suppression of the MAPK signaling pathway by targeting GRN, thus inhibiting EMT, invasion, and metastasis in HCC.     

Glutenase and collagenase activities of wheat cysteine protease Triticain-α: Feasibility for enzymatic therapy assays

Insufficient and/or improper protein degradation is associated with the development of various human pathologies. Enzymatic therapy with proteolytic enzymes aimed to improve insufficient proteolytic activity was suggested as a treatment of protease deficiency-induced disorders. Since in many cases human degradome is incapable of degrading the entire target protein(s), other organisms can be used as a source of proteases exhibiting activities distinct from human enzymes, and plants are perspective candidates for this source. In this study recombinant wheat cysteine protease Triticain-α was shown to refold in vitro into an autocatalytically activated proteolytic enzyme possessing glutenase and collagenase activities at acidic (or close to neutral) pH levels at the temperature of human body. Mass-spectrometry analysis of the products of Triticain-α-catalyzed gluten hydrolysis revealed multiple cleavage sites within the sequences of gliadin toxic peptides, in particular, in the major toxic 33-mer α-gliadin-derived peptide initiating inflammatory responses to gluten in celiac disease (CD) patients. Triticain-α was found to be relatively stable in the conditions simulating stomach environment. We conclude that Triticain-α can be exploited as a basic compound for development of (i) pharmaceuticals for oral administration aimed at release of the active enzyme into the gastric lumen for CD treatment, and (ii) topically active pharmaceuticals for wound debridement applications.     

The miR-15/107 Group of MicroRNA Genes: Evolutionary Biology, Cellular Functions, and Roles in Human Diseases

The miR-15/107 group of microRNA (miRNA) gene is increasingly appreciated to serve key functions in humans. These miRNAs regulate gene expression involved in cell division, metabolism, stress response, and angiogenesis in vertebrate species. The miR-15/107 group has also been implicated in human cancers, cardiovascular disease and neurodegenerative disease, including Alzheimer's disease. Here we provide an overview of the following: (1) the evolution of miR-15/107 group member genes; (2) the expression levels of miRNAs in mammalian tissues; (3) evidence for overlapping gene-regulatory functions by different miRNAs; (4) the normal biochemical pathways regulated by miR-15/107 group miRNAs; and (5) the roles played by these miRNAs in human diseases. Membership in this group is defined based on sequence similarity near the mature miRNAs' 5′ end: all include the sequence AGCAGC. Phylogeny of this group of miRNAs is incomplete; thus, a definitive taxonomic classification (e.g., designation as a “superfamily”) is currently not possible. While all vertebrates studied to date express miR-15a, miR-15b, miR-16, miR-103, and miR-107, mammals alone are known to express miR-195, miR-424, miR-497, miR-503, and miR-646. Multiple different miRNAs in the miR-15/107 group are expressed at moderate to high levels in human tissues. We present data on the expression of all known miR-15/107 group members in human cerebral cortical gray matter and white matter using new miRNA profiling microarrays. There is extensive overlap in the mRNAs targeted by miR-15/107 group members. We show new data from cultured H4 cancer cells that demonstrate similarities in mRNAs targeted by miR-16 and miR-103 and also support the importance of the mature miRNAs' 5′ seed region in mRNA target recognition. In conclusion, the miR-15/107 group of miRNA genes is a fascinating topic of study for evolutionary biologists, miRNA biochemists, and clinically oriented translational researchers alike.