Large-scale production of a therapeutic protein in transgenic tobacco plants: effect of subcellular targeting on quality of a recombinant dog gastric lipase

A recombinant dog gastric lipase with therapeutic potential for the treatment of exocrine pancreatic insufficiency was expressed in transgenic tobacco plants. We targeted the protein using two different signal sequences for either vacuolar retention or secretion. In both cases, an active glycosylated recombinant protein was obtained. The recombinant enzymes and the native enzyme displayed similar properties including acid resistance and acidic optimum pH. The proteolytic maturation and the specific activity of the recombinant proteins, however, were found to be dependent on subcellular compartmentalization. Expression levels of recombinant dog gastric lipase were about 5% and 7% of acid extractable plant proteins for vacuolar retention and secretion respectively. This expression system already has allowed the production of tens of grams of purified lipase through open-field culture of transgenic tobacco plants.     

Isolation and characterization of Bacillus sp. BP-6 LipA,a ubiquitous lipase among mesophilic Bacillus species

AIMS: The aim of this study was to perform the isolation, cloning and characterization of a lipase from Bacillus sp. BP-6 bearing the features of a biotechnologically important group of enzymes. METHODS AND RESULTS: Strain Bacillus sp. BP-6, showing activity on tributyrin plates, was used for isolation of lipase-coding gene lipA by means of inverse and direct PCR. The complete 633 nucleotide ORF isolated was cloned in Escherichia coli for further characterization. The amino acid sequence of the cloned protein was 98% identical to B. subtilis and B. megaterium lipases, the enzyme also showing similar molecular and biochemical features. CONCLUSIONS: The gene coding for Bacillus sp. BP-6 LipA was found in all mesophilic Bacillus species assayed, indicating its ubiquity in the genus. The cloned enzyme displayed the same properties as those of homologous lipases. SIGNIFICANCE AND IMPACT OF THE STUDY: The overall profile of Bacillus sp. BP-6 LipA was found to be that of a ubiquitous and highly conserved subfamily I.4 bacterial lipase. Previously described lipases within this family have shown to be well suited for biotechnological applications, suggesting that the cloned enzyme could be used accordingly.     

Tertiary motifs as building blocks for the design of protein‐binding peptides

Despite advances in protein engineering, the de novo design of small proteins or peptides that bind to a desired target remains a difficult task. Most computational methods search for binder structures in a library of candidate scaffolds, which can lead to designs with poor target complementarity and low success rates. Instead of choosing from pre‐defined scaffolds, we propose that custom peptide structures can be constructed to complement a target surface. Our method mines tertiary motifs (TERMs) from known structures to identify surface‐complementing fragments or “seeds.” We combine seeds that satisfy geometric overlap criteria to generate peptide backbones and score the backbones to identify the most likely binding structures. We found that TERM‐based seeds can describe known binding structures with high resolution: the vast majority of peptide binders from 486 peptide‐protein complexes can be covered by seeds generated from single‐chain structures. Furthermore, we demonstrate that known peptide structures can be reconstructed with high accuracy from peptide‐covering seeds. As a proof of concept, we used our method to design 100 peptide binders of TRAF6, seven of which were predicted by Rosetta to form higher‐quality interfaces than a native binder. The designed peptides interact with distinct sites on TRAF6, including the native peptide‐binding site. These results demonstrate that known peptide‐binding structures can be constructed from TERMs in single‐chain structures and suggest that TERM information can be applied to efficiently design novel target‐complementing binders.     

Hysteresis in poly‐2′‐deoxycytidine i‐motif folding is impacted by the method of analysis as well as loop and stem lengths

In DNA, i‐motif (iM) folds occur under slightly acidic conditions when sequences rich in 2′‐deoxycytidine (dC) nucleotides adopt consecutive dC self base pairs. The pH stability of an iM is defined by the midpoint in the pH transition (pH_T) between the folded and unfolded states. Two different experiments to determine pH_T values via circular dichroism (CD) spectroscopy were performed on poly‐dC iMs of length 15, 19, or 23 nucleotides. These experiments demonstrate two points: (1) pH_T values were dependent on the titration experiment performed, and (2) pH‐induced denaturing or annealing processes produced isothermal hysteresis in the pH_T values. These results in tandem with model iMs with judicious mutations of dC to thymidine to favor particular folds found the hysteresis was maximal for the shorter poly‐dC iMs and those with an even number of base pairs, while the hysteresis was minimal for longer poly‐dC iMs and those with an odd number of base pairs. Experiments to follow the iM folding via thermal changes identified thermal hysteresis between the denaturing and annealing cycles. Similar trends were found to those observed in the CD experiments. The results demonstrate that the method of iM analysis can impact the pH_T parameter measured, and hysteresis was observed in the pH_T and T_m values.     

A Fifth of the Protein World: Rossmann-like Proteins as an Evolutionarily Successful Structural unit

The Rossmann-like fold is the most prevalent and diversified doubly-wound superfold of ancient evolutionary origin. Rossmann-like domains are present in a variety of metabolic enzymes and are capable of binding diverse ligands. Discerning evolutionary relationships among these domains is challenging because of their diverse functions and ancient origin. We defined a minimal Rossmann-like structural motif (RLM), identified RLM-containing domains among known 3D structures (20%) and classified them according to their homologous relationships. New classifications were incorporated into our Evolutionary Classification of protein Domains (ECOD) database. We defined 156 homology groups (H-groups), which were further clustered into 123 possible homology groups (X-groups). Our analysis revealed that RLM-containing proteins constitute approximately 15% of the human proteome. We found that disease-causing mutations are more frequent within RLM domains than within non-RLM domains of these proteins, highlighting the importance of RLM-containing proteins for human health.     

In silico annotation of unreviewed acetylcholinesterase (AChE) in some lepidopteran insect pest species reveals the causes of insecticide resistance

Lepidoptera is the second most diverse insect order outnumbered only by the Coeleptera. Acetylcholinesterase (AChE) is the major target site for insecticides. Extensive use of insecticides, to inhibit the function of this enzyme, have resulted in the development of insecticide resistance. Complete knowledge of the target proteins is very important to know the cause of resistance. Computational annotation of insect acetylcholinesterase can be helpful for the characterization of this important protein. Acetylcholinesterase of fourteen lepidopteran insect pest species was annotated by using different bioinformatics tools. AChE in all the species was hydrophilic and thermostable. All the species showed lower values for instability index except L. orbonalis, S. exigua and T. absoluta. Highest percentage of Arg, Asp, Asn, Gln and Cys were recorded in P. rapae. High percentage of Cys and Gln might be reason for insecticide resistance development in P. rapae. Phylogenetic analysis revealed the AChE in T. absoluta, L. orbonalis and S. exigua are closely related and emerged from same primary branch. Three functional motifs were predicted in eleven species while only two were found in L. orbonalis, S. exigua and T. absoluta. AChE in eleven species followed secretory pathway and have signal peptides. No signal peptides were predicted for S. exigua, L. orbonalis and T. absoluta and follow non secretory pathway. Arginine methylation and cysteine palmotylation was found in all species except S. exigua, L. orbonalis and T. absoluta. Glycosylphosphatidylinositol (GPI) anchor was predicted in only nine species.     

TRIM59 predicts poor prognosis and promotes pancreatic cancer progression via the PI3K/AKT/mTOR-glycolysis signaling axis

Aberrant expression of the tripartite motif containing 59 (TRIM59) has been reported to participate in the development and progression of various human cancers. However, its expression pattern and cellular roles in pancreatic cancer (PC) remains unclear. In our study, we found that TRIM59 expression was significantly increased in PC tissues and was positively correlated with several malignant behaviors and poor overall survival of PC patients based on bioinformatics analysis and immunohistochemistry staining. Functionally, small interfering RNA–mediated TRIM59 depletion inhibited cell proliferation and migration in vitro, while TRIM59 overexpression promoted cell proliferation and migration in vitro and drove tumor growth and liver metastasis in vivo. Mechanically, TRIM59 was found to enhance glycolysis through activating the PI3K/AKT/mTOR pathway, ultimately contributing to PC progression. Taken together, our results demonstrate that TRIM59 may be a potential predictor for PC and promotes PC progression via the PI3K/AKT/mTOR-glycolysis signaling pathway, which establishes the rationale for targeting the TRIM59-related pathways to treat PC.