As a potent autophagy inducer, Beclin 1 is essential for the initiation of autophagic cell death, and triggering extensive autophagy by targeted delivery of Beclin 1 to tumors has enormous potential to inhibit tumor growth. Yet, the therapeutic application of Beclin 1 is hampered by its inability to internalize into cells and nonselective biodistribution in vivo. To tackle this challenge, we employed a novel Beclin 1 delivery manner by constructing a functional protein (Trx-pHLIP-Beclin 1, TpB) composed of a thioredoxin (Trx) tag, a pH low insertion peptide (pHLIP), and an evolutionarily conserved motif of Beclin 1. This protein could effectively transport Beclin 1 to breast and ovarian cancer cell lines under weakly acidic conditions (pH 6.5), markedly inhibit tumor cell growth and proliferation, and induce obvious autophagy. Furthermore, the in vivo antitumor efficacy of the functional Beclin 1 against an SKOV3 xenograft tumor mouse model was tested via intravenous injection. TpB preferentially accumulated in tumors and exhibited a significantly higher tumor growth inhibition than the nontargeted Beclin 1 control, whereas no overt side effects were observed. Taken together, this study sheds light on the potential application of TpB as a highly efficient yet safe antitumor agent for cancer treatment.

Gelonin is a plant toxin that exerts potent cytotoxic activity by inactivation of the 60S ribosomal subunit. The high-level expression of soluble gelonin still remains a great challenge and there was no detailed biophysical analysis of gelonin from Escherichia coli (E. coli) yet. In this study, the soluble and high-yield expression of recombinant gelonin (rGel) was achieved in E. coli BL21 (DE3) for the first time, with a yield of 6.03 mg/L medium. Circular dichroism (CD) analysis indicated that rGel consisted of 21.7% α-helix, 26.3% β-sheet, 18.5% β-turn, and 32.3% random coil, and it could maintain its secondary structure up to 60 °C. The antitumor activity of rGel was evaluated in two colon cancer cell lines-HCT116 and HCT-8, and it was clearly demonstrated that rGel exerted antiproliferative activity against these two cell lines by inhibiting cellular protein synthesis. These findings provide insights for researchers involved in the expression of similar biotoxins, and the biophysical characterizations of gelonin will favor its further therapeutic applications.

Glucose-regulated protein of 78 kDa (GRP78) has become an attractive and novel target for tumor therapy. Design and construction of powerful delivery systems that could efficiently transport doxorubicin (DOX) to a tumor-cell nucleus remains a formidable challenge for improving the tumor therapeutic index and mitigating side effects to normal tissues. Herein, a novel doxorubicin prodrug (NDP) with GRP78 recognition and nucleus-targeting ability was synthesized by a facile chemical route. NDP exhibited an enhanced antiproliferative activity against colorectal cancer cells and could efficiently enter the cell nucleus. Furthermore, it is inspiring to note that NDP displayed a much stronger inhibitory efficacy against the growth of colorectal cancer xenografts in nude mice than free DOX and showed superior in vivo safety. Together, the work provides a novel GRP78 and nucleus-targeting strategy, and the NDP holds great promise to be used as a potent and safe chemotherapeutic agent.

Flexible NC-loops between the catalytic domain and the cap domain of the α/β hydrolase fold enzymes show remarkable diversity in length, sequence, and configuration. Recent investigations have suggested that the NC-loop might be involved in catalysis and substrate recognition in many enzymes from the α/β hydrolase fold superfamily. To foster a deep understanding of its role in catalysis, stability, and divergent evolution, we here systemically investigated the function of the NC-loop (residues 131–151) in a lipase (FClip1) from thermophilic bacterium Fervidobacterium changbaicum by loop deletion, alanine-scanning mutagenesis and site-directed mutagenesis. We found that the upper part of the NC-loop (residues 131–138) was of great importance to enzyme catalysis. Single substitutions in this region could fine-tune the activity of FClip1 as much as 41-fold, and any deletions from this region rendered the enzyme completely inactive. The lower part of the NC-loop (residues 139–151) was capable of enduring extensive deletions without loss of activity. The shortened mutants in this region were found to show both improved activity and increased stability simultaneously. We therefore speculated that the NC-loop, especially the lower part, would be a perfect target for enzyme engineering to optimize the enzymatic properties, and might present a hot zone for the divergent evolution of α/β hydrolases. Our findings may provide an opportunity for better understanding of the mechanism of divergent evolution in the α/β hydrolase fold superfamily, and may also guide the design of novel biocatalysts for industrial applications.