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Low barrier hydrogen bond (LBHB) is a special type of hydrogen bond which occurs where two heteroatoms with similar pKa values share a single proton resulting in an unusually strong and short hydrogen bond. LBHBs in protein play important roles in enzyme catalysis and maintaining protein structural integrity but its other biochemical roles are unknown. Here we report a novel function of LBHB in selectively inducing tubulin protein degradation. A tubulin inhibitor, 3-(3-Phenoxybenzyl) amino-β-carboline (PAC), promotes selective degradation of αβ-tubulin heterodimers by binding to the colchicine site of β-tubulin. Biochemical studies have revealed that PAC specifically destabilizes tubulin, making it prone to aggregation that then predisposes it to ubiquitinylation and then degradation. Structural activity analyses have indicated that the destabilization is mediated by a single hydrogen bond formed between the pyridine nitrogen of PAC and βGlu198, which is identified as a LBHB. In contrast, another two tubulin inhibitors only forming normal hydrogen bonds with βGlu198 exhibit no degradation effect. Thus, the LBHB accounts for the degradation. Most importantly, we screened for compounds capable of forming LBHB with βGlu198 and demonstrated that BML284, a Wnt signaling activator, also promotes tubulin heterodimers degradation in a PAC-like manner as expected. Our study has identified a novel approach for designing tubulin degraders, providing a unique example of LBHB function and suggests that designing small molecules to form LBHBs with protein residues resulting in the highly specific degradation of a target protein could be a new strategy for drug development.

A novel binding site on α-tubulin shows promise as a target for a new generation of anticancer drugs., Microtubules, composed of αβ-tubulin heterodimers, have remained popular anticancer targets for decades. Six known binding sites on tubulin dimers have been identified thus far, with five sites on β-tubulin and only one site on α-tubulin, hinting that compounds binding to α-tubulin are less well characterized. Cevipabulin, a microtubule-active antitumor clinical candidate, is widely accepted as a microtubule-stabilizing agent by binding to the vinblastine site. Our x-ray crystallography study reveals that, in addition to binding to the vinblastine site, cevipabulin also binds to a new site on α-tubulin. We find that cevipabulin at this site pushes the αT5 loop outward, making the nonexchangeable GTP exchangeable, which reduces the stability of tubulin, leading to its destabilization and degradation. Our results confirm the existence of a new agent binding site on α-tubulin and shed light on the development of tubulin degraders as a new generation of antimicrotubule drugs targeting this novel site.

Background Porcine cytomegalovirus (PCMV) is an immunosuppressive virus that mainly inhibits T-lymphocyte and macrophage immune functions; it has significantly damaged the farming industry. Although recent studies have shown that miRNAs play important roles in immune responses, the regulatory mechanisms of miRNAs during immunosuppressive virus infection remain unclear. Methods In this study, porcine small-RNA transcriptomes of PCMV-infected and uninfected vital organs were first characterised by high-throughput sequencing. miRDeep2 software was used to predict novel pig-encoded miRNAs. To verify the accuracy of the high-throughput sequencing results, stem-loop qRT-PCR was performed on 12 significantly DE miRNAs. The physical and functional interactions between the immune-related target genes of the DE miRNAs in PCMV-infected organs were analysed using the STRING database. Results In total, 306 annotated and 295 novel miRNAs were identified from PCMV-infected and uninfected porcine organs, respectively, through alignment with known Sus scrofa pre-miRNAs. Overall, 92, 107, 95, 77 and 111 miRNAs were significantly differentially expressed in lung, liver, spleen, kidney and thymus after PCMV infection, respectively. According to Gene Ontology enrichment analysis, target genes of the differentially expressed miRNAs associated with immune system processes, regulation of biological processes and metabolic processes were enriched in every sample. Integrated expression analysis of the differentially expressed miRNAs and their target mRNAs in PCMV-infected thymus showed that the significant differential expression of specific miRNAs under the pressure of PCMV infection in central immune organs interfered with the expression of genes involved in important immune-related signalling pathways, thus promoting the viral infection. Conclusions This is the first comprehensive analysis of the responses of host small-RNA transcriptomes to PCMV infection in vital porcine organs. It provides new insights into the regulatory mechanisms of miRNAs during infection by immunosuppressive viruses. Electronic supplementary material The online version of this article (10.1186/s12985-018-0922-x) contains supplementary material, which is available to authorized users.

Liver fibrosis is an abnormal wound healing response and a common consequence of chronic liver diseases from infection or alcohol/xenobiotic exposure. At the cellular level, liver fibrosis is mediated by trans-differentiation of hepatic stellate cells (HSCs), which is driven by persistent hepatic and systemic inflammation. However, impaired enterohepatic circulation and gut dysbiosis may indirectly contribute to the liver fibrogenesis. The composition of the gut microbiota depends on diet composition and host factors. In this study, we examined chlorophyllin, derived from green pigment chlorophyll, on gut microbiota, the intestinal mucosal barrier, and liver fibrosis. BALB/c mice received carbon tetrachloride through intraperitoneal injection to induce liver fibrosis and chlorophyllin was administrated in drinking water. The effects of chlorophyllin on liver fibrosis were evaluated for (1) survival rate, (2) hepatic morphologic analysis, (3) inflammatory factors in both the small intestine and liver, and (4) gut microbiota. Our results indicate that oral administration of chlorophyllin could attenuate intestinal and hepatic inflammation and ameliorate liver fibrosis. Importantly, oral administration of chlorophyllin promptly rebalanced the gut microbiota, exhibiting down-regulation of the phylum Firmicutes and up-regulation of the phylum Bacteroidetes. In vitro experiments on intestinal epithelial cells showed that chlorophyllin exposure could inhibit NF-κB pathway via IKK-phosphorylation suppression. In conclusion, this study demonstrates potential application of chlorophyllin to regulate the intestinal microbiota and ameliorate hepatic fibrosis.

Background Porcine torovirus (PToV) is a member of the genus Torovirus which is responsible for gastrointestinal disease in both human beings and animals with particular prevalence in youth. Torovirus infections are generally asymptomatic, however, their presence may worsen disease consequences in concurrent infections with other enteric pathogens. Methods A total of 872 diarrheic fecal samples from pigs of different ages were collected from 12 districts of Sichuan Province in the southwest of China. RT-PCR was done with PToV S gene specific primers to detect the presence of PToV positive samples. M gene specific primers were used with the PToV positive samples and the genes were sequenced. A phylogenetic tree was constructed based on the M gene nucleotide sequences from the 19 selected novel Sichuan strains and 21 PToV and BToV M gene sequences from GenBank. Results A total of 331 (37.96%, 331/872) samples were found to be positive for PToV and the highest prevalence was observed in piglets aged from 1 to 3 weeks old. Through phylogenetic inference the 40 PToV M gene containing sequences were placed into two genotypes (I & II). The 19 novel Sichuan strains of genotype I showed strong correlations to two Korean gene sequences (GU-07-56-11 and GU-07-56-22). Amino-acid sequence analysis of the 40 PToV M gene strains revealed that the M gene protein was highly conserved. Conclusions This study uncovered the presence of PToV in Sichuan Province, and demonstrated the need for continuous surveillance PToV of epidemiology.

Background Torque teno sus virus (TTSuV), infecting domestic swine and wild boar, is a non-enveloped virus with a circular, single-stranded DNA genome. which has been classified into the genera Iotatorquevirus (TTSuV1) and Kappatorquevirus (TTSuV2) of the family Anelloviridae. A molecular study was conducted to detect evidence of a phylogenic relationship between these two porcine TTSuV genogroups from the sera of 244 infected pigs located in 21 subordinate prefectures and/or cities of Sichuan. Results Both genogroups of TTSuV were detected in pig sera collected from all 21 regions examined. Of the 244 samples, virus from either genogroup was detected in 203 (83.2%), while 44 animals (18.0%) were co-infected with viruses of both genogroups. Moreover, TTSuV2 (186/244, 76.2%) was more prevalent than TTSuV1 (61/244, 25%). There was statistically significant difference between the prevalence of genogroups 1 infection alone (9.4%, 23/244) and 2 alone (64.8%, 158/244), and between the prevalence of genogroups 2 (76.2%, 186/244) and both genogroups co-infection (18.0%, 44/244). The untranslated region of the swine TTSuV genome was found to be an adequate molecular marker of the virus for detection and surveillance. Phylogenetic analysis indicated that both genogroups 1 and 2 could be further divided into two subtypes, subtype a and b. TTSuV1 subtype b and the two TTSuV2 subtypes are more prevalent in Sichuan Province. Conclusions Our study presents detailed geographical evidence of TTSuV infection in China.