A micro-evolutionary change in target binding sites as key determinant of Ultrabithorax function in Drosophila.

All Hox proteins are known to recognize, in vitro, similar DNA-binding sites containing a TAAT core sequence. This poor DNA-binding specificity is in sharp contrast with their specific functions in vivo. Here we report a new binding motif with TAAAT core sequence to which the Hox protein Ultrabithorax (Ubx) binds with higher affinity and specificity. Using transgenic and luciferase assays, we show that this new motif is critical for Ubx-mediated regulation of a target gene in Drosophila melanogaster. Interestingly, this new motif with TAAAT core sequences is not associated with the targets of Ubx in the honeybee, Apis mellifera, wherein hindwings are nearly identical to the forewings. We show that introduction of TAAAT motif in the place of TAAT motif is sufficient to bring an enhancer of a wing-promoting gene of A. mellifera under the regulation of Ubx. Our results, thus, suggest that binding motifs with a TAAAT core sequence may help identify functionally relevant direct targets of Ubx in D. melanogaster and the emergence of these binding sites may be crucial for Hox-mediated morphological changes during insect evolution.

Virtual Evolution of HVEM Segment for Checkpoint Inhibitor Discovery

Immune therapy has emerged as an effective treatment against cancers. Inspired by the PD-1/PD-L1 antibodies, which have achieved great success in clinical, other immune checkpoint proteins have drawn increasing attention in cancer research. B and T lymphocyte attenuator (BTLA) and herpes virus entry mediator (HVEM) are potential targets for drug development. The co-crystal structure of BTLA/HVEM have revealed that HVEM (26–38) fragment is the core sequence which directly involved on the interface. Herein, we conducted virtual evolution with this sequence by using saturation mutagenesis in silico and mutants with lower binding energy were selected. Wet-lab experiments confirmed that several of them possessed higher affinity with BTLA. Based on the best mutant of the core sequence, extended peptides with better efficacy were obtained. Furthermore, the mechanism of the effects of mutations was revealed by computational analysis. The mutated peptide discovered here can be a potent inhibitor to block BTLA/HVEM interaction and its mechanism may extend people’s view on inhibitor discovery for the checkpoint pair.

Characterizing Transcriptional Regulatory Sequences in Coronaviruses and Their Role in Recombination

Novel coronaviruses, including SARS-CoV-2, SARS, and MERS, often originate from recombination events. The mechanism of recombination in RNA viruses is template switching. Coronavirus transcription also involves template switching at specific regions, called transcriptional regulatory sequences (TRS). It is hypothesized but not yet verified that TRS sites are prone to recombination events. Here, we developed a tool called SuPER to systematically identify TRS in coronavirus genomes and then investigated whether recombination is more common at TRS. We ran SuPER on 506 coronavirus genomes and identified 465 TRS-L and 3,509 TRS-B. We found that the TRS-L core sequence (CS) and the secondary structure of the leader sequence are generally conserved within coronavirus genera but different between genera. By examining the location of recombination breakpoints with respect to TRS-B CS, we observed that recombination hotspots are more frequently colocated with TRS-B sites than expected.

Characterizing transcriptional regulatory sequences in coronaviruses and their role in recombination

ABSTRACTNovel coronaviruses, including SARS-CoV-2, SARS, and MERS, often originate from recombination events. The mechanism of recombination in RNA viruses is template switching. Coronavirus transcription also involves template switching at specific regions, called transcriptional regulatory sequences (TRS). It is hypothesized but not yet verified that TRS sites are prone to recombination events. Here, we developed a tool called SuPER to systematically identify TRS in coronavirus genomes and then investigated whether recombination is more common at TRS. We ran SuPER on 506 coronavirus genomes and identified 465 TRS-L and 3509 TRS-B. We found that the TRS-L core sequence (CS) and the secondary structure of the leader sequence are generally conserved within coronavirus genera but different between genera. By examining the location of recombination breakpoints with respect to TRS-B CS, we observed that recombination hotspots are more frequently co-located with TRS-B sites than expected.

AsWRKY44 represses the wound-induced sesquiterpene biosynthetic gene ASS1 expression in Aquilaria sinensis (Lour.) Gilg

Agarwood is derived from wounded Aquilaria trees and is widely used in traditional medicine, incense, and perfume. Sesquiterpenes are one of the main active components in agarwood and are known to be induced by wounding or injury. The molecular mechanism by which wounding leads to sesquiterpene formation remains largely unknown. ASS1 is one of key enzymes responsible for the biosynthesis of sesquiterpenes and is a pivotal jasmonate (JA)-responsive wound-inducible synthase. However, why ASS1 does not express in healthy trees and how its expression is induced as a result of wounding remains unexplored. Here, we report that ASS1 is a wound-induced gene with a promoter in which the 242-bp (-973 to -731bp) region is identified as the core sequence for responding to wound signals. AsWRKY44 binds directly to this region and represses ASS1 promoter activity. Downregulation or disruption of AsWRKY44 can relieve the inhibition and activate ASS1 expression. Further, it is found that in response to the exogenous MeJA, AsWRKY44 is degraded and the expression of ASS1 is significantly upregulated. These findings confirm AsWRKY44 is a crucial negative regulator involved in the regulation of wound-induced ASS1 transcription, which reveals the core mechanism of agarwood sesquiterpenes biosynthesis.

An Amyloid Core Sequence in the Major Candida albicans Adhesin Als1p Mediates Cell-Cell Adhesion

ABSTRACT The human fungal commensal Candida albicans can become a serious opportunistic pathogen in immunocompromised hosts. The C. albicans cell adhesion protein Als1p is a highly expressed member of a large family of paralogous adhesins. Als1p can mediate binding to epithelial and endothelial cells, is upregulated in infections, and is important for biofilm formation. Als1p includes an amyloid-forming sequence at amino acids 325 to 331, identical to the sequence in the paralogs Als5p and Als3p. Therefore, we mutated Val326 to test whether this sequence is important for activity. Wild-type Als1p (Als1pWT) and Als1p with the V326N mutation (Als1pV326N) were expressed at similar levels in a Saccharomyces cerevisiae surface display model. Als1pV326N cells adhered to bovine serum albumin (BSA)-coated beads similarly to Als1pWT cells. However, cells displaying Als1pV326N showed visibly smaller aggregates and did not fluoresce in the presence of the amyloid-binding dye Thioflavin-T. A new analysis tool for single-molecule force spectroscopy-derived surface mapping showed that statistically significant force-dependent Als1p clustering occurred in Als1pWT cells but was absent in Als1pV326N cells. In single-cell force spectroscopy experiments, strong cell-cell adhesion was dependent on an intact amyloid core sequence on both interacting cells. Thus, the major adhesin Als1p interacts through amyloid-like β-aggregation to cluster adhesin molecules in cis on the cell surface as well as in trans to form cell-cell bonds. IMPORTANCE Microbial cell surface adhesins control essential processes such as adhesion, colonization, and biofilm formation. In the opportunistic fungal pathogen Candida albicans, the agglutinin-like sequence (ALS) gene family encodes eight cell surface glycoproteins that mediate adherence to biotic and abiotic surfaces and cell-cell aggregation. Als proteins are critical for commensalism and virulence. Their activities include attachment and invasion of endothelial and epithelial cells, morphogenesis, and formation of biofilms on host tissue and indwelling medical catheters. At the molecular level, Als5p-mediated cell-cell aggregation is dependent on the formation of amyloid-like nanodomains between Als5p-expressing cells. A single-site mutation to valine 326 abolishes cellular aggregation and amyloid formation. Our results show that the binding characteristics of Als1p follow a mechanistic model similar to Als5p, despite its differential expression and biological roles.

Sequence variability of HCV 3a isolates based on core gene in patients from Lahore, Pakistan

Aim: To investigate the HCV 3a core sequence variation and amino acid substitutions of patients from Lahore, Pakistan. Materials & methods: Blood samples from HCV positive patients (n = 232) were collected for viral genotypes. Moreover, the nucleotide sequencing was performed for core gene of 20 samples. Results: Viral genotyping showed that 69.82% (n = 162) belonged to 3a genotype, 9.05% (1a; n = 21), 2.15% (3b; n = 5) and 18.98% were untypable (n = 44). Phylogenetic analyses suggest majority of our isolates clustered with previously reported reference isolates from Pakistan. The remaining isolates clustered with HCV-core sequences reported from Vietnam, Japan, Thailand, Iran, USA, Bangladesh, Malaysia and Morocco. Conclusion: We report HCV-core substitutions (G60E, R70Q, C91A, A94Q and Q63E/D) that could be associated with treatment response in Pakistani patients.