Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection

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Bombesin-like peptide recruits disinhibitory cortical circuits and enhances fear memories

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Evolutionary assembly of cooperating cell types in an animal chemical defense system

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Orientational Ambiguity in Septin Coiled Coils and its Structural Basis

Septins are an example of subtle molecular recognition whereby different paralogues must correctly assemble into functional filaments important for essential cellular events such as cytokinesis. Most possess C-terminal domains capable of forming coiled coils which are believed to be involved in filament formation and bundling. Here, we report an integrated structural approach which aims to unravel their architectural diversity and in so doing provide direct structural information for the coiled-coil regions of five human septins. Unexpectedly, we encounter dimeric structures presenting both parallel and antiparallel arrangements which are in consonance with molecular modelling suggesting that both are energetically accessible. These sequences therefore code for two metastable states of different orientations which employ different but overlapping interfaces. The antiparallel structures present a mixed coiled-coil interface, one side of which is dominated by a continuous chain of core hydrophilic residues. This unusual type of coiled coil could be used to expand the toolkit currently available to the protein engineer for the design of previously unforeseen coiled-coil based assemblies. Within a physiological context, our data provide the first atomic details related to the assumption that the parallel orientation is likely formed between septin monomers from the same filament whilst antiparallelism may participate in the widely described interfilament cross bridges necessary for higher order structures and thereby septin function.     

Structure Basis for Shaping the Nse4 Protein by the Nse1 and Nse3 Dimer within the Smc5/6 Complex

The Smc5/6 complex facilitates chromosome replication and DNA break repair. Within this complex, a subcomplex composed of Nse1, Nse3 and Nse4 is thought to play multiple roles through DNA binding and regulating ATP-dependent activities of the complex. However, how the Nse1-Nse3-Nse4 subcomplex carries out these multiple functions remain unclear. To address this question, we determine the crystal structure of the Xenopus laevis Nse1-Nse3-Nse4 subcomplex at 1.7 Å resolution and examine how it interacts with DNA. Our structural analyses show that the Nse1-Nse3 dimer adopts a closed conformation and forms three interfaces with a segment of Nse4, forcing it into a Z-shaped conformation. The Nse1-Nse3-Nse4 structure provides an explanation for how the lung disease immunodeficiency and chromosome breakage syndrome-causing mutations could dislodge Nse4 from Nse1-Nse3. Our DNA binding and mutational analyses reveal that the N-terminal and the middle region of Nse4 contribute to DNA interaction and cell viability. Integrating our data with previous crosslink mass spectrometry data, we propose potential roles of the Nse1-Nse3-Nse4 complex in binding DNA within the Smc5/6 complex.     

Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

Malathion biodegradation by a psychrotolerant bacteria Ochrobactrum sp. M1D and metabolic pathway analysis

An organophosphorus pesticide malathion biodegradation was investigated by using the bacteria Ochrobactrum sp. M1D isolated from a soil sample of peach orchards in Palampur, District Kangra, Himachal Pradesh (India). The bacterium was able to utilize malathion as the sole source of carbon and energy. The isolated bacterium was found psychrotolerant and could degrade 100% of 100 mg l⁻¹ malathion in minimal salt medium at 20°C, pH 7·0 within 12 days with no major significant metabolites left at the end of the study. Through GCMS analysis, methyl phosphate, diethyl maleate, and diethyl 2-mercaptosuccinate were detected and identified as the major pathway metabolites. Based on the GCMS profile, three probable degradation pathways were interpreted. The present study is the first report of malathion biodegradation at both the psychrophilic and mesophilic conditions by any psychrotolerant strain and also through multiple degradation pathways. In the future, the strain can be explored to bio-remediate the malathion contaminated soil in the cold climatic region and to utilize the enzymatic systems for advanced biotechnology applications.     

High glucose protects cardiomyocytes against ischaemia/reperfusion injury by suppressing myocardiocyte apoptosis via circHIPK3/miR-29b/AKT3 signalling

High glucose promoted expression of AKT3, a direct target gene of miR-29b, by regulating circHIPK3 that functioned as ceRNA to sponge and down-regulate miR-29b. As a potential target gene of miR-29b, AKT3 plays a crucial role in the pathogenesis of myocardial ischaemia/reperfusion (I/R) injury, and this study aimed to investigate the potential role of high glucose in the outcome of I/R injury. qPCR and luciferase assay were carried out to investigate the relationship between the expression of circHIPK3, miR-29b and ATK3 mRNA. Immunohistochemistry and TUNEL were performed to analyse the relationship between AKT3 expression and apoptosis of myocardiocytes in vivo. No obvious difference in myocardial functions was observed between I/R and control rats under hyperglycaemia (HG) and normal glucose (NG) conditions, except that the infarct size/area at risk (IS/AR) ratio and the amount of h-FABP expression were different under HG and NG conditions. The expression of circHIPK3 and ATK3 was significantly elevated in the rats preconditioned by NG, whereas the expression of miR-29a was remarkably decreased. Meanwhile, the apoptosis of myocardial tissue was reduced in the rats preconditioned by NG. Luciferase assay confirmed that miR-29a played a repressive role in the expression of circHIPK3 and ATK3. And subsequent study indicated that the over-expressed AKT3 could rescue the increased cell apoptosis rate induced by the knockdown of circHIPK3. In this study, we demonstrated that high glucose protects cardiomyocytes against I/R associated injury by suppressing apoptosis and high glucose promoted the expression of AKT3 by regulating the expression of circHIPK3/miR-29b.     

GDF11 prevents the formation of thoracic aortic dissection in mice: Promotion of contractile transition of aortic SMCs

Thoracic aortic dissection (TAD) is an aortic disease associated with dysregulated extracellular matrix composition and de-differentiation of vascular smooth muscle cells (SMCs). Growth Differentiation Factor 11 (GDF11) is a member of transforming growth factor β (TGF-β) superfamily associated with cardiovascular diseases. The present study attempted to investigate the expression of GDF11 in TAD and its effects on aortic SMC phenotype transition. GDF11 level was found lower in the ascending thoracic aortas of TAD patients than healthy aortas. The mouse model of TAD was established by β-aminopropionitrile monofumarate (BAPN) combined with angiotensin II (Ang II). The expression of GDF11 was also decreased in thoracic aortic tissues accompanied with increased inflammation, arteriectasis and elastin degradation in TAD mice. Administration of GDF11 mitigated these aortic lesions and improved the survival rate of mice. Exogenous GDF11 and adeno-associated virus type 2 (AAV-2)-mediated GDF11 overexpression increased the expression of contractile proteins including ACTA2, SM22α and myosin heavy chain 11 (MYH11) and decreased synthetic markers including osteopontin and fibronectin 1 (FN1), indicating that GDF11 might inhibit SMC phenotype transition and maintain its contractile state. Moreover, GDF11 inhibited the production of matrix metalloproteinase (MMP)-2, 3, 9 in aortic SMCs. The canonical TGF-β (Smad2/3) signalling was enhanced by GDF11, while its inhibition suppressed the inhibitory effects of GDF11 on SMC de-differentiation and MMP production in vitro. Therefore, we demonstrate that GDF11 may contribute to TAD alleviation via inhibiting inflammation and MMP activity, and promoting the transition of aortic SMCs towards a contractile phenotype, which provides a therapeutic target for TAD.