Intrinsically disordered interaction network in an RNA chaperone revealed by native mass spectrometry.

RNA-binding proteins contain intrinsically disordered regions whose functions in RNA recognition are poorly understood. The RNA chaperone Hfq is a homohexamer that contains six flexible C-terminal domains (CTDs). The effect of the CTDs on Hfq's integrity and RNA binding has been challenging to study because of their sequence identity and inherent disorder. We used native mass spectrometry coupled with surface-induced dissociation and molecular dynamics simulations to disentangle the arrangement of the CTDs and their impact on the stability of Escherichia coli Hfq with and without RNA. The results show that the CTDs stabilize the Hfq hexamer through multiple interactions with the core and between CTDs. RNA binding perturbs this network of CTD interactions, destabilizing the Hfq ring. This destabilization is partially compensated by binding of RNAs that contact multiple surfaces of Hfq. By contrast, binding of short RNAs that only contact one or two subunits results in net destabilization of the complex. Together, the results show that a network of intrinsically disordered interactions integrate RNA contacts with the six subunits of Hfq. We propose that this CTD network raises the selectivity of RNA binding.

Gut Parabacteroides merdae protects against cardiovascular damage by enhancing branched-chain amino acid catabolism.

Obesity, dyslipidemia and gut dysbiosis are all linked to cardiovascular diseases. A Ganoderma meroterpene derivative (GMD) has been shown to alleviate obesity and hyperlipidemia through modulating the gut microbiota in obese mice. Here we show that GMD protects against obesity-associated atherosclerosis by increasing the abundance of Parabacteroides merdae in the gut and enhancing branched-chain amino acid (BCAA) catabolism. Administration of live P. merdae to high-fat-diet-fed ApoE-null male mice reduces atherosclerotic lesions and enhances intestinal BCAA degradation. The degradation of BCAAs is mediated by the porA gene expressed in P. merdae. Deletion of porA from P. merdae blunts its capacity to degrade BCAAs and leads to inefficacy in fighting against atherosclerosis. We further show that P. merdae inhibits the mTORC1 pathway in atherosclerotic plaques. In support of our preclinical findings, an in silico analysis of human gut metagenomic studies indicates that P. merdae and porA genes are depleted in the gut microbiomes of individuals with atherosclerosis. Our results provide mechanistic insights into the therapeutic potential of GMD through P. merdae in treating obesity-associated cardiovascular diseases.

Computational prediction and validation of specific EmbR binding site on PknH.

Tuberculosis drug resistance continues to threaten global health but the underline molecular mechanisms are not clear. Ethambutol (EMB), one of the well-known first - line drugs in tuberculosis treatment is, unfortunately, not free from drug resistance problems. Genomic studies have shown that some genetic mutations in Mycobacterium tuberculosis (Mtb) EmbR, and EmbC/A/B genes cause EMB resistance. EmbR-PknH pair controls embC/A/B operon, which encodes EmbC/A/B genes, and EMB interacts with EmbA/B proteins. However, the EmbR binding site on PknH was unknown. We conducted molecular simulation on the EmbR- peptides binding structures and discovered phosphorylated PknH 273-280 (N'-HEALSPDPD-C') makes ß strand with the EmbR FHA domain, as ß-MoRF (MoRF; molecular recognition feature) does at its binding site. Hydrogen bond number analysis also supported the peptides' ß-MoRF forming activity at the EmbR FHA domain. Also, we discovered that previously known phosphorylation residues might have their chronological order according to the phosphorylation status. The discovery validated that Mtb PknH 273-280 (N'-HEALSDPD-C') has reliable EmbR binding affinity. This approach is revolutionary in the computer-aided drug discovery field, because it is the first trial to discover the protein-protein interaction site, and find binding partner in nature from this site.

The gut commensal fungus, Candida parapsilosis, promotes high fat-diet induced obesity in mice.

Gut fungi is known to play many important roles in human health regulations. Herein, we investigate the anti-obesity efficacy of the antifungal antibiotics (amphotericin B, fluconazole and 5-fluorocytosine) in the high fat diet-fed (HFD) mice. Supplementation of amphotericin B or fluconazole in water can effectively inhibit obesity and its related disorders, whereas 5-fluorocytosine exhibit little effects. The gut fungus Candida parapsilosis is identified as a key commensal fungus related to the diet-induced obesity by the culture-dependent method and the inoculation assay with C. parapsilosis in the fungi-free mice. In addition, the increase of free fatty acids in the gut due to the production of fungal lipases from C. parapsilosis is confirmed as one mechanism by which C. parapsilosis promotes obesity. The current study demonstrates the gut C. parapsilosis as a causal fungus for the development of diet-induced obesity in mice and highlights the therapeutic strategy targeting the gut fungi.

Hyper-Synergistic Antifungal Activity of Rapamycin and Peptide-Like Compounds against Candida albicans Orthogonally via Tor1 Kinase.

Candida albicans is a life-threatening, opportunistic fungal pathogen with a high mortality rate, especially within the immunocompromised populations. Multidrug resistance combined with limited antifungal drugs even worsens the situation. Given the facts that the current drug discovery strategies fail to deliver sufficient antifungals for the emerging multidrug resistance, we urgently need to develop novel approaches. By systematically investigating what caused the different antifungal activity of rapamycin in RPMI 1640 and YPD, we discovered that peptide-like compounds can generate a hyper-synergistic antifungal effect with rapamycin on both azole-resistant and sensitive clinical C. albicans isolates. The minimum inhibitory concentration (MIC) of rapamycin reaches as low as 2.14 nM (2-9 µg/mL), distinguishing this drug combination as a hyper-synergism by having a fractional inhibitory concentration (FIC) index ≤ 0.05 from the traditional defined synergism with an FIC index < 0.5. Further studies reveal that this hyper-synergism orthogonally targets the protein Tor1 and affects the TOR signaling pathway in C. albicans, very likely without crosstalk to the stress response, Ras/cAMP/PKA, or calcineurin signaling pathways. These results lead to a novel strategy of controlling drug resistant C. albicans infection in the immunocompromised populations. Instead of prophylactically administering other antifungals with undesirable side-effects for extended durations, we now only need to coadminister some nontoxic peptide additives. The novel antifungal strategy approached in this study not only provides a new therapeutic method to control fungal infections in rapamycin-taking immunocompromised patients but also mitigates the immunosuppressive side-effects of rapamycin, repurposing rapamycin as an antifungal agent with wide applications.