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Mass spectrometry-based sample multiplexing with isobaric tags permits the development of high-throughput and precise quantitative biological assays with proteome-wide coverage and minimal missing values. Here, we nearly doubled the multiplexing capability of the TMTpro reagent set to a 35-plex through the incorporation of one deuterium isotope into the reporter group. Substituting deuterium frequently results in suboptimal peak coelution, which can compromise the accuracy of reporter ion-based quantification. To counteract the deuterium effect on quantitation, we implemented a strategy that necessitated the segregation of nondeuterium and deuterium-containing channels into distinct subplexes during normalization procedures, with reassembly through a common bridge channel. This multiplexing strategy of "design independent sub-plexes but acquire together" (DISAT) was used to compare protein expression differences between human cell lines and in a cysteine-profiling (i.e., chemoproteomics) experiment to identify compounds binding to cysteine-113 of Pin1.

Cofilin, a key actin-binding protein, orchestrates the dynamics of the actomyosin network through its actin-severing activity and by promoting the recycling of actin monomers. Recent experiments suggest that cofilin forms functionally distinct oligomers via thiol post-translational modifications (PTMs) that promote actin nucleation and assembly. Despite these advances, the structural conformations of cofilin oligomers that modulate actin activity remain elusive because there are combinatorial ways to oxidize thiols in cysteines to form disulfide bonds rapidly. This study employs molecular dynamics simulations to investigate human cofilin 1 as a case study for exploring cofilin dimers via disulfide bond formation. Utilizing a biasing scheme in simulations, we focus on analyzing dimer conformations conducive to disulfide bond formation. Additionally, we explore potential PTMs arising from the examined conformational ensemble. Using the free energy profiling, our simulations unveil a range of probable cofilin dimer structures not represented in current Protein Data Bank entries. These candidate dimers are characterized by their distinct population distributions and relative free energies. Of particular note is a dimer featuring an interface between cysteines 139 and 147 residues, which demonstrates stable free energy characteristics and intriguingly symmetrical geometry. In contrast, the experimentally proposed dimer structure exhibits a less stable free energy profile. We also evaluate frustration quantification based on the energy landscape theory in the protein-protein interactions at the dimer interfaces. Notably, the 39-39 dimer configuration emerges as a promising candidate for forming cofilin tetramers, as substantiated by frustration analysis. Additionally, docking simulations with actin filaments further evaluate the stability of these cofilin dimer-actin complexes. Our findings thus offer a computational framework for understanding the role of thiol PTM of cofilin proteins in regulating oligomerization, and the subsequent cofilin-mediated actin dynamics in the actomyosin network.

The compound FR901379, a sulfated echinocandin produced by the filamentous fungus Coleophoma empetri F-11899, is an important intermediate for the synthesis of the antifungal drug micafungin. In this study, we established an efficient clustered regularly interspaced short palindromic repeats/Cas9-based gene editing tool for the industrial production strain C. empetri SIPI1284. With this method, the efficiency of gene mutagenesis in the target locus is up to 84%, which enables the rapid gene disruption for the analysis of FR901379 biosynthetic genes. Next, we verified the putative functional genes of the FR901379 biosynthetic gene cluster via gene disruption and gene complementation in vivo. These core functional genes included the nonribosomal peptide synthetase gene ( CEnrps ), the fatty-acyl-AMP ligase gene ( CEligase ) responsible for the formation of the activated form of palmitic acid and its transfer to CEnrps , four nonheme mononuclear iron oxygenase genes ( CEoxy1 , CEoxy2 , CEoxy3 , and CEoxy4 ) responsible for the synthesis of nonproteinogenic amino acids, l-homotyrosine biosynthesis genes ( CEhtyA-D ), two cytochrome P450 enzyme genes ( CEp450-1 and CEp450-2 ), and a transcription regulator gene ( CEhyp ). In addition, by screening the whole genome, we identified two unknown genes ( CEp450-3 and CEsul ) responsible for the sulfonyloxy group of FR901379, which were separated from the core FR901379 biosynthetic cluster. Furthermore, during gene disruptions in the research, we obtained a series of FR901379 analogues and elucidated the relationship between the groups and antifungal activities.

Glarea lozoyensis is an important industrial fungus that produces the pneumocandin B 0 , which is used for the synthesis of antifungal drug caspofungin. However, because of the limitations and complications of traditional genetic tools, G. lozoyensis strain engineering has been hindered. In this study, we established an efficient CRISPR/Cas9-based gene editing tool in G. lozoyensis SIPI1208. With this method, gene mutagenesis efficiency in the target locus can be up to 80%, which enables the rapid gene knockout. According to the reports, GloF and Ap-HtyE, proline hydroxylases involved in pneumocandin and Echinocandin B biosynthesis, respectively, can catalyze the proline to generate different ratios of trans -3-hydroxy-l-proline to trans -4-hydroxy-l-proline. Heterologous expression of Ap-HtyE in G. lozoyensis decreased the ratio of pneumocandin C 0 to (pneumocandin B 0 + pneumocandin C 0 ) from 33.5% to 11% without the addition of proline to the fermentation medium. Furthermore, the gloF was replaced by ap-htyE to study the production of pneumocandin C 0 . However, the gene replacement has been hampered by traditional gene tools since gloF and gloG , two contiguous genes indispensable in the biosynthesis of pneumocandins, are cotranscribed into one mRNA. With the CRISPR/Cas9 strategy, ap-htyE was knocked in and successfully replaced gloF , and results showed that the knock-in strain retained the ability to produce pneumocandin B 0 , but the production of pneumocandin C 0 was abolished. Thus, this strain displayed a competitive advantage in the industrial production of pneumocandin B 0 . In summary, this study showed that the CRISPR/Cas9-based gene editing tool is efficient for manipulating genes in G. lozoyensis .

Voriconazole (VCZ) is a widely used triazole drug for the treatment of serious incidence of invasive fungal infections (IFIs), and its most commonly reported clinical side effect is hepatotoxicity. The mechanism of VCZ-induced hepatotoxicity is unclear, and no specific marker can be used for prediction and diagnosis. This study aims to apply the targeted metabolomics approach to identify specific VCZ-induced metabolites related to hepatotoxicity via liquid chromatography-triple quadrupole mass spectrometry (LC-QqQ-MS) in a C57BL/6 mouse model. Mice treated with three repeated doses of 40 mg/kg VCZ by tail vein injection to induce hepatotoxicity (VCZ-induced hepatotoxicity group, n = 8) were compared with mice without treatment (control group, n = 10). Both liver tissue and plasma were collected and analyzed to propose underlying mechanisms associated with VCZ-induced hepatotoxicity. The results indicated that the metabolites associated with oxidative stress were altered, and alterations in the metabolites involved in glutathione biosynthesis were noticed. The ratio of glutamine to glutamate showed a significant reduction in the VCZ-induced hepatotoxicity group compared to the control group, suggesting that glutamine might be transformed into glutamate for glutathione biosynthesis. Accordingly, we proposed that VCZ-induced hepatotoxicity is associated with oxidative stress to cause cell dysfunction, leading to alterations in energy metabolism, the urea cycle, and nucleoside metabolism. To the best of our knowledge, this is the first study to apply metabolomics for investigating the mechanism of VCZ-induced hepatotoxicity.

Type I protein arginine methyltransferases (PRMTs) catalyze asymmetric dimethylation of various proteins, and their dysregulations often correlate with tumorigenesis or developmental deficiency. Recent studies have focused on the in vivo substrate identification and the enzyme mechanism with peptide substrates. However, how PRMTs recognize substrates at the protein level remains unknown. PRMT8 is one of the least characterized type I PRMTs, and its crystal structure has not been reported. Here, we report the crystal structure of the PRMT8:SAH complex, identify a new non-histone protein substrate NIFK, and uncover a previously unknown regulatory region specifically required for recognizing NIFK. Instead of the canonical dimeric structure for other type I PRMTs, PRMT8 exists as a tetramer in solution. Using X-ray crystallography in combination with small-angle X-ray scattering experiments, the dimer of dimers architecture in which two PRMT8 dimers are held together mainly by β strand interactions was proposed. Mutation of PRMT8-β15 impedes the methylation of NIFK but still allows methylation of the histone H2A/H2B dimer or a peptide substrate, suggesting a possible structural basis for recognition of protein substrates. Lastly, we observed two PRMT8 dimer orientations resulting in open (without SAH) and closed (with SAH bound) conformations. The comparison between open and closed conformations may provide useful information for PRMT1/8 inhibitor design.

Forkhead-associated (FHA) domain is the only signaling domain that recognizes phosphothreonine (pThr) specifically. TRAF-interacting protein with an FHA domain (TIFA) was shown to be involved in immune responses by binding with TRAF2 and TRAF6. We recently reported that TIFA is a dimer in solution and that, upon stimulation by TNF-α, TIFA is phosphorylated at Thr9, which triggers TIFA oligomerization via pThr9-FHA domain binding and activates nuclear factor κB (NF-κB). However, the structural mechanism for the functionally important TIFA oligomerization remains to be established. While FHA domain-pThr binding is known to mediate protein dimerization, its role in oligomerization has not been demonstrated at the structural level. Here we report the crystal structures of TIFA (residues 1-150, with the unstructured C-terminal tail truncated) and its complex with the N-terminal pThr9 peptide (residues 1-15), which show unique features in the FHA structure (intrinsic dimer and extra β-strand) and in its interaction with the pThr peptide (with residues preceding rather than following pThr). These structural features support previous and additional functional analyses. Furthermore, the structure of the complex suggests that the pThr9-FHA domain interaction can occur only between different sets of dimers rather than between the two protomers within a dimer, providing the structural mechanism for TIFA oligomerization. Our results uncover the mechanism of FHA domain-mediated oligomerization in a key step of immune responses and expand the paradigm of FHA domain structure and function.

A new single nanowire based nanosensor is demonstrated for illustrating its ultrahigh sensitivity for gas sensing. The device is composed of a single ZnO nanowire mounted on Pt electrodes with one end in Ohmic contact and the other end in Schottky contact. The Schottky contact functions as a "gate" that controls the current flowing through the entire system. By tuning the Schottky barrier height through the responsive variation of the surface chemisorbed gases and the amplification role played by the nanowire to Schottky barrier effect, an ultrahigh sensitivity of 32,000% was achieved using the Schottky contacted device operated in reverse bias mode at 275 degrees C for detection of 400 ppm CO, which is 4 orders of magnitude higher than that obtained using an Ohmic contact device under the same conditions. In addition, the response time and reset time have been shortened by a factor of 7. The methodology and principle illustrated in the paper present a new sensing mechanism that can be readily and extensively applied to other gas sensing systems.

A facile one-step polymer-incorporation sol-gel process, together with a surface modification and an ambient pressure drying processes, was developed to prepare silica-poly(vinylpyrrolidone) composite aerogels. These composite aerogels are with high hydrophobicity (static contact angle >120 degrees), good mechanical strength (Young's modulus of bending >30 MPa), and low high-temperature thermal conductivity (0.063 W/m-K at 300 degrees C), which are critical characteristics for practical applications of aerogels, particularly in energy saving areas, for long-term usage and large scale production.