A new family of highly active ethylene tri-/tetramerization catalysts based on N-phosphinoamidinechromium complexes has been investigated. The 1-hexene to 1-octene molar ratio can be tuned from 140 ...to 1.5 by varying the steric environment around the chromium center, and product purities are very good to excellent. Precatalyst tridentate coordination effectively shuts down catalytic activity, suggesting that THF abstraction from the chromium center by the Lewis acidic aluminum activator is necessary to achieve an active catalyst system.
Proanthocyanidins (PAs) are natural polymers of flavan-3-ols, commonly (+)-catechin and (-)-epicatechin. However, exactly how PA oligomerization proceeds is poorly understood. Here we show, both ...biochemically and genetically, that ascorbate (AsA) is an alternative "starter unit" to flavan-3-ol monomers for leucocyanidin-derived (+)-catechin subunit extension in the Arabidopsis thaliana anthocyanidin synthase (ans) mutant. These (catechin)
:ascorbate conjugates (AsA-C
) also accumulate throughout the phase of active PA biosynthesis in wild-type grape flowers, berry skins and seeds. In the presence of (-)-epicatechin, AsA-C
can further provide monomeric or oligomeric PA extension units for non-enzymatic polymerization in vitro, and their role in vivo is inferred from analysis of relative metabolite levels in both Arabidopsis and grape. Our findings advance the knowledge of (+)-catechin-type PA extension and indicate that PA oligomerization does not necessarily proceed by sequential addition of a single extension unit. AsA-C
defines a new type of PA intermediate which we term "sub-PAs".
DNA replication requires coordination between replication fork progression and deoxynucleotide triphosphate (dNTP)–generating metabolic pathways. We find that perturbation of ribonucleotide reductase ...(RNR) in humans elevates reactive oxygen species (ROS) that are detected by peroxiredoxin 2 (PRDX2). In the oligomeric state, PRDX2 forms a replisome-associated ROS sensor, which binds the fork accelerator TIMELESS when exposed to low levels of ROS. Elevated ROS levels generated by RNR attenuation disrupt oligomerized PRDX2 to smaller subunits, whose dissociation from chromatin enforces the displacement of TIMELESS from the replisome. This process instantly slows replication fork progression, which mitigates pathological consequences of replication stress. Thus, redox signaling couples fluctuations of dNTP biogenesis with replisome activity to reduce stress during genome duplication. We propose that cancer cells exploit this pathway to increase their adaptability to adverse metabolic conditions.
Polyhydroxyalkanoates (PHAs) are natural biodegradable polyesters that are produced by numerous prokaryotic microorganisms primarily as a carbon- and energy reserve. The PhaC enzyme catalyzes the ...last step in the PHA biosynthesis pathway and synthesizes PHA polymers from hydroxyalkanoic acids. A type I PhaC from a PHA-producing marine bacterium Brevundimonas sp. KH11J01 (BrPhaC) was identified, produced recombinantly and characterized. Its properties were compared with its homolog from C. necator H16 (RePhaC). Unlike other PhaCs, it was found that BrPhaC is a lag-phase free enzyme organized as a trimer, even without the presence of a substrate. The enzymatic reaction is initiated instantly irrespective of temperature, in contrast to RePhaC in which the duration of the lag-phase was highly affected by temperature. At 10 °C BrPhaC was 40% active whereas RePhaC was barely active. The significance of using marine microorganisms, harboring cold-active PHA biosynthesis enzymes, for energy efficient PHA production, is also discussed briefly. The unique trimeric organization of BrPhaC challenges our understanding of the PhaC reaction mechanisms, which is mainly based on the crystal structures of the inactive forms of the enzyme.
•A type I PHA synthase from Brevundimonas sp. (BrPhaC) uniquely exists as trimer in absence of substrate.•Enzymatic reaction of BrPhaC starts instantly without a lag-phase unlike many other PhaCs.•Duration of the lag-phase of PHA synthase from Cupriavidus necator H16 (RePhaC) was highly affected by temperature.•Optimal function of BrPhaC at ambient temperature is beneficial for energy efficient PHA production.
Summary
The NLRP3 inflammasome, an intracellular sensor consisting of the nucleotide‐binding oligomerization domain‐like receptor family, pyrin domain containing 3 (NLRP3), the adaptor protein ...apoptosis‐associated speck‐like protein containing a caspase‐recruitment domain (ASC), and procaspase‐1, plays critical roles in host defense against microbial pathogens by inducing production of interleukin‐1β (IL‐1β) and IL‐18. Mycoplasma salivarium and Mycoplasma pneumoniae cells activated murine bone marrow‐derived macrophages (BMMs) to induce production of IL‐1α, IL‐1β, and IL‐18. The IL‐1β production‐inducing activities of these mycoplasmas toward BMMs from Toll‐like receptor 2 (TLR2)‐deficient mice were significantly attenuated compared with those from C57BL/6 mice (B6BMMs). This result suggests the possibility that their lipoproteins as TLR2 agonists are involved in the activity. Lipoproteins of M. salivarium and M. pneumoniae (MsLP and MpLP), and the M. salivarium‐derived lipopeptide FSL‐1 induced IL‐1β production by B6BMMs, but not by BMMs from caspase‐1‐, NLRP3‐ or ASC‐deficient mice. The activities of MsLP and MpLP were not downregulated by the proteinase K treatment, suggesting that the active sites are their N‐terminal lipopeptide moieties. B6BMMs internalized the mycoplasmal N‐terminal lipopeptide FSL‐1 at least 30 min after incubation, FSL‐1‐containing endosomes started to fuse with the lysosomes around 2 hours, and then FSL‐1 translocated into the cytosol from LAMP‐1+ endosomes. The artificial delivery of FSL‐1 into the cytosol of B6BMMs drastically enhanced the IL‐1β production‐inducing activity. FSL‐1 as well as the representative NLRP3 inflammasome activator nigericin induced the NLRP3/ASC speck, but FSL‐1 located in a compartment different from the NLRP3/ASC speck.
It is important to assess the identity and purity of proteins and protein complexes during and after protein purification to ensure that samples are of sufficient quality for further biochemical and ...structural characterization, as well as for use in consumer products, chemical processes and therapeutics. Native mass spectrometry (nMS) has become an important tool in protein analysis due to its ability to retain non-covalent interactions during measurements, making it possible to obtain protein structural information with high sensitivity and at high speed. Interferences from the presence of non-volatiles are typically alleviated by offline buffer exchange, which is time-consuming and difficult to automate. We provide a protocol for rapid online buffer exchange (OBE) nMS to directly screen structural features of pre-purified proteins, protein complexes or clarified cell lysates. In the liquid chromatography coupled to mass spectrometry (LC-MS) approach described in this protocol, samples in MS-incompatible conditions are injected onto a short size-exclusion chromatography column. Proteins and protein complexes are separated from small molecule non-volatile buffer components using an aqueous, non-denaturing mobile phase. Eluted proteins and protein complexes are detected by the mass spectrometer after electrospray ionization. Mass spectra can inform regarding protein sample purity and oligomerization, and additional tandem mass spectra can help to further obtain information on protein complex subunits. Information obtained by OBE nMS can be used for fast (<5 min) quality control and can further guide protein expression and purification optimization.
African swine fever (ASF) is a highly contagious and acute hemorrhagic viral disease in domestic pigs and wild boars. Domestic pigs infected with virulent African swine fever virus (ASFV) isolates ...have a high mortality, approaching 100%. Identification of ASFV genes related to virulence/pathogenicity and deletion of them are considered to be key steps in the development of live attenuated vaccines, because the ability of ASFV to escape host innate immune responses is related to viral pathogenicity. However, the relationship between the host antiviral innate immune responses and the pathogenic genes of ASFV has not been fully understood. In this study, the ASFV H240R protein (pH240R), a capsid protein of ASFV, was found to inhibit type I interferon (IFN) production. Mechanistically, pH240R interacted with the N-terminal transmembrane domain of stimulator of interferon genes (STING) and inhibited its oligomerization and translocation from the endoplasmic reticulum to the Golgi apparatus. Additionally, pH240R inhibited the phosphorylation of interferon regulatory factor 3 (IRF3) and TANK binding kinase 1 (TBK1), leading to reduced production of type I IFN. Consistent with these results, infection with
-deficient ASFV (ASFV-ΔH240R) induced more type I IFN than infection with its parental strain, ASFV HLJ/18. We also found that pH240R may enhance viral replication via inhibition of type I IFN production and the antiviral effect of interferon alpha (IFN-α). Taken together, our findings provide a new explanation for the reduction of ASFV's replication ability by knockout of the
gene and a clue for the development of live attenuated ASFV vaccines.
African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious and acute hemorrhagic viral disease with a high mortality, approaching 100% in domestic pigs. However, the relationship between viral pathogenicity and immune evasion of ASFV is not fully understood, which limits the development of safe and effective ASF vaccines, specifically, live attenuated vaccines. In this study, we found that pH240R, as a potent antagonist, inhibited type I IFN production by targeting STING and inhibiting its oligomerization and translocation from the endoplasmic reticulum to the Golgi apparatus. Furthermore, we also found that deletion of the
gene reduced viral pathogenicity by enhancing type I IFN production, which decreases ASFV replication. Taken together, our findings provide a clue for the development of an ASFV live attenuated vaccine via deleting the
gene.
Stress-induced cleavage of transfer RNAs (tRNAs) into tRNA-derived fragments (tRFs) occurs across organisms from yeast to humans; yet, its mechanistic underpinnings and pathological consequences ...remain poorly defined. Small RNA profiling revealed increased abundance of a cysteine tRNA fragment (5'-tRF
) during breast cancer metastatic progression. 5'-tRF
was required for efficient breast cancer metastatic lung colonization and cancer cell survival. We identified Nucleolin as the direct binding partner of 5'-tRF
. 5'-tRF
promoted the oligomerization of Nucleolin and its bound metabolic transcripts Mthfd1l and Pafah1b1 into a higher-order transcript stabilizing ribonucleoprotein complex, which protected these transcripts from exonucleolytic degradation. Consistent with this, Mthfd1l and Pafah1b1 mediated pro-metastatic and metabolic effects downstream of 5'-tRF
-impacting folate, one-carbon, and phosphatidylcholine metabolism. Our findings reveal that a tRF can promote oligomerization of an RNA-binding protein into a transcript stabilizing ribonucleoprotein complex, thereby driving specific metabolic pathways underlying cancer progression.
Cr(III) catalysts supported by asymmetic N,N-diphospholylamine ligands bearing a phenoxaphosphine group are highly active for ethylene tri-/tetramerization with considerable selectivity upon ...activation with MMAO-3A. Asymmetric PNP ligand with a N-cyclohexyl group achieved the highest activity of 282.2kg/(gCr/h) with a high total selectivity of 83.2% toward valuable 1-hexene (28.7%) and 1-octene (54.5%) at 35 bar under 80°C.
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We have developed novel Cr(III) catalysts supported by asymmetic N,N-diphospholylamine ligands bearing a phenoxaphosphine group. Upon activation with MMAO-3A, the Cr(III) catalysts supported by the PNP ligands are highly active for ethylene tri-/tetramerization with considerable selectivity. The ligand substitution and oligomerization conditions are found to be essential to achieve high activity and controllable selectivity. The catalytic system with asymmetric diphospholylamine ligands exhibited higher activity than that supported by symmetric ligands. Asymmetric diphospholylamine ligand with a N-cyclohexyl group achieved the highest activity of 282.2kg/(gCr/h) with a high total selectivity of 83.2% toward valuable 1-hexene (28.7%) and 1-octent (54.5%) at 35bar under 80°C.
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