Proceedings of the National Academy of Sciences of the United States of America最新文献

The ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) and its regulatory protein Cdc20 play important roles in the control of different stages of mitosis. APC/C associated with Cdc20 is active and promotes metaphase-anaphase transition by targeting for degradation inhibitors of anaphase initiation. Earlier in mitosis, premature action of APC/C is prevented by the mitotic checkpoint (or spindle assembly checkpoint) system, which ensures that anaphase is not initiated until all chromosomes are properly attached to the mitotic spindle. The active mitotic checkpoint system promotes the assembly of a Mitotic Checkpoint Complex (MCC), which binds to APC/C and inhibits its activity. The interaction of MCC with APC/C is strongly enhanced by Cdc20 bound to APC/C. While the association of Cdc20 with APC/C was known to be essential for both these stages of mitosis, it was not known how Cdc20 remains bound in spite of ongoing processes, phosphorylation and ubiquitylation, that stimulate its release from APC/C. We find that MCC strongly inhibits the release of Cdc20 from APC/C by the action of mitotic protein kinase Cdk1-cyclin B. This is not due to protection from phosphorylation of specific sites in Cdc20 that affect its interaction with APC/C. Rather, MCC stabilizes the binding to APC/C of partially phosphorylated forms of Cdc20. MCC also inhibits the autoubiquitylation of APC/C-bound Cdc20 and its ubiquitylation-promoted release from APC/C. We propose that these actions of MCC to maintain Cdc20 bound to APC/C in mitosis are essential for the control of mitosis during active mitotic checkpoint and in subsequent anaphase initiation.

Prolyl-hydroxylation is an oxygen-dependent posttranslational modification (PTM) that is known to regulate fibril formation of collagenous proteins and modulate cellular expression of hypoxia-inducible factor (HIF) α subunits. However, our understanding of this important but relatively rare PTM has remained incomplete due to the lack of biophysical methodologies that can directly measure multiple prolyl-hydroxylation events within intrinsically disordered proteins. Here, we describe a real-time ¹³C-direct detection NMR-based assay for studying the hydroxylation of two evolutionarily conserved prolines (P402 and P564) simultaneously in the intrinsically disordered oxygen-dependent degradation domain of hypoxic-inducible factor 1α by exploiting the "proton-less" nature of prolines. We show unambiguously that P564 is rapidly hydroxylated in a time-resolved manner while P402 hydroxylation lags significantly behind that of P564. The differential hydroxylation rate was negligibly influenced by the binding affinity to prolyl-hydroxylase enzyme, but rather by the surrounding amino acid composition, particularly the conserved tyrosine residue at the +1 position to P564. These findings support the unanticipated notion that the evolutionarily conserved P402 seemingly has a minimal impact in normal oxygen-sensing pathway.

Oral delivery of proteins faces challenges due to the harsh conditions of the gastrointestinal (GI) tract, including gastric acid and intestinal enzyme degradation. Permeation enhancers are limited in their ability to deliver proteins with high molecular weight and can potentially cause toxicity by opening tight junctions. To overcome these challenges, we propose the use of montmorillonite (MMT) as an adjuvant that possesses both inflammation-oriented abilities and the ability to regulate gut microbiota. This adjuvant can be used as a universal protein oral delivery technology by fusing with advantageous binding amino acid sequences. We demonstrated that anti-TNF-α nanobody (V_II) can be intercalated into the MMT interlayer space. The carboxylate groups (-COOH) of aspartic acid (D) and glutamic acid (E) interact with the MMT surface through electrostatic interactions with sodium ions (Na⁺). The amino groups (NH₂) of asparagine (N) and glutamine (Q) are primarily attracted to the MMT layers through hydrogen bonding with oxygen atoms on the surface. This binding mechanism protects V_II from degradation and ensures its release in the intestinal tract, as well as retaining biological activity, leading to significantly enhanced therapeutic effects on colitis. Furthermore, V_II@MMT increases the abundance of short-chain fatty acids (SCFAs)-producing strains, including Clostridia, Prevotellaceae, Alloprevotella, Oscillospiraceae, Clostridia_vadinBB60_group, and Ruminococcaceae, therefore enhance the production of SCFAs and butyrate, inducing regulatory T cells (Tregs) production to modulate local and systemic immune homeostasis. Overall, the MMT adjuvant provides a promising universal strategy for protein oral delivery by rational designed protein.

The factors that contribute to pain after nerve injury remain incompletely understood. Laser-assisted in situ keratomileusis (LASIK) and photorefractive keratectomy (PRK) are common surgical techniques to correct refractive errors. After LASIK or PRK, a subset of patients suffers intense and persistent pain, of unknown origin, described by patients as feeling like shards of glass in their eye. Here, we evaluated a TRPV1 variant, p.V527M, found in a 49-y-old woman who developed corneal pain after LASIK and subsequent PRK enhancement, reporting an Ocular Surface Disease Index score of 100. Using patch-clamp and Ca²⁺ imaging, we found that the V527M mutation enhances the response to acidic pH. Increasing proton concentration induced a stronger leftward shift in the activation curve of V527M compared to WT, resulting in channel activity of the mutant in acidic pH at more physiological membrane potentials. Finally, comparing the responses to consecutive applications of different agonists, we found in V527M channels a reduced capsaicin-induced desensitization and increased sensitization by the arachidonic acid metabolite 12-hydroxyeicosatetraenoic acid (12-HETE). We hypothesize that the increased response in V527M channels to protons and enhanced sensitization by 12-HETE, two inflammatory mediators released in the cornea after tissue damage, may contribute to the pathogenesis of corneal neuralgia after refractive surgery.

High forest low deforestation jurisdictions (HFLDs) contain many of the world's last intact forests with historically low deforestation. Since carbon financing typically uses historical deforestation rates as baselines, HFLDs facing the prospect of future threats may receive insufficient incentives to be protected. We found that from 2002 to 2020, HFLDs (n = 310) experienced 44% higher deforestation rates than their historical baselines, and 60 HFLDs underwent periods of high deforestation (deforestation rate > 0.501%) at 0.983 ± 0.649% (mean ± SD)-a rate 7.5 times higher than the 10-y historical baseline of all HFLDs. For HFLDs to receive sufficient carbon finance requires baselines that can better reflect future deforestation trajectories of HFLDs. Using an empirical multifactorial model, we show that most contemporary HFLDs are expected to undergo higher deforestation from 2020 to 2038 than their historical baselines, with 72 HFLDs likely (>66% probability) to undergo high deforestation. Over the next 18 y, HFLDs are expected to lose 2.16 Mha y^-1 of forests corresponding to 585 ± 74 MtCO₂e y^-1 (mean ± SE) of emissions. Efforts to protect HFLD forests from future threats will be crucial. In particular, improving baselining methods is key to ensuring that sufficient financing can flow to HFLDs to prevent deforestation.

We present Einstein coefficient spectra and a detailed-balance derivation of generalized Einstein relations between them that is based on the connection between spontaneous and stimulated emission. If two broadened levels or bands overlap in energy, transitions between them need not be purely absorptive or emissive. Consequently, spontaneous emission can occur in both transition directions, and four Einstein coefficient spectra replace the three Einstein coefficients for a line. At equilibrium, the four different spectra obey five pairwise relationships and one lineshape generates all four. These relationships are independent of molecular quantum statistics and predict the Stokes' shift between forward and reverse transitions required by equilibrium with blackbody radiation. For Boltzmann statistics, the relative strengths of forward and reverse transitions depend on the formal chemical potential difference between the initial and final bands, which becomes the standard chemical potential difference for ideal solutes. The formal chemical potential of a band replaces both the energy and degeneracy of a quantum level. Like the energies of quantum levels, the formal chemical potentials of bands obey the Rydberg-Ritz combination principle. Each stimulated Einstein coefficient spectrum gives a frequency-dependent transition cross-section. Transition cross-sections obey causality and a detailed-balance condition with spontaneous emission, but do not directly obey generalized Einstein relations. Even with an energetic width much less than the photon energy, a predominantly absorptive forward transition with an energetic width much greater than the thermal energy can have such an extreme Stokes' shift that its reverse transition cross-section becomes predominantly absorptive rather than emissive.

Human C-reactive protein (CRP) is a pentameric complex involved in immune defense and regulation of autoimmunity. CRP is also a therapeutic target, with both administration and depletion of serum CRP being pursued as a possible treatment for autoimmune and cardiovascular diseases, among others. CRP binds to phosphocholine (PC) moieties on membranes to activate the complement system via the C1 complex, but it is unknown how CRP, or any pentraxin, binds to C1. Here, we present a cryoelectron tomography (cryoET)-derived structure of CRP bound to PC ligands and the C1 complex. To gain control of CRP binding, a synthetic mimotope of PC was synthesized and used to decorate cell-mimetic liposome surfaces. Structure-guided mutagenesis of CRP yielded a fully active complex able to bind PC-coated liposomes that was ideal for cryoET and subtomogram averaging. In contrast to antibodies, which form Fc-mediated hexameric platforms to bind and activate the C1 complex, CRP formed rectangular platforms assembled from four laterally associated CRP pentamers that bind only four of the six available globular C1 head groups. Potential residues mediating lateral association of CRP were identified from interactions between unit cells in existing crystal structures, which rationalized previously unexplained mutagenesis data regarding CRP-mediated complement activation. The structure also enabled interpretation of existing biochemical data regarding interactions mediating C1 binding and identified additional residues for further mutagenesis studies. These structural data therefore provide a possible mechanism for regulation of complement by CRP, which limits complement progression and has consequences for how the innate immune system influences autoimmunity.

Immigrants are highly entrepreneurial. But, what is the broader relationship between high-skilled immigration and regional entrepreneurship activity beyond the ventures that immigrants establish themselves? Using administrative data on newly awarded H-1B visas in the United States, we document a positive relationship between high-skilled immigration and regional entrepreneurship. A doubling of immigrants to a metropolitan statistical area is followed by a 6% increase in entrepreneurship within three years. In contrast, continuing H-1Bs and the arrival of unskilled immigrants (H-2B visas) do not increase regional entrepreneurship. Focusing on Indian immigrants (representing about 70% of all H-1B visas), we find the effect is stronger in metropolitan statistical areas with a larger local Indian population, but not other nationalities, suggesting that presence of conationals facilitates the relationship between high-skilled immigration and regional entrepreneurship. We present this and other evidence as consistent with a knowledge transfer mechanism.