Search

The 26S proteasome is the macromolecular machine responsible for ATP/ubiquitin dependent degradation. As aberration in proteasomal degradation has been implicated in many human diseases, structural analysis of the human 26S proteasome complex is essential to advance our understanding of its action and regulation mechanisms. In recent years, cross-linking mass spectrometry (XL-MS) has emerged as a powerful tool for elucidating structural topologies of large protein assemblies, with its unique capability of studying protein complexes in cells. To facilitate the identification of cross-linked peptides, we have previously developed a robust amine reactive sulfoxide-containing MS-cleavable cross-linker, disuccinimidyl sulfoxide (DSSO). To better understand the structure and regulation of the human 26S proteasome, we have established new DSSO-based in vivo and in vitro XL-MS workflows by coupling with HB-tag based affinity purification to comprehensively examine protein-protein interactions within the 26S proteasome. In total, we have identified 447 unique lysine-to-lysine linkages delineating 67 interprotein and 26 intraprotein interactions, representing the largest cross-link dataset for proteasome complexes. In combination with EM maps and computational modeling, the architecture of the 26S proteasome was determined to infer its structural dynamics. In particular, three proteasome subunits Rpn1, Rpn6, and Rpt6 displayed multiple conformations that have not been previously reported. Additionally, cross-links between proteasome subunits and 15 proteasome interacting proteins including 9 known and 6 novel ones have been determined to demonstrate their physical interactions at the amino acid level. Our results have provided new insights on the dynamics of the 26S human proteasome and the methodologies presented here can be applied to study other protein complexes.

Neutrophil-to-lymphocyte ratio (NLR) has been suggested to be a prognostic marker for various diseases, but whether NLR dynamics (ΔNLR) is related to mortality and disease severity in patients with septic acute kidney injury (AKI) has not been determined. Between August 2013 and August 2021, septic AKI patients at our center were retrospectively enrolled. ΔNLR was defined as the difference between the NLR at septic AKI diagnosis and at hospital admission. The relationship between the ΔNLR and mortality was evaluated by Kaplan–Meier curves, Cox proportional hazards, and cubic spline analyses. The prediction values were compared by area under the receiver-operating characteristic curve (AUROC), net reclassification improvement (NRI), and integrated discrimination improvement (IDI) analyses. Of the 413 participants, the mean age was 63 ± 17 years, and 134 were female (32.4%). According to the median value, patients in the high-ΔNLR group had significantly greater 90-d mortality (74.4% vs. 46.6%, p < 0.001). After adjustment for potential confounders, high ΔNLR remained an independent predictor of 90-d mortality (HR = 2.80; 95% CI = 1.74–4.49, p < 0.001). Furthermore, ΔNLR had the highest AUROC for 90-d mortality (0.685) among the various biomarkers and exhibited an improved NRI (0.314) and IDI (0.027) when incorporated with PCT and CRP. For secondary outcomes, patients with high ΔNLR had increased risk of 30-d mortality (p = 0.004), need for renal replacement therapy (p = 0.011), and developing stage-3 AKI (p = 0.040) according to the adjusted models. High ΔNLR is independently associated with increased risk of patient mortality and adverse outcomes. ΔNLR might be utilized to facilitate risk stratification and optimize septic AKI management. [ABSTRACT FROM AUTHOR]

Metal‐halide perovskite solar cells (PSCs) have emerged as a promising photovoltaic technology. Fabricating PSCs in ambient air can accelerate their low‐cost commercialization, since it can remove the reliance on atmosphere‐controlled equipment. However, the power conversion efficiency (PCE) of air‐fabricated PSCs still lags behind those fabricated in glovebox. Here, based on a technology to fabricate high‐quality perovskite film in ambient air, a compatible optimization is performed on electron transport layer (ETL) to further enhance the photovoltaic performance of PSCs. A soft‐templated deposition strategy is proposed that utilizes tetrasodium glutamate diacetate (GLDA) to finely regulate the chemical bath deposition process, leading to an ideal SnO2 ETL with no additive residual. Adopting this feature of no residual, a molecular bridge using β‐guanidinopropionic acid (βA) is constructed at the buried interface (SnO2/perovskite), which effectively enhances the electron extraction and decreases electron losses. The resulting PSCs (0.08 cm2) achieve an impressive PCE of 25.74% (certificated 25.43%), which is the highest among the air‐fabricated PSCs reported to date. A PCE of 24.61% in 1 cm2‐PSCs is also obtained, exhibiting the scalable potential of the technology. In addition, the excellent operational stability of these PSCs is also demonstrated. [ABSTRACT FROM AUTHOR]

The doped organic hole transport layer (HTL) is crucial for achieving high‐efficiency perovskite solar cells (PSCs). However, the traditional doping strategy undergoes a time‐consuming and environment‐dependent oxidation process, which hinders the technology upgrades and commercialization of PSCs. Here, we reported a new strategy by introducing a cascade reaction in traditional doped Spiro‐OMeTAD, which can simultaneously achieve rapid oxidation and overcome the erosion of perovskite by 4‐tert‐butylpyridine (tBP) in organic HTL. The ideal dopant iodobenzene diacetate was utilized as the initiator that can react with Spiro to generate Spiro⋅+ radicals quickly and efficiently without the participation of ambient air, with the byproduct of iodobenzene (DB). Then, the DB can coordinate with tBP through a halogen bond to form a tBP‐DB complex, minimizing the sustained erosion from tBP to perovskite. Based on the above cascade reaction, the resulting Spiro‐based PSCs have a champion PCE of 25.76 % (certificated of 25.38 %). This new oxidation process of HTL is less environment‐dependent and produces PSCs with higher reproducibility. Moreover, the PTAA‐based PSCs obtain a PCE of 23.76 %, demonstrating the excellent applicability of this doping strategy on organic HTL. [ABSTRACT FROM AUTHOR]

In response to the growing demand for railway obstacle monitoring, lidar technology has emerged as an up-and-coming solution. In this study, we developed a mechanical 3D lidar system and meticulously calibrated the point cloud transformation to monitor specific areas precisely. Based on this foundation, we have devised a novel set of algorithms for obstacle detection within point clouds. These algorithms encompass three key steps: (a) the segmentation of ground point clouds and extraction of track point clouds using our RS-Lo-RANSAC (region select Lo-RANSAC) algorithm; (b) the registration of the BP (background point cloud) and FP (foreground point cloud) via an improved Robust ICP algorithm; and (c) obstacle recognition based on the VFOR (voxel-based feature obstacle recognition) algorithm from the fused point clouds. This set of algorithms has demonstrated robustness and operational efficiency in our experiments on a dataset obtained from an experimental field. Notably, it enables monitoring obstacles with dimensions of 15 cm × 15 cm × 15 cm. Overall, our study showcases the immense potential of lidar technology in railway obstacle monitoring, presenting a promising solution to enhance safety in this field. [ABSTRACT FROM AUTHOR]

Proteasomes are protein degradation machines that exist in cells as heterogeneous and dynamic populations. A group of proteins function as ubiquitin receptors (UbRs) that can recognize and deliver ubiquitinated substrates to proteasome complexes for degradation. Defining composition of proteasome complexes engaged with UbRs is critical to understand proteasome function. However, because of the dynamic nature of UbR interactions with the proteasome, it remains technically challenging to capture and isolate UbR-proteasome subcomplexes using conventional purification strategies. As a result, distinguishing the molecular differences among these subcomplexes remains elusive. We have developed a novel affinity purification strategy, in vivo cross-linking (X) assisted bimolecular tandem affinity purification strategy (XBAP), to effectively isolate dynamic UbR-proteasome subcomplexes and define their subunit compositions using label-free quantitative mass spectrometry. In this work, we have analyzed seven distinctive UbR-proteasome complexes and found that all of them contain the same type of the 26S holocomplex. However, selected UbRs interact with a group of proteasome interacting proteins that may link each UbR to specific cellular pathways. The compositional similarities and differences among the seven UbR-proteasome subcomplexes have provided new insights on functional entities of proteasomal degradation machineries. The strategy described here represents a general and useful proteomic tool for isolating and studying dynamic and heterogeneous protein subcomplexes in cells that have not been fully characterized.

Adduction of a nitric oxide moiety (NO•) to cysteine(s), termed S-nitrosylation (SNO), is a novel mechanism for NO to regulate protein function directly. However, the endothelial SNO-protein network that is affected by endogenous and exogenous NO is obscure. This study was designed to develop a quantitative proteomics approach using stable isotope labeling by amino acids in cell culture for comparing vascular endothelial growth factor (VEGFA)- and NO donor-responsive endothelial nitroso-proteomes. Primary placental endothelial cells were labeled with "light" (L-(12)C6 (14)N4-Arg and L-(12)C6 (14)N2-Lys) or "heavy" (L-(13)C6 (15)N4-Arg and L-(13)C6 (15)N2-Lys) amino acids. The light cells were treated with an NO donor nitrosoglutathione (GSNO, 1 mM) or VEGFA (10 ng/ml) for 30 min, while the heavy cells received vehicle as control. Equal amounts of cellular proteins from the light (GSNO or VEGFA treated) and heavy cells were mixed for labeling SNO-proteins by the biotin switch technique and then trypsin digested. Biotinylated SNO-peptides were purified for identifying SNO-proteins by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ratios of light to heavy SNO-peptides were calculated for determining the changes of the VEGFA- and GSNO-responsive endothelial nitroso-proteomes. A total of 387 light/heavy pairs of SNO-peptides were identified, corresponding to 213 SNO-proteins that include 125 common and 27 VEGFA- and 61 GSNO-responsive SNO-proteins. The specific SNO-cysteine(s) in each SNO-protein were simultaneously identified. Pathway analysis revealed that SNO-proteins are involved in various endothelial functions, including proliferation, motility, metabolism, and protein synthesis. We collectively conclude that endogenous NO on VEGFA stimulation and exogenous NO from GSNO affect common and different SNO-protein networks, implicating SNO as a critical mechanism for VEGFA stimulation of angiogenesis.

GSK3β regulates some functions of the brain, but the mechanisms involved in the maintenance of GSK3β protein stability remain ambiguous. REGγ, an important proteasome activator for ubiquitin-independent protein degradation, has been shown to degrade certain intact proteins and is involved in the regulation of important biological processes. Here we demonstrate that REGγ promotes the degradation of GSK3β protein in vitro and in vivo. With increased GSK3β activity, REGγ knockout (REGγ-/-) mice exhibit late-onset sensorimotor gating and cognitive deficiencies including decreased working memory, hyperlocomotion, increased stereotype, defective prepulse inhibition (PPI), and disability in nest building, at the age of 8 months or older. Inhibition of GSK3β rescued the compromised PPI phenotypes and working memory deficiency in the knockout mice. Also, we found an age-dependent decrease in the trypsin-like proteasomal activity in REGγ-/- mice brains, which may be reflective of a lack of degradation of GSK3β. Collectively, our findings reveal a novel regulatory pathway in which the REGγ-proteasome controls the steady-state level of GSK3β protein. Dysfunction in this non-canonical proteasome degradation pathway may contribute to the sensorimotor gating deficiency and cognitive disorders in aging mice.