Profiling Protein-Protein Interactions in the Human Brain by Refined Cofractionation Mass Spectrometry.

Proteins usually execute their biological functions through interactions with other proteins and by forming macromolecular complexes, but global profiling of protein complexes directly from human tissue samples has been limited. In this study, we utilized cofractionation mass spectrometry (CF-MS) to map protein complexes within the postmortem human brain with experimental replicates. First, we used concatenated anion and cation Ion Exchange Chromatography (IEX) to separate native protein complexes in 192 fractions and then proceeded with Data-Independent Acquisition (DIA) mass spectrometry to analyze the proteins in each fraction, quantifying a total of 4,804 proteins with 3,260 overlapping in both replicates. We improved the DIA's quantitative accuracy by implementing a constant amount of bovine serum albumin (BSA) in each fraction as an internal standard. Next, advanced computational pipelines, which integrate both a database-based complex analysis and an unbiased protein-protein interaction (PPI) search, were applied to identify protein complexes and construct protein-protein interaction networks in the human brain. Our study led to the identification of 486 protein complexes and 10054 binary protein-protein interactions, which represents the first global profiling of human brain PPIs using CF-MS. Overall, this study offers a resource and tool for a wide range of human brain research, including the identification of disease-specific protein complexes in the future.

Synthesis of a Triangle-Fused Six-Pointed Star and Its Electrocatalytic CO2 Reduction Activity.

As electrocatalysts, molecular catalysts with large aromatic systems (such as terpyridine, porphyrin, or phthalocyanine) have been widely applied in the CO2 reduction reaction (CO2RR). However, these monomeric catalysts tend to aggregate due to strong π-π interactions, resulting in limited accessibility of the active site. In light of these challenges, we present a novel strategy of active site isolation for enhancing the CO2RR. Six Ru(Tpy)2 were integrated into the skeleton of a metallo-organic supramolecule by stepwise self-assembly in order to form a rhombus-fused six-pointed star R1 with active site isolation. The turnover frequency (TOF) of R1 was as high as 10.73 s-1 at -0.6 V versus reversible hydrogen electrode (vs RHE), which is the best reported value so far at the same potential to our knowledge. Furthermore, by increasing the connector density on R1's skeleton, a more stable triangle-fused six-pointed star T1 was successfully synthesized. T1 exhibits exceptional stability up to 126 h at -0.4 V vs RHE and excellent TOF values of CO. The strategy of active site isolation and connector density increment significantly enhanced the catalytic activity by increasing the exposure of the active site. This work provides a starting point for the design of molecular catalysts and facilitates the development of a new generation of catalysts with a high catalytic performance.

TsMS combined with EA promotes functional recovery and axonal regeneration via mediating the miR-539-5p/Sema3A/PlexinA1 signalling axis in sciatic nerve-injured rats.

Enhancing axonal regeneration is one of the most important processes in treating nerve injuries. Both magnetic and electrical stimulation have the effect of promoting nerve axon regeneration. But few study has investigated the effects of trans-spinal magnetic stimulation (TsMS) combined with electroacupuncture (EA) on nerve regeneration in rats with sciatic nerve injury. In this study, we compared the improvement of neurological function in rats with sciatic nerve crush injuries after 4 weeks of different interventions (EA, TsMS, or TsMS combined with EA). We further explored the morphological and molecular biological alterations following sciatic nerve injury by HE, Masson, RT-PCR, western blotting, immunofluorescence staining and small RNA transcriptome sequencing. The results showed that TsMS combined with EA treatment significantly promoted axonal regeneration, increased the survival rate of neurons, and suppressed denervation atrophy of the gastrocnemius muscle. Subsequent experiments suggested that the combination treatment may play an active role by mediating the miR-539-5p/Sema3A/PlexinA1 signaling axis.

Treadmill training improves lung function and inhibits alveolar cell apoptosis in spinal cord injured rats.

Secondary lung injury after SCI is a major cause of patient mortality, with apoptosis playing a key role. This study aimed to explore the impact of treadmill training and miR145-5p on the MAPK/Erk signaling pathway and apoptosis in rats with complete SCI. SD rats were used to establish T10 segmental complete SCI models and underwent treadmill training 3, 7, or 14 days postinjury. Various techniques including arterial blood gas analysis, lung wet/dry weight ratio, HE staining, immunofluorescence staining, immunohistochemical staining, qRT-PCR, and Western blotting were employed to assess alterations in lung function and the expression levels of crucial apoptosis-related factors. In order to elucidate the specific mechanism, the impact of miR145-5p on the MAPK/Erk pathway and its role in apoptosis in lung cells were confirmed through miR145-5p overexpression and knockdown experiments. Following spinal cord injury (SCI), an increase in apoptosis, activation of the MAPK/Erk pathway, and impairment of lung function were observed in SCI rats. Conversely, treadmill training resulted in a reduction in alveolar cell apoptosis, suppression of the MAPK/Erk pathway, and enhancement of lung function. The gene MAP3K3 was identified as a target of miR145-5p. The influence of miR145-5p on the MAPK/Erk pathway and its impact on apoptosis in alveolar cells were confirmed through the manipulation of miR145-5p expression levels. The upregulation of miR145-5p in spinal cord injury (SCI) rats led to a reduction in MAP3K3 protein expression within lung tissues, thereby inhibiting the MAPK/Erk signaling pathway and decreasing apoptosis. Contrarily, rats with miR145-5p knockdown undergoing treadmill training exhibited an increase in miR145-5p expression levels, resulting in the inhibition of MAP3K3 protein expression in lung tissues, suppression of the MAPK/Erk pathway, and mitigation of lung cell apoptosis. Ultimately, the findings suggest that treadmill training may attenuate apoptosis in lung cells post-spinal cord injury by modulating the MAP3K3 protein through miR145-5p to regulate the MAPK/Erk signaling pathway.

Electric Field Generated at the Millisecond Pulse-Polarized Interface Facilitates the Electrolytic Conversion of SO2 into H2S.

Interfacial electric field holds significant importance in determining both the polar molecular configuration and surface coverage during electrocatalysis. This study introduces a methodology leveraging the varying electric dipole moment of SO2 under distinct interfacial electric field strengths to enhance the selectivity of the SO2 electroreduction process. This approach presented the first attempt to utilize pulsed voltage application to the Au/PTFE membrane electrode for the control of the molecular configuration and coverage of SO2 on the electrode surface. Remarkably, the modulation of pulse duration resulted in a substantial inhibition of the hydrogen evolution reaction (HER) (FEH2 < 3%) under millisecond pulse conditions (ta = 10 ms, tc = 300 ms, Ea = -0.8 V (vs Hg/Hg2SO4), Ec = -1.8 V (vs Hg/Hg2SO4)), concomitant with a noteworthy enhancement in H2S selectivity (FEH2S > 97%). A comprehensive analysis, incorporating in situ Raman spectroscopy, electrochemical quartz crystal microbalance, COMSOL simulations, and DFT calculations, corroborated the increased selectivity of H2S products was primarily associated with the inherently large dipole moment of the SO2 molecule. The enhancement of the interfacial electric field induced by millisecond pulses was instrumental in amplifying SO2 coverage, activating SO2, facilitating the formation of the pivotal intermediate product *SOH, and effectively reducing the reaction energy barrier in the SO2 reduction process. These findings provide novel insights into the influences of ion and molecular transport dynamics, as well as the temporal intricacies of competitive pathways during the SO2 electroreduction process. Moreover, it underscores the intrinsic correlation between the electric dipole moment and surface-molecule interaction of the catalyst.

Evaluation of Protein Identification and Quantification by the diaPASEF Method on timsTOF SCP.

Accurate and precise quantification is crucial in modern proteomics, particularly in the context of exploring low-amount samples. While the innovative 4D-data-independent acquisition (DIA) quantitative proteomics facilitated by timsTOF mass spectrometers gives enhanced sensitivity and selectivity for protein identification, the diaPASEF (parallel accumulation-serial fragmentation combined with data-independent acquisition) parameters have not been systematically optimized, and a comprehensive evaluation of the quantification is currently lacking. In this study, we conducted a thorough optimization of key parameters on a timsTOF SCP instrument, including sample loading amount (50 ng), ramp/accumulation time (140 ms), isolation window width (20 m/z), and gradient time (60 min). To further improve the identification of proteins in low-amount samples, we utilized different column settings and introduced 0.02% n-dodecyl-ß-d-maltoside (DDM) in the sample reconstitution solution, resulting in a remarkable 19-fold increase in protein identification at the single-cell-equivalent level. Moreover, a comprehensive comparison of protein quantification using a tandem mass tag reporter (TMT-reporter), complement TMT ions (TMTc), and diaPASEF revealed a strong correlation between these methods. Both diaPASEF and TMTc have effectively addressed the issue of ratio compression, highlighting the diaPASEF method's effectiveness in achieving accurate quantification data compared to TMT reporter quantification. Additionally, an in-depth analysis of in-group variation positioned diaPASEF between the TMT-reporter and TMTc methods. Therefore, diaPASEF quantification on the timsTOF SCP instrument emerges as a precise and accurate methodology for quantitative proteomics, especially for samples with small amounts.

Integrated proteomics reveals autophagy landscape and an autophagy receptor controlling PKA-RI complex homeostasis in neurons.

Autophagy is a conserved, catabolic process essential for maintaining cellular homeostasis. Malfunctional autophagy contributes to neurodevelopmental and neurodegenerative diseases. However, the exact role and targets of autophagy in human neurons remain elusive. Here we report a systematic investigation of neuronal autophagy targets through integrated proteomics. Deep proteomic profiling of multiple autophagy-deficient lines of human induced neurons, mouse brains, and brain LC3-interactome reveals roles of neuronal autophagy in targeting proteins of multiple cellular organelles/pathways, including endoplasmic reticulum (ER), mitochondria, endosome, Golgi apparatus, synaptic vesicle (SV) for degradation. By combining phosphoproteomics and functional analysis in human and mouse neurons, we uncovered a function of neuronal autophagy in controlling cAMP-PKA and c-FOS-mediated neuronal activity through selective degradation of the protein kinase A - cAMP-binding regulatory (R)-subunit I (PKA-RI) complex. Lack of AKAP11 causes accumulation of the PKA-RI complex in the soma and neurites, demonstrating a constant clearance of PKA-RI complex through AKAP11-mediated degradation in neurons. Our study thus reveals the landscape of autophagy degradation in human neurons and identifies a physiological function of autophagy in controlling homeostasis of PKA-RI complex and specific PKA activity in neurons.