Protein Species Research Articles

Cardiolipin prolongs the lifetimes of respiratory proteins in Drosophila flight muscle

Respiratory complexes and cardiolipins have exceptionally long lifetimes. The fact that they co-localize in mitochondrial cristae raises the questions of whether their longevities have a common cause and whether the longevity of OXPHOS proteins is dependent on cardiolipin. To address these questions, we developed a method to measure side-by-side the half-lives of proteins and lipids in wildtype Drosophila and cardiolipin deficient mutants. We fed adult flies with stable isotope-labeled precursors (13C615N2-lysine or 13C6-glucose) and determined the relative abundance of heavy isotopomers in protein and lipid species by mass spectrometry. To minimize confounding effects of tissue regeneration, we restricted our analysis to the thorax, the bulk of which consists of post-mitotic flight muscles. Analysis of 680 protein and 45 lipid species showed that the subunits of respiratory complexes I-V and the carriers for phosphate and ADP/ATP were among the longest-lived proteins (average half-life of 48±16 days) while the molecular species of cardiolipin were the longest-lived lipids (average half-life of 27±6 days). The remarkable longevity of these crista residents was not shared by all mitochondrial proteins, especially not by those residing in the matrix and the inner boundary membrane. Ablation of cardiolipin synthase, which causes replacement of cardiolipin by phosphatidylglycerol, and ablation of tafazzin, which causes partial replacement of cardiolipin by monolyso-cardiolipin, decreased the lifetimes of the respiratory complexes. Ablation of tafazzin also decreased the lifetimes of the remaining cardiolipin species. These data suggest that an important function of cardiolipin in mitochondria is to protect respiratory complexes from degradation.

A multifunctional integrated biomimetic spore nanoplatform for successively overcoming oral biological barriers

The biological barriers have seriously restricted the efficacious responses of oral delivery system in diseases treatment. Utilizing a carrier based on the single construction means is hard to overcome these obstacles simultaneously because the complex gastrointestinal tract environment requires carrier to have different or even contradictory properties. Interestingly, spore capsid (SC) integrates many unique biological characteristics, such as high resistance, good stability etc. This fact offers a boundless source of inspiration for the construction of multi-functional oral nanoplatform based on SC without further modification. Herein, we develop a type of biomimetic spore nanoplatform (SC@DS NPs) to successively overcome oral biological barriers. Firstly, doxorubicin (DOX) and sorafenib (SOR) are self-assembled to form carrier-free nanoparticles (DS NPs). Subsequently, SC is effectively separated from probiotic spores and served as a functional vehicle for delivering DS NPs. As expect, SC@DS NPs can efficaciously pass through the rugged stomach environment after oral administration and further be transported to the intestine. Surprisingly, we find that SC@DS NPs exhibit a significant improvement in the aspects of mucus penetration and transepithelial transport, which is related to the protein species of SC. This study demonstrates that SC@DS NPs can efficiently overcome multiple biological barriers and improve the therapeutic effect.

In situ synchrotron quantitative analysis of competitive adsorption tendency of human serum protein to different clinical hemodialysis membranes and assessment of potential impacts

Protein adsorption on hemodialysis (HD) membrane matrices and surfaces is a highly undesirable process, as it triggers complement activation and leads to severe health complications for HD patients. However, there is a lack of systematic research investigating the correlation between membrane characteristics and their performance in the dialysis process. This study aims to provide a comprehensive understanding of the competitive adsorption tendencies of three human serum proteins (albumin (HSA), fibrinogen (FB), and transferrin (TRF)) on clinical dialysis membranes composed of polyethersulfone (PES), polyacrylonitrile (PAN), and polyvinyl fluoride (PVDF) polymers. To assess membrane morphology across the membrane cross-section, in situ synchrotron radiation micro-computed tomography (SR-µCT) imaging was conducted at the Canadian Light Source (CLS). Protein adsorption was analyzed qualitatively and quantitatively using an innovative synchrotron-based X-ray tomography technique. To determine the mutual influence of protein species on overall protein adsorption, adsorption from single-protein solutions was compared to adsorption from a protein mixture. Regarding single-protein adsorption, the PVDF membrane exhibited 40% less adsorption of HSA compared to the PES membrane. Conversely, FB adsorption was approximately 25% higher on the PVDF membrane compared to the PES membrane. The PAN membrane showed similar levels of HSA adsorption as the PES membrane and similar levels of FB adsorption as the PVDF membrane. In the case of adsorption from the protein mixture, a suppression of HSA adsorption and replacement of HSA with FB were observed. Consequently, the initial solution with a HSA content of 92% resulted in an adsorbed layer containing approximately 75–80% HSA across all the studied membranes. TRF adsorption remained unaffected by the presence of other proteins, consistent across all three membranes. Notably, the PES membrane exhibited the most significant change in the adsorbed protein composition between single-protein and multi-protein adsorption. In addition, this study comprehensively assesses the impact of fibrinogen adsorption on dialysis, including its consequences, blood activation, and implications for quality of life.

Small-angle scattering from flat bilayers containing correlated scattering length density inhomogeneities.

Model lipid bilayers have been widely employed as a minimal system to investigate the structural properties of biological membranes by small-angle X-ray (SAXS) and neutron scattering (SANS) techniques. These have nanometre resolution and can give information regarding membrane thickness and scattering length densities (SLDs) of polar and apolar regions. However, biological membranes are complex systems containing different lipids and protein species, in which lipid domains can be dynamically assembled and disassembled. Therefore, SLD variations can occur within the biomembrane. In this work, a novel method has been developed to simulate SAXS and SANS profiles obtained from large unilamellar vesicles containing SLD inhomogeneities that are spatially correlated over the membrane surface. Such inhomogeneities are represented by cylindrical entities with equivalent SLDs. Stacking of bilayers is also included in the model, with no correlation between horizontal and vertical order. The model is applied to a lipid bilayer containing SLD inhomogeneities representing pores, lipid domains, and transmembrane, partially immersed and anchored proteins. It is demonstrated that all the structural information from the host lipid bilayer and from the SLD inhomogeneity can be consistently retrieved by a combined analysis of experimental SAXS and SANS data through the methodology proposed here.

Molecular characteristics and potential source of urban PM2.5-bound water-soluble organic matter in Shanghai during springtime

Water-soluble organic matter (WSOM) is complicated component in atmospheric fine particulate matter (PM2.5) and has attracted increasing attentions due to their effects on air quality, climate change, and human health. In this study, the molecular characteristics and potential source of PM2.5-bound water-soluble organic matter in Shanghai during springtime were investigated using ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) coupled with electrospray ionization (ESI) source in negative ion mode. A total of 2537 monoisotopic molecular formulas were assigned in WSOM, and CHO, CHON, CHOS and CHNOS compounds were main components. Highly unsaturated CHO (DBE = 5.01−7.30) and CHON (DBE = 3.66−10.1) compounds were found in good weather days, while the highly oxygenated multifunctional CHOS compounds (O/C = 0.50−0.88) increased in slightly polluted days. Van Krevelen diagrams revealed that carbohydrates, lignin species, and protein species were the most abundant classes in all samples, which their relative abundance accounted for 81.8%. Comparison of our results with previously published data on atmospheric PM2.5 indicated that the biogenic aerosol and local industrial emission presented a larger impact on molecular compositions of WSOM in this region. These findings present a comprehensive overview of WSOM in Shanghai, which helps to understand their characteristics and sources.

Probing the Biosafety of Implantable Artificial Secretory Granules for the Sustained Release of Bioactive Proteins.

Among bio-inspired protein materials, secretory protein microparticles are of clinical interest as self-contained, slow protein delivery platforms that mimic secretory granules of the human endocrine system, in which the protein is both the drug and the scaffold. Upon subcutaneous injection, their progressive disintegration results in the sustained release of the building block polypeptides, which reach the bloodstream for systemic distribution and subsequent biological effects. Such entities are easily fabricated in vitro by Zn-assisted cross-molecular coordination of histidine residues. Using cationic Zn for the assembly of selected pure protein species and in the absence of any heterologous holding material, these granules are expected to be nontoxic and therefore adequate for different clinical uses. However, such presumed biosafety has not been so far confirmed and the potential protein dosage threshold not probed yet. By selecting the receptor binding domain (RBD) from the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein as a model protein and using a mouse lab model, we have explored the toxicity of RBD-made secretory granules at increasing doses up to ∼100 mg/kg of animal weight. By monitoring body weight and biochemical blood markers and through the histological scrutiny of main tissues and organs, we have not observed systemic toxicity. Otherwise, the bioavailability of the material was demonstrated by the induction of specific antibody responses. The presented data confirm the intrinsic biosafety of artificial secretory granules made by recombinant proteins and prompt their further clinical development as self-contained and dynamic protein reservoirs.

Mass spectrometry-based proteomic characterization of the middle-aged mouse brain for animal model research of neuromuscular diseases.

Neuromuscular diseases with primary muscle wasting symptoms may also display multi-systemic changes in the body and exhibit secondary pathophysiological alterations in various non-muscle tissues. In some cases, this includes proteome-wide alterations and/or adaptations in the central nervous system. Thus, in order to provide an improved bioanalytical basis for the comprehensive evaluation of animal models that are routinely used in muscle research, this report describes the mass spectrometry-based proteomic characterization of the mouse brain. Crude tissue extracts were examined by bottom-up proteomics and detected 4558 distinct protein species. The detailed analysis of the brain proteome revealed the presence of abundant cellular proteoforms in the neuronal cytoskeleton, as well as various brain region enriched proteins, including markers of the cerebral cortex, cerebellum, hippocampus and the olfactory bulb. Neuroproteomic markers of specific cell types in the brain were identified in association with various types of neurons and glia cells. Markers of subcellular structures were established for the plasmalemma, nucleus, endoplasmic reticulum, mitochondria and other crucial organelles, as well as synaptic components that are involved in presynaptic vesicle docking, neurotransmitter release and synapse remodelling.

STUDI BIOGEOGRAFIS IKAN PUYAU (osteochilus SP) BERBASIS PROFIL PROTEIN DARI DANAU ROWO IJO, SITUS BUKIT BANGKAI

This Bukit Bangkai would like be became a candidate for place evolution rock art heritage unit (PERAHU). In this site, the Rowo Ijo lake as a habitat, where it was found biogeographically, a Puyau fish (Osteochilus sp). Furthermore, the method of precipitation fraction of ammonium sulfate ((NH4)2SO4) with Lowry and SDS-PAGE elaboration was used, as the main method to obtain the protein profile of fish protein. Analysis of kinship relations was carried out using the UPGMA method based on the profile.. Based on the results of the fraction (NH4)2SO4 and Lowry methods, the optimum protein content was obtained, namely 68.93; 116.08; 6.72; 7.96 (mg/ml) in the 70-80% fraction and 4 samples from Banjar Regency 19.17; 16.93; 20.04; and 20.04 mg/mL in the 40-70% fraction, respectively. Based on the results of SDS-PAGE at the optimum level of protein; Puyau Rowo Ijo fish have 64 bands, with a profile of 11 bands on P.1R code, 9 bands on P.2R code, 10 bands on P.3R code and 9 bands on P.4R. For Puyau Banjar fish there are 25 bands with the following profile; each code P.1B and P.2B has 6 bands, while P.3B has 10 bands and P.4B has 3 bands. The types of proteins found were thought to include Actin Protein, Carbonic Anhydrase, Trypsin Inhibitor, -lactalbumin, and Lysozyme. The results of the UPGMA analysis based on the distribution of protein species and weights were that there was related closely to two groups form (clusters) between puyau from the Lake Rowo Ijo and Banjar Regency.

Variations in the Protein Hydration and Hydrogen-Bond Network of Water Molecules Induced by the Changes in the Secondary Structures of Proteins Studied through Near-Infrared Spectroscopy.

This study investigated how the secondary structural changes of proteins in aqueous solutions affect their hydration and the hydrogen-bond network of water molecules using near-infrared (NIR) spectroscopy. The aqueous solutions of three types of proteins, i.e., ovalbumin, β-lactoglobulin, and bovine serum albumin, were denatured by heating, and changes in the NIR bands of water reflecting the states of hydrogen bonds induced via protein secondary structural changes were investigated. On heating, the intermolecular hydrogen bonds between water molecules as well as between water and protein molecules were broken, and protein molecules were no longer strongly bound by the surrounding water molecules. Consequently, the denaturation was observed to proceed depending on the thermodynamic properties of the proteins. When the aqueous solutions of proteins were cooled after denaturation, the hydrogen-bond network was reformed. However, the state of protein hydration was changed owing to the secondary structural changes of proteins, and the variation patterns were different depending on the protein species. These changes in protein hydration may be derived from the differences in the surface charges of proteins. The elucidation of the mechanism of protein hydration and the formation of the hydrogen-bond network of water molecules will afford a comprehensive understanding of the protein functioning and dysfunctioning derived from the structural changes in proteins.

Evaluation of the contents and ratios of five fractionated proteins to elucidate the protein composition in soybean seeds

AbstractSelecting suitable soybean cultivars is important for food processing and exploring their endowment with desirable physiological functions. We applied the fractionation method previously established to compare soy protein composition among cultivars to promote such a selection. More than 95% of the proteins in soybean cotyledons were extracted from 13 soybean cultivars using a high‐concentration salt solution. The extracted proteins were fractionated into five fractions, namely oil body‐associated protein (OBAP), polar lipid‐associated protein (PLAP), globulins (11S and 7S), and whey by centrifugation after tuning the solubility behavior of the proteins with various solutions. Protein species in each fraction were analyzed by SDS‐PAGE. Protein content in the total extract and five fractions was quantified to characterize the protein composition of soybean cultivars. The correlation between the protein content of each fraction and the total protein in cotyledon was investigated. A strong positive correlation was found only for the 11S fraction (r = 0.82), followed by a positive correlation in the 7S fraction (r = 0.65). Thus, we surmised that the increased protein content in soybean was due to increased globulin content. Furthermore, the calculation of the average ratio of protein content in each fraction indicated the globulin fraction (7S and 11S) to be 52%, the lipophilic protein fraction (OBAP and PLAP) to be 33%, and the whey fraction to be 13%. The preparation method employed in this study is a promising tool for efficiently comparing the protein composition of soybean cultivars to evaluate the potential use of cultivars for food production.

Mutated Toll-like receptor 9 increases Alzheimer’s disease risk by compromising innate immunity protection

The development of Alzheimer’s disease (AD) involves central and peripheral immune deregulation. Gene identification and studies of AD genetic variants of peripheral immune components may aid understanding of peripheral-central immune crosstalk and facilitate new opportunities for therapeutic intervention. In this study, we have identified in a Flanders-Belgian family a novel variant p.E317D in the Toll-like receptor 9 gene (TLR9), co-segregating with EOAD in an autosomal dominant manner. In human, TLR9 is an essential innate and adaptive immune component predominantly expressed in peripheral immune cells. The p.E317D variant caused 50% reduction in TLR9 activation in the NF-κB luciferase assay suggesting that p.E317D is a loss-of-function mutation. Cytokine profiling of human PBMCs upon TLR9 activation revealed a predominantly anti-inflammatory response in contrast to the inflammatory responses from TLR7/8 activation. The cytokines released upon TLR9 activation suppressed inflammation and promoted phagocytosis of Aβ42 oligomers in human iPSC-derived microglia. Transcriptome analysis identified upregulation of AXL, RUBICON and associated signaling pathways, which may underline the effects of TLR9 signaling-induced cytokines in regulating the inflammatory status and phagocytic property of microglia. Our data suggest a protective role of TLR9 signaling in AD pathogenesis, and we propose that TLR9 loss-of-function may disrupt a peripheral-central immune crosstalk that promotes dampening of inflammation and clearance of toxic protein species, leading to the build-up of neuroinflammation and pathogenic protein aggregates in AD development.

Endocytic pathways of pathogenic protein aggregates in neurodegenerative diseases.

Endocytosis is the fundamental uptake process through which cells internalize extracellular materials and species. Neurodegenerative diseases (NDs) are characterized by a progressive accumulation of intrinsically disordered protein species, leading to neuronal death. Misfolding in many proteins leads to various NDs such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS) and other disorders. Despite the significance of disordered protein species in neurodegeneration, their spread between cells and the cellular uptake of extracellular species is not entirely understood. This review discusses the major internalization mechanisms of the different conformer species of these proteins and their endocytic mechanisms. We briefly introduce the broad types of endocytic mechanisms found in cells and then summarize what is known about the endocytosis of monomeric, oligomeric and aggregated conformations of tau, Aβ, α-Syn, Huntingtin, Prions, SOD1, TDP-43 and other proteins associated with neurodegeneration. We also highlight the key players involved in internalizing these disordered proteins and the several techniques and approaches to identify their endocytic mechanisms. Finally, we discuss the obstacles involved in studying the endocytosis of these protein species and the need to develop better techniques to elucidate the uptake mechanisms of a particular disordered protein species.

Proteomic analysis by mass spectrometry of postmortem muscle protein degradation for PMI estimation: A pilot study

The determination of the postmortem interval is a topic of great forensic interest. The possibility of using new technologies has allowed the study of postmortem decay of biomolecules in the determination of PMI. Skeletal muscle proteins are promising candidates because skeletal muscle exhibits slower postmortem decay compared to other internal organs and nervous tissues, while its degradation is faster than cartilage and bone. In this pilot study, skeletal muscle tissue from pigs was degraded at two different controlled temperatures, 21 °C and 6 °C, and analysed at predefined times points: 0, 24, 48, 72, 96, and 120 h. The obtained samples were analysed by mass spectrometry proteomics approach for qualitative and quantitative evaluation of proteins and peptides. Immunoblotting validation was performed for the candidate proteins. The results obtained appeared significant and identified several proteins useful for possible postmortem interval estimation. Of these proteins, PDLIM7, TPM1, and ATP2A2 were validated by immunoblotting at a larger number of experimental points and at different temperatures. The results obtained are in agreement with those observed in similar works. In addition, the use of a mass spectrometry approach increased the number of protein species identified, providing a larger panel of proteins for PMI assessment.

Influence of apolipoprotein E (apoE) on ADAM10 secretase in cerebrospinal fluid of older adults with Alzheimer’s disease

AbstractBackgroundThe ε4 allele of APOE is the strongest genetic risk for sporadic late‐onset Alzheimer’s disease (AD), and is associated with an increase in the levels of β‐amyloid (Aβ) deposition. The main α‐secretase that inhibits the Aβ deposition is the ADAM10, which is altered in the blood and cerebrospinal fluid (CSF) of older adults with AD.MethodTo assess whether the levels of ADAM10 forms are altered in CSF of AD and non‐dementia control (NDC) individuals with different APOE genotype, we analysed a total of 13 samples from NDC (mean age = 66.3 ±9.8; T‐tau = 305 ±135 ng/L; Aβ42 = 780 ±253 ng/L) and 41 from AD (mean age 77.0 ±6.2; T‐tau = 845 ±356.1 ng/L; Aβ42 = 456 ± 66 ng/L), subdivided into NDC APOE ε3/ε3 (n = 8), NDC APOE ε3/ε4 (n = 5), AD APOE ε3/ε3 (n = 13), AD APOE ε3/ε4 (n = 14) and AD APOE ε4/ε4 (n = 14). ADAM10 was characterized in human CSF samples by western blotting.ResultAn antibody raised against to the ectodomain region of ADAM10, common to all species, recognized three bands, which might be the proADAM10 (immature form retaining the prodomain), ADAM10f (mature unprocessed full‐length form) and sADAM10 (truncated fragment). Regarding proADAM10, no differences were found among NDC with different APOE genotype. However, the AD APOE ε4/ε4 showed a reduction, which was significantly lower than AD APOE ε3/ε3 (p = 0.0011), NDC APOE ε3/ε3 (p = 0.0460) and NDC APOE ε3/ε3 (p = 0.0012), suggesting the impact of being homozygous for APOE ε4 allele on proADAM10 levels. In the ADAM10f and sADAM10, we observed similar findings. For both species of protein, AD groups carriers of APOE ε4 allele, that is, AD APOE ε3/ε4 and AD APOE ε4/ε4, presented significant lower levels of ADAM10f compared with both non‐carriers subgroups (AD APOE ε3/ε3 and NDC APOE ε3/ε3).ConclusionThe results indicated that being a carrier of the APOE ε4 allele and having developed AD are associated with lower levels of ADAM10f and sADAM10. This study contributes to a better understanding the impact of apoE on the disease.

Anle138b interaction in α-synuclein aggregates by dynamic nuclear polarization NMR.

Small molecules that bind to oligomeric protein species such as membrane proteins and fibrils are of clinical interest for development of therapeutics and diagnostics. Definition of the binding site at atomic resolution via NMR is often challenging due to low binding stoichiometry of the small molecule. For fibrils and aggregation intermediates grown in the presence of lipids, we report atomic-resolution contacts to the small molecule at sub nm distance via solid-state NMR using dynamic nuclear polarization (DNP) and orthogonally labelled samples of the protein and the small molecule. We apply this approach to α-synuclein (αS) aggregates in complex with the small molecule anle138b, which is a clinical drug candidate for disease modifying therapy. The small central pyrazole moiety of anle138b is detected in close proximity to the protein backbone and differences in the contacts between fibrils and early intermediates are observed. For intermediate species, the 100K condition for DNP helps to preserve the aggregation state, while for both fibrils and oligomers, the DNP enhancement is essential to obtain sufficient sensitivity.

Can MSH3 lowering stop HTT repeat expansion in its CAG tract?

Huntington’s disease (HD) is a neurodegenerative repeat expansion disorder caused by an expansion of the CAG trinucleotide repeat present in exon 1 of the huntingtin (HTT) gene. The expanded repeat exhibits instability in somatic tissues, resulting in an inexorable increase in length over the HD gene carrier’s lifetime. Although the length of the expanded CAG repeat is the strongest predictor of age at onset and progression, potential genetic modifiers have also been identified. 1 Moss D.J.H. Pardiñas A.F. Langbehn D. Lo K. Leavitt B.R. Roos R. Durr A. Mead S. TRACK-HD investigators REGISTRY investigators et al. Identification of genetic variants associated with Huntington’s disease progression: a genome-wide association study. Lancet Neurol. 2017; 16: 701-711 Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar ,2 Genetic Modifiers of Huntington’s Disease GeM-HD Consortium Lee J.-M. Correia K. Loupe J. Kim K.-H. Barker D. Hong E.P. Chao M.J. Long J.D. Lucente D. et al. CAG repeat not polyglutamine length determines timing of huntington’s disease onset. Cell. 2019; 178: 887-900.e14 Abstract Full Text Full Text PDF Scopus (183) Google Scholar Many of these modifiers are within DNA repair genes and have been implicated in repeat expansion, including the mismatch repair protein MSH3. The process of somatic repeat expansion in the brain appears to be a major factor in driving disease progression, leading to production of more toxic HTT protein species. As a monogenic disease, many genetic therapeutic approaches past and present aim to ameliorate the effects of the mutant HTT protein by lowering its levels. In this issue of Molecular Therapy, O’Reilly and colleagues 3 O'Reilly D. Belgrad J. Ferguson C. Summers A. Sapp E. McHugh C. Mathews E. Boudi A. Buchwald J. Ly S. et al. Di-valent siRNA mediated silencing of MSH3 blocks somatic repeat expansion in mouse models of Huntington’s disease. Mol. Ther. 2023; https://doi.org/10.1016/j.ymthe.2023.05.006 Abstract Full Text Full Text PDF Google Scholar demonstrate the potential of an alternative therapeutic intervention to slow the expansion of the CAG repeat by targeting MSH3. They achieve suppression of somatic expansion in two different but complementary HD mouse models by instead lowering the expression of MSH3, using a nucleic acid therapeutic modality established previously by the Khvorova group, the di-valent siRNA (di-siRNA). 4 Alterman J.F. Godinho B.M.D.C. Hassler M.R. Ferguson C.M. Echeverria D. Sapp E. Haraszti R.A. Coles A.H. Conroy F. Miller R. et al. A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system. Nat. Biotechnol. 2019; 37: 884-894 Crossref PubMed Scopus (81) Google Scholar

Transcriptomic analyses reveal the potential antibacterial mechanism of citral against Staphylococcus aureus.

The emergence of multi-drug resistant Staphylococcus aureus (S. aureus) has posed a challenging clinical problem for treating its infection. The development of novel or new antibacterial agents becomes one of the useful methods to solve this problem, and has received more attention over the past decade. Citral is reported to have antibacterial activity against S. aureus, but its mechanism is yet entirely clear. To reveal the antibacterial mechanism of citral against S. aureus, comparative transcriptomic analysis was carried out to analyze the gene expression differences between the citral-treated and untreated groups. The changes of protein, adenosine triphosphate (ATP) and reactive oxygen species (ROS) content in S. aureus caused by citral were also examined. Six hundred and fifty-nine differentially expressed genes were obtained according to the comparative transcriptomic analysis, including 287 up-regulated genes and 372 down-regulated genes. The oxidoreductase activity and fatty acid degradation pathway were enriched in up-regulated genes, and ribosome and S. aureus infection pathway were enriched in down-regulated genes. Meanwhile, physiological trials revealed a decline in ATP and protein levels, but an increase in ROS content within the citral-treated group. Thus, it can be inferred that the antibacterial effects of citral against S. aureus were likely due to its ability to decrease ATP content by down-regulating ATP synthase genes (atpD and atpG), reduce protein content, induce cell membrane and cell wall damages, accumulate ROS, and down-regulate virulence factor genes to reduce pathogenicity. These findings revealed the antibacterial mechanism of citral was likely a type of multi-target mode that affected multiple molecular processes in S. aureus, which lays the groundwork for further exploitation of citral as a therapeutic candidate against S. aureus infections.

Mass Spectrometry of RNA-Binding Proteins during Liquid-Liquid Phase Separation Reveals Distinct Assembly Mechanisms and Droplet Architectures.

Liquid-liquid phase separation (LLPS) of heterogeneous ribonucleoproteins (hnRNPs) drives the formation of membraneless organelles, but structural information about their assembled states is still lacking. Here, we address this challenge through a combination of protein engineering, native ion mobility mass spectrometry, and molecular dynamics simulations. We used an LLPS-compatible spider silk domain and pH changes to control the self-assembly of the hnRNPs FUS, TDP-43, and hCPEB3, which are implicated in neurodegeneration, cancer, and memory storage. By releasing the proteins inside the mass spectrometer from their native assemblies, we could monitor conformational changes associated with liquid-liquid phase separation. We find that FUS monomers undergo an unfolded-to-globular transition, whereas TDP-43 oligomerizes into partially disordered dimers and trimers. hCPEB3, on the other hand, remains fully disordered with a preference for fibrillar aggregation over LLPS. The divergent assembly mechanisms revealed by ion mobility mass spectrometry of soluble protein species that exist under LLPS conditions suggest structurally distinct complexes inside liquid droplets that may impact RNA processing and translation depending on biological context.

Novel therapeutic approaches to target neurodegeneration.

Ageing is the main risk factor common to most primary neurodegenerative disorders. Indeed, age-related brain alterations have been long considered to predispose to neurodegeneration. Although protein misfolding and the accumulation of toxic protein aggregates have been considered as causative events in neurodegeneration, several other biological pathways affected by brain ageing also contribute to pathogenesis. Here, we discuss the evidence showing the involvement of the mechanisms controlling neuronal structure, gene expression, autophagy, cell metabolism and neuroinflammation in the onset and progression of neurodegenerative disorders. Furthermore, we review the therapeutic strategies currently under development or as future approaches designed to normalize these pathways, which may then increase brain resilience to cope with toxic protein species. In addition to therapies targeting the insoluble protein aggregates specifically associated with each neurodegenerative disorder, these novel pharmacological approaches may be part of combined therapies designed to rescue brain function.

Apolipoprotein E O-glycosylation is associated with amyloid plaques and APOE genotype

Although the APOE ε4 allele is the strongest genetic risk factor for sporadic Alzheimer's disease (AD), the relationship between apolipoprotein (apoE) and AD pathophysiology is not yet fully understood. Relatively little is known about the apoE protein species, including post-translational modifications, that exist in the human periphery and CNS. To better understand these apoE species, we developed a LC-MS/MS assay that simultaneously quantifies both unmodified and O-glycosylated apoE peptides. The study cohort included 47 older individuals (age 75.6 ± 5.7 years [mean ± standard deviation]), including 23 individuals (49%) with cognitive impairment. Paired plasma and cerebrospinal fluid samples underwent analysis. We quantified O-glycosylation of two apoE protein residues – one in the hinge region and one in the C-terminal region – and found that glycosylation occupancy of the hinge region in the plasma was significantly correlated with plasma total apoE levels, APOE genotype and amyloid status as determined by CSF Aβ42/Aβ40. A model with plasma glycosylation occupancy, plasma total apoE concentration, and APOE genotype distinguished amyloid status with an AUROC of 0.89. These results suggest that plasma apoE glycosylation levels could be a marker of brain amyloidosis, and that apoE glycosylation may play a role in the pathophysiology of AD.