Cell Membrane Components Research Articles

A Novel Cell Membrane-Associated RNA Extraction Method and Its Application in the Discovery of Breast Cancer Markers.

Cell membrane-associated RNA (mem-RNA) has been demonstrated to be cell-specific and disease-related and are considered as potential biomarkers for disease diagnostics, drug delivery, and cell screening. However, there is still a lack of methods specifically designed to extract mem-RNA from cells, limiting the discovery and applications of mem-RNA. In this study, we propose the first all-in-one solution for high-purity mem-RNA isolation based on two types of magnetic nanoparticles, named MREMB (Membrane-associated RNA Extraction based on Magnetic Beads), which achieved ten times enrichment of cell membrane components and over 90% recovery rate of RNA extraction. To demonstrate MREMB's potential in clinical research, we extracted and sequenced mem-RNA of typical breast cancer MCF-7, MDA-MB-231, and SKBR-3 cell lines and non-neoplastic breast epithelial cell MCF-10A. Compared to total RNA, sequencing results revealed that membrane/secreted protein-encoding mRNAs and long noncoding RNAs (lncRNAs) were enriched in the mem-RNA, some of which were significantly overexpressed in the three cancer cell lines, including extracellular matrix-related genes COL5A1 and lncRNA TALAM1. The results indicated that MREMB could enrich membrane/secreted protein-coding RNA and amplify the expression differences of related RNAs between cancer and non-neoplastic cells, promising for cancer biomarker discovery.

Mechanism analysis of calcium-induced low freeze-drying survival of probiotic encapsulated in alginate

Calcium alginate is widely used for probiotics encapsulation. In our previous report, low freeze-drying survival of probiotics encapsulated in calcium alginate was presented due to the damage by calcium with increased concentration during freeze-drying. The damage makes freeze-dried probiotic-loaded calcium alginate difficult to use in commercial foods. Therefore, it is urgent to analyze the mechanism of calcium-induced low survival of probiotics. In the present work, this mechanism was analyzed using Lactobacillus rhamnosus GG (LGG) as a research model. The results showed that calcium led to the emergence of 491 differential genes (300 up-regulated genes), 309 (175 up-regulated metabolites, positive ion) and 129 differential metabolites (33 up-regulated metabolites, negative ion). For one of the mechanisms, GO and KEGG analysis of differential genes indicated that calcium increased cardiolipin by downregulating glpO and upregulating LGG-RS07795 and cls genes in the glycerophospholipid metabolism pathway, increasing the fluidity of cell membrane, thereby causing the catabolism of cell membrane components. Additionally, the disturbances of differential genes/metabolites co-enrichment pathways by calcium were other mechanisms. Specifically, the correlation analysis of differential genes/metabolites that had upstream/downstream relationships in co-enrichment KEGG pathways showed consistently strong correlations, among which hisC gene regulated histidine metabolism pathway and phenylalanine, tyrosine and tryptophan biosynthesis pathway, ndk gene regulated pyrimidine metabolism pathway and purine metabolism pathway, and 3-phosphoserine, phenylalanine, and tyrosine were involved in multiple pathways including other differential genes/metabolites co-enrichment KEGG pathways. Based on the above mechanism analysis, the addition of small molecule sugar/sugar alcohol and down-regulated differential metabolites (ergothioneine and cytidine) in the co-enrichment pathway were effective strategies to improve the freeze-drying survival of LGG encapsulated in calcium alginate. Overall, these findings are expected to promote the application of dried calcium-based probiotics foods in the food industry.

Random forest and live single-cell metabolomics reveal metabolic profiles of human macrophages upon polarization.

Human macrophages are innate immune cells with diverse, functionally distinct phenotypes, namely, pro-inflammatory M1 and anti-inflammatory M2 macrophages. Both are involved in multiple physiological and pathological processes, including would healing, infection, and cancer. However, the metabolic differences between these phenotypes are largely unexplored at single-cell resolution. To address this knowledge gap, an untargeted live single-cell mass spectrometry-based metabolomic profiling coupled with a machine-learning data analysis approach was developed to investigate the metabolic profile of each phenotype at the single-cell level. Results show that M1 and M2 macrophages have distinct metabolic profiles, with differential levels of fatty acyls, glycerophospholipids, and sterol lipids, which are important components of plasma membrane and involved in multiple biological processes. Furthermore, we could discern several putatively annotated molecules that contribute to inflammatory response of macrophages. The combination of random forest and live single-cell metabolomics provided an in-depth profile of the metabolome of primary human M1 and M2 macrophages at the single-cell level for the first time, which will pave the way for future studies targeting the differentiation of other immune cells.

SREBP-1 upregulates lipophagy to maintain cholesterol homeostasis in brain tumor cells

Cholesterol is a structural component of cell membranes. How rapidly growing tumor cells maintain membrane cholesterol homeostasis is poorly understood. Here, we found that glioblastoma (GBM), the most lethal brain tumor, maintains normal levels of membrane cholesterol but with an abundant presence of cholesteryl esters (CEs) in its lipid droplets (LDs). Mechanistically, SREBP-1 (sterol regulatory element-binding protein 1), a master transcription factor that is activated upon cholesterol depletion, upregulates critical autophagic genes, including ATG9B, ATG4A, and LC3B, as well as lysosome cholesterol transporter NPC2. This upregulation promotes LD lipophagy, resulting in the hydrolysis of CEs and the liberation of cholesterol from the lysosomes, thus maintaining plasma membrane cholesterol homeostasis. When this pathway is blocked, GBM cells become quite sensitive to cholesterol deficiency with poor growth invitro. Our study unravels an SREBP-1-autophagy-LD-CE hydrolysis pathway that plays an important role in maintaining membrane cholesterol homeostasis while providing a potential therapeutic avenue for GBM.

Cholesterol-Controlled Interaction of Ionic Liquids with Model Cellular Membranes.

While ionic liquids (ILs) are considered as prospective ingredients of new antimicrobial agents, it is important to understand the adverse effects of these molecules on human cells. Since cholesterol is the essential component of a human cell membrane, in the present study, the effect of an imidazolium-based IL has been investigated on the model membrane in the presence of cholesterol. The area per sphingomyelin lipid is found to reduce in the presence of the IL, which is quantified by the area-surface pressure isotherm of the lipid monolayer formed at the air-water interface. The effect is considerably diminished in the cholesterol-containing monolayer. Further, the IL is observed to decrease the rigidity of the cholesterol-free monolayer. Interestingly, the presence of cholesterol does not allow any change in this property of the layer at lower surface pressure. However, at a higher surface pressure, the IL increases the elasticity in the cholesterol-induced condensed phase of the lipid layer. The X-ray reflectivity measurement on a stack of cholesterol-free lipid bilayers proved the formation of IL-induced phase-separated domains in the matrix of a pure lipid phase. These domains are found to be formed by interdigitating the chains of the lipids, producing a thinner membrane. Such a phase is less intense in the cholesterol-containing membrane. All of these results indicate that the IL molecules may deform the cholesterol-free membrane of a bacterial cell, but the same may not be harmful to human beings as cholesterol could restrict the insertion in the cellular membrane of a human cell.

Role of microbiome in development of systemic inflammatory response syndrome and sepsis (review)

Sepsis is a life-threatening organ dysfunction caused by a disturbed response to infection. Its development is preceded by systemic inflammatory response syndrome, which is the overall inflammatory response of the body to severe lesions. The role of opportunistic pathogens in the development of systemic inflammatory response syndrome and sepsis may be known, but the value of the intestinal microbiome remains underestimated in this context.
 Experimental models are widely employed to study the role of the microbiome in the development of sepsis. Animal models of sepsis are created by disrupting the barrier function of the host intestine through cecal ligation/puncture, installation of an ascending bowel stent, and intraperitoneal feces injection. Toxemia is reproduced by the injection of lipopolysaccharides, peptidoglycans, lipoteichoic acid, CpG DNA, zymosan, and synthetic lipopeptides.
 The review systematized data on the role of the cell wall or membrane components of gram-positive and gram-negative bacteria, which are representatives of the intestinal microbiome in the pathogenesis of systemic inflammatory response syndrome and sepsis.

The Chemical Profile, and Antidermatophytic, Anti-Inflammatory, Antioxidant and Antitumor Activities of Withania chevalieri A.E. Gonç. Ethanolic Extract

Withania chevalieri, endogenous from Cape Verde, is a medicinal plant used in ethnomedicine with a large spectrum of applications, such as treating skin fungal infections caused by dermatophytes. The aim of this work was to chemically characterize the W. chevalieri crude ethanolic extract (WcCEE), and evaluate its bioactivities as antidermatophytic, antioxidant, anti-inflammatory and anticancer, as well as its cytotoxicity. WcCEE was chemically characterized via HPLC–MS. The minimal inhibitory concentration, minimal fungicidal concentration, time-kill and checkerboard assays were used to study the antidermatophytic activity of WcCEE. As an approach to the mechanism of action, the cell wall components, β-1,3-glucan and chitin, and cell membrane ergosterol were quantified. Transmission electron microscopy (TEM) allowed for the study of the fungal ultrastructure. WcCEE contained phenolic acids, flavonoids and terpenes. It had a concentration-dependent fungicidal activity, not inducing relevant resistance, and was endowed with synergistic effects, especially terbinafine. TEM showed severely damaged fungi; the cell membrane and cell wall components levels had slight modifications. The extract had antioxidant, anti-inflammatory and anti-cancer activities, with low toxicity to non-tumoral cell lines. The results demonstrated the potential of WcCEE as an antidermatophytic agent, with antioxidant, anti-inflammatory and anticancer activity, to be safely used in pharmaceutical and dermocosmetic applications.

Glycosphingolipids in human parasites.

Glycosphingolipids (GSLs) are comprised of glycans (oligosaccharides) linked to a lipid containing a sphingosine moiety. They are major membrane components in cells of most animals, and importantly, they also occur in parasitic protozoans and worms that infect people. While the endogenous functions of the GSLs in most parasites are elusive, many of these GSLs are recognized by antibodies in infected human and animal hosts, and thus, their structures, biosynthesis, and functions are of great interest. Such knowledge of GSLs could lead to new drugs and diagnostics for treating infections, as well as novel vaccine strategies. The diversity of GSLs recently identified in such infectious organisms and aspects of their immune recognition are major topics of this review. It is not intended to be exhaustive but to highlight aspects of GSL glycans in human parasites.

Impacts of polypropylene microplastics on lipid profiles of mouse liver uncovered by lipidomics analysis and Raman spectroscopy

Microplastics (MPs) are emerging contaminants, and there are only limited studies reporting the impacts of some MPs on liver lipid metabolism in animals. In this study, we investigated the accumulation of polypropylene-MPs in mouse liver and unraveled the change in lipid metabolic profiles by both lipidomics and Raman spectroscopy. Polypropylene-MP exposure did not cause obvious health symptoms, but hematoxylin-eosin staining showed pathological changes that polypropylene-MPs induced lipid droplet accumulation in liver. Lipidomics results showed a significant change in lipid metabolic profiles and the most influenced categories were triglycerides, fatty acids, free fatty acids and lysophosphatidylcholine, implying the effects of polypropylene-MPs on the hemostasis of lipid droplet biogenesis and catabolism. Most altered lipids contained unsaturated bonds and polyunsaturated phospholipids, possibly affecting the fluidity and curvature of membrane surfaces. Raman spectroscopy confirmed that the major spectral alterations of liver tissues were related to lipids, evidencing the altered lipid metabolism and cell membrane components in the presence of polypropylene-MPs. Our findings firstly disclosed the impacts of polypropylene-MPs on lipid metabolisms in mouse liver and hinted at their detrimental disturbance on membrane properties, cellular lipid storage and oxidation regulation, helping our deeper understanding on the toxicities and corresponding risks of polypropylene-MPs to mammals.

Surface-enhanced fluorescence for lipopolysaccharide analysis based on shell-isolated nanoparticle

Lipopolysaccharide (LPS) as the component of cell membrane on gram-negative bacteria played a central role on inflammatory inducer to stimulate a multi-system host response. Herein, a surface-enhanced fluorescent (SEF) sensor was developed for LPS analysis based on shell-isolated nanoparticles (SHINs). The fluorescent signal of CdTe quantum dots (QDs) was amplified by silica shell-coated Au nanoparticles (Au NPs). The 3D finite-difference time-domain (3D-FDTD) simulation revealed that this enhancement was due to local electric field amplification. This method has a linear detection range of 0.1–20 μg/mL and a detection limit of 64 ng/mL for LPS. Furthermore, the developed method was successfully applied for LPS analysis in milk and human serum sample. The results indicated that the as-prepared sensor has significant potential for selective detection of LPS in biomedical diagnosis and food safety.

NK Cells from Human Cytomegalovirus-Seropositive Individuals Have a Distinct Metabolic Profile That Correlates with Elevated mTOR Signaling.

CMV can elicit adaptive immune features in both mouse and human NK cells. Mouse Ly49H+ NK cells expand 100- to 1000-fold in response to mouse CMV infection and persist for months after exposure. Human NKG2C+ NK cells also expand after human CMV (HCMV) infection and persist for months. The clonal expansion of adaptive NK cells is likely an energy-intensive process, and the metabolic requirements that support adaptive NK cell expansion and persistence remain largely uncharacterized. We previously reported that NK cells from HCMV-seropositive donors had increased maximum capacity for both glycolysis and mitochondrial oxidative phosphorylation relative to NK cells from HCMV-seronegative donors. In this article, we report an extension of this work in which we analyzed the metabolomes of NK cells from HCMV-seropositive donors with NKG2C+ expansions and NK cells from HCMV seronegative donors without such expansions. NK cells from HCMV+ donors exhibited striking elevations in purine and pyrimidine deoxyribonucleotides, along with moderate increases in plasma membrane components. Mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase that, as a part of mTOR complex 1 (mTORC1), bridges nutrient signaling to metabolic processes necessary for cell growth. Signaling through mTORC1 induces both nucleotide and lipid synthesis. We observed elevated mTORC1 signaling on activation in both NKG2C- and NKG2C+ NK cells from HCMV+ donors relative to those from HCMV- donors, demonstrating a correlation between higher mTORC1 activity and synthesis of key metabolites for cell growth and division.

Regulation of TORC2 Function and Localization in Yeast.

Every eukaryotic cell contains two distinct multisubunit protein kinase complexes that each contain a TOR (target of rapamycin) protein as the catalytic subunit. These ensembles, designated TORC1 and TORC2, serve as nutrient and stress sensors, signal integrators, and regulators of cell growth and homeostasis, but they differ in their composition, localization, and function. TORC1, activated on the cytosolic surface of the vacuole (or, in mammalian cells, on the cytosolic surface of the lysosome), promotes biosynthesis and suppresses autophagy. TORC2, located primarily at the plasma membrane (PM), maintains the proper levels and bilayer distribution of all PM components (sphingolipids, glycerophospholipids, sterols, and integral membrane proteins), which are needed for the membrane expansion that accompanies cell growth and division and for combating insults to PM integrity. This review summarizes our current understanding of the assembly, structural features, subcellular distribution, and function and regulation of TORC2, obtained largely through studies conducted with Saccharomyces cerevisiae.

Doxorubicin Interaction with Lipid Monolayers Leads to Decreased Membrane Stiffness when Experiencing Compression-Expansion Dynamics.

Physical membrane models permit to study and quantify the interactions of many external molecules with monitored and simplified systems. In this work, we have constructed artificial Langmuir single-lipid monolayers with dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylserine (DPPS), or sphingomyelin to resemble the main lipid components of the mammalian cell membranes. We determined the collapse pressure, minimum area per molecule, and maximum compression modulus (Cs-1) from surface pressure measurements in a Langmuir trough. Also, from compression/expansion isotherms, we estimated the viscoelastic properties of the monolayers. With this model, we explored the membrane molecular mechanism of toxicity of the well-known anticancer drug doxorubicin, with particular emphasis in cardiotoxicity. The results showed that doxorubicin intercalates mainly between DPPS and sphingomyelin, and less between DPPE, inducing a change in the Cs-1 of up to 34% for DPPS. The isotherm experiments suggested that doxorubicin had little effect on DPPC, partially solubilized DPPS lipids toward the bulk of the subphase, and caused a slight or large expansion in the DPPE and sphingomyelin monolayers, respectively. Furthermore, the dynamic viscoelasticity of the DPPE and DPPS membranes was greatly reduced (by 43 and 23%, respectively), while the reduction amounted only to 12% for sphingomyelin and DPPC models. In conclusion, doxorubicin intercalates into the DPPS, DPPE, and sphingomyelin, but not into the DPPC, membrane lipids, inducing a structural distortion that leads to decreased membrane stiffness and reduced compressibility modulus. These alterations may constitute a novel, early step in explaining the doxorubicin mechanism of action in mammalian cancer cells or its toxicity in non-cancer cells, with relevance to explain its cardiotoxicity.

Formation and Tandem Mass Spectrometry of Doubly Charged Lipid-Metal Ion Complexes.

Phospholipids are major components of most eukaryotic cell membranes. Changes in metabolic states are often accompanied by phospholipid structure variations. The structural changes of phospholipids are the hallmark of disease states, or specific lipid structures have been associated with distinct organisms. Prime examples are microorganisms that synthesize phospholipids with, for example, different branched chain fatty acids. Assignment and relative quantitation of structural isomers of phospholipids that arise from attachment of different fatty acids to the glycerophospholipid backbone are difficult with routine tandem mass spectrometry or with liquid chromatography without authentic standards. In this work, we report on the observation that all investigated phospholipid classes form doubly charged lipid-metal ion complexes during electrospray ionization (ESI) and show that these complexes can be used to assign lipid classes and fatty acid moieties, distinguish isomers of branched chain fatty acids, and relatively quantify these isomers in positive-ion mode. Use of water free methanol and addition of divalent metal salts (100 mol %) to ESI spray solutions afford highly abundant doubly charged lipid-metal ion complexes (up to 70 times of protonated compounds). Higher-energy collisional dissociation and collision-induced dissociation of doubly charged complexes yield a diverse set of lipid class-dependent fragment ions. In common for all lipid classes is the liberation of fatty acid-metal adducts that yield fragment ions from the fatty acid hydrocarbon chain upon activation. This ability is used to pinpoint sites of branching in saturated fatty acids and is showcased for free fatty acids as well as glycerophospholipids. The analytical utility of doubly charged phospholipid-metal ion complexes is demonstrated by distinguishing fatty acid branching-site isomers in phospholipid mixtures and relatively quantifying the corresponding isomeric compounds.

Microbial Surfactants to the Rescue

Microbial Surfactants to the Rescue

Dual mechanisms of cholesterol-GPCR interactions that depend on membrane phospholipid composition

Cholesterol is a critical component of mammalian cell membranes and an allosteric modulator of G protein-coupled receptors (GPCRs), but divergent views exist on the mechanisms by which cholesterol influences receptor functions. Leveraging the benefits of lipid nanodiscs, i.e., quantitative control of lipid composition, we observe distinct impacts of cholesterol in the presence and absence of anionic phospholipids on the function-related conformational dynamics of the human A2A adenosine receptor (A2AAR). Direct receptor-cholesterol interactions drive activation of agonist-bound A2AAR in membranes containing zwitterionic phospholipids. Intriguingly, the presence of anionic lipids attenuates cholesterol's impact through direct interactions with the receptor, highlighting a more complex role for cholesterol that depends on membrane phospholipid composition. Targeted amino acid replacements at two frequently predicted cholesterol interaction sites showed distinct impacts of cholesterol at different receptor locations, demonstrating the ability to delineate different roles of cholesterol in modulating receptor signaling and maintaining receptor structural integrity.

A new insight into the polar lipid composition in mature breast milk and ewe milk with comparative lipidomics analysis

Polar lipids play essential biological functions in energy storage, both as structural components of cell membranes and as signaling molecules. In this study, a comprehensive UHPLC-QTRAP-MS-based lipidomic analysis of mature breast milk (BM) and ewe milk (EM) was conducted. Through the analysis, a total of 362 polar lipid species from 14 subclasses were characterized, including 60 phosphatidylethanolamines (PEs), 59 phosphatidylcholines (PCs), 38 phosphatidylinositols (PIs), 35 sphingomyelins (SMs), and 34 ceramides (Cers). Of these, 139 lipid molecules were screened as significantly differentially expressed polar lipids (SDPLs) between the two kinds of milk based on the following criteria: a variable importance in projection (VIP) value > 1.0, a false discovery rate-adjusted P-value < 0.0001, and a fold change (FC) of either > 2.0 or < 0.5; these included 111 upregulated and 28 downregulated SDPLs in EM compared to BM. Among these SDPLs, the content of PE (16:1_18:0) was found to be significantly higher in EM compared to BM (FC = 69.5853, P < 0.0001). Moreover, sphingolipid metabolism and glycerophospholipid metabolism were determined to be vital metabolic pathways. This was derived from the finding that PE, PC, SM, and PI were key lipid metabolites in the two kinds of milk that were related to these two metabolic pathways. This study provides new insights into the characterization of SDPLs in mammalian milk, and also provides a theoretical basis for optimizing infant formula.

Eukaryotic splicing machinery in the plant-virus battleground.

Plant virual infections are mainly caused by plant-virus parasitism which affects ecological communities. Some viruses are highly pathogen specific that can infect only specific plants, while some cancause widespread harm, such as tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV). After a virus infects the host, undergoes a series of harmful effects, including the destruction of host cell membrane receptors, changes in cell membrane components, cell fusion, and the production of neoantigens on the cell surface. Therefore, competition between the host and the virus arises. The virus starts gaining control of critical cellular functions of the host cells and ultimately affects the fate of the targetedhost plants. Among these critical cellular processes, alternative splicing (AS) is an essential posttranscriptional regulation process in RNA maturation, which amplify host protein diversity and manipulates transcript abundance in response to plant pathogens. AS is widespread in nearly all human genes and critical in regulating animal-virus interactions. In particular, an animal virus can hijack the host splicing machinery to re-organize its compartments for propagation. Changes in AS are known to cause human disease, and various AS events have been reported to regulate tissue specificity, development, tumour proliferation, and multi-functionality. However, the mechanisms underlying plant-virus interactions are poorly understood. Here, we summarize the current understanding of how viruses interact with their plant hosts compared with humans, analyze currently used and putative candidate agrochemicals to treat plant-viral infections, and finally discussed the potential research hotspots in the future. This article is categorized under: RNA Processing > Splicing Mechanisms RNA Processing > Splicing Regulation/Alternative Splicing.

Structural basis of NINJ1-mediated plasma membrane rupture in cell death

Eukaryotic cells can undergo different forms of programmed cell death, many of which culminate in plasma membrane rupture as the defining terminal event1–7. Plasma membrane rupture was long thought to be driven by osmotic pressure, but it has recently been shown to be in many cases an active process, mediated by the protein ninjurin-18 (NINJ1). Here we resolve the structure of NINJ1 and the mechanism by which it ruptures membranes. Super-resolution microscopy reveals that NINJ1 clusters into structurally diverse assemblies in the membranes of dying cells, in particular large, filamentous assemblies with branched morphology. A cryo-electron microscopy structure of NINJ1 filaments shows a tightly packed fence-like array of transmembrane α-helices. Filament directionality and stability is defined by two amphipathic α-helices that interlink adjacent filament subunits. The NINJ1 filament features a hydrophilic side and a hydrophobic side, and molecular dynamics simulations show that it can stably cap membrane edges. The function of the resulting supramolecular arrangement was validated by site-directed mutagenesis. Our data thus suggest that, during lytic cell death, the extracellular α-helices of NINJ1 insert into the plasma membrane to polymerize NINJ1 monomers into amphipathic filaments that rupture the plasma membrane. The membrane protein NINJ1 is therefore an interactive component of the eukaryotic cell membrane that functions as an in-built breaking point in response to activation of cell death.

Proteostatic modulation in brain aging without associated Alzheimer's disease-and age-related neuropathological changes.

(Phospho)proteomics of old-aged subjects without cognitive or behavioral symptoms, and without AD-neuropathological changes and lacking any other neurodegenerative alteration will increase understanding about the physiological state of human brain aging without associate neurological deficits and neuropathological lesions. (Phospho)proteomics using conventional label-free- and SWATH-MS (Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry) has been assessed in the frontal cortex (FC) of individuals without NFTs, senile plaques (SPs) and age-related co-morbidities classified by age (years) in four groups; group 1 (young, 30-44); group 2 (middle-aged: MA, 45-52); group 3 (early-elderly, 64-70); and group 4 (late-elderly, 75-85). Protein levels and deregulated protein phosphorylation linked to similar biological terms/functions, but involving different individual proteins, are found in FC with age. The modified expression occurs in cytoskeleton proteins, membranes, synapses, vesicles, myelin, membrane transport and ion channels, DNA and RNA metabolism, ubiquitin-proteasome-system (UPS), kinases and phosphatases, fatty acid metabolism, and mitochondria. Dysregulated phosphoproteins are associated with the cytoskeleton, including microfilaments, actin-binding proteins, intermediate filaments of neurons and glial cells, and microtubules; membrane proteins, synapses, and dense core vesicles; kinases and phosphatases; proteins linked to DNA and RNA; members of the UPS; GTPase regulation; inflammation; and lipid metabolism. Noteworthy, protein levels of large clusters of hierarchically-related protein expression levels are stable until 70. However, protein levels of components of cell membranes, vesicles and synapses, RNA modulation, and cellular structures (including tau and tubulin filaments) are markedly altered from the age of 75. Similarly, marked modifications occur in the larger phosphoprotein clusters involving cytoskeleton and neuronal structures, membrane stabilization, and kinase regulation in the late elderly. Present findings may increase understanding of human brain proteostasis modifications in the elderly in the subpopulation of individuals not having AD neuropathological change and any other neurodegenerative change in any telencephalon region.