Detergent-insoluble Fraction Research Articles

Biochemical Properties of Synaptic Proteins Are Dependent on Tissue Preparation: NMDA Receptor Solubility Is Regulated by the C-Terminal Tail.

Synaptic proteins are essential for neuronal development, synaptic transmission, and synaptic plasticity. The postsynaptic density (PSD) is a membrane-associated structure at excitatory synapses, which is composed of a huge protein complex. To understand the interactions and functions of PSD proteins, researchers have employed a variety of imaging and biochemical approaches including sophisticated mass spectrometry. However, the field is lacking a systematic comparison of different experimental conditions and how they might influence the study of the PSD interactome isolated from various tissue preparations. To evaluate the efficiency of several common solubilization conditions, we isolated receptors, scaffolding proteins, and adhesion molecules from brain tissue or primary cultured neurons or human forebrain neurons differentiated from induced pluripotent stem cells (iPSCs). We observed some striking differences in solubility. We found that N-methyl-d-aspartate receptors (NMDARs) and PSD-95 are relatively insoluble in brain tissue, cultured neurons, and human forebrain neurons compared to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptors (AMPARs) or SAP102. In general, synaptic proteins were more soluble in primary neuronal cultures and human forebrain neurons compared to brain tissue. Interestingly, NMDARs are relatively insoluble in HEK293T cells suggesting that insolubility does not directly represent the synaptic fraction but rather it is related to a detergent-insoluble fraction such as lipid rafts. Surprisingly, truncation of the intracellular carboxyl-terminal tail (C-tail) of NMDAR subunits increased NMDAR solubility in HEK293T cells. Our findings show that detergent, pH, and temperature are important for protein preparations to study PSD protein complexes, and NMDAR solubility is regulated by its C-tail, thus providing a technical guide to study synaptic interactomes and subcellular localization of synaptic proteins.

Adult-onset deactivation of autophagy leads to loss of synapse homeostasis and cognitive impairment, with implications for alzheimer disease

ABSTRACT A growing number of studies link dysfunction of macroautophagy/autophagy to the pathogenesis of diseases such as Alzheimer disease (AD). Given the global importance of autophagy for homeostasis, how its dysfunction can lead to specific neurological changes is puzzling. To examine this further, we compared the global deactivation of autophagy in the adult mouse using the atg7iKO with the impact of AD-associated pathogenic changes in autophagic processing of synaptic proteins. Isolated forebrain synaptosomes, rather than total homogenates, from atg7iKO mice demonstrated accumulation of synaptic proteins, suggesting that the synapse might be a vulnerable site for protein homeostasis disruption. Moreover, the deactivation of autophagy resulted in impaired cognitive performance over time, whereas gross locomotor skills remained intact. Despite deactivation of autophagy for 6.5 weeks, changes in cognition were in the absence of cell death or synapse loss. In the symptomatic APP PSEN1 double-transgenic mouse model of AD, we found that the impairment in autophagosome maturation coupled with diminished presence of discrete synaptic proteins in autophagosomes isolated from these mice, leading to the accumulation of one of these proteins in the detergent insoluble protein fraction. This protein, SLC17A7/Vglut, also accumulated in atg7iKO mouse synaptosomes. Taken together, we conclude that synaptic autophagy plays a role in maintaining protein homeostasis, and that while decreasing autophagy interrupts normal cognitive function, the preservation of locomotion suggests that not all circuits are affected similarly. Our data suggest that the disruption of autophagic activity in AD may have relevance for the cognitive impairment in this adult-onset neurodegenerative disease. Abbreviations: 2dRAWM: 2-day radial arm water maze; AD: Alzheimer disease; Aβ: amyloid-beta; AIF1/Iba1: allograft inflammatory factor 1; APP: amyloid beta precursor protein; ATG7: autophagy related 7; AV: autophagic vacuole; CCV: cargo capture value; Ctrl: control; DLG4/PSD-95: discs large MAGUK scaffold protein 4; GFAP: glial fibrillary acidic protein; GRIN2B/NMDAR2b: glutamate ionotropic receptor NMDA type subunit 2B; LTD: long-term depression; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; m/o: months-old; PNS: post-nuclear supernatant; PSEN1/PS1: presenilin 1; SHB: sucrose homogenization buffer; SLC32A1/Vgat: solute carrier family 32 member 1; SLC17A7/Vglut1: solute carrier family 17 member 7; SNAP25: synaptosome associated protein 25; SQSTM1/p62: sequestosome 1; SYN1: synapsin I; SYP: synaptophysin ; SYT1: synaptotagmin 1; Tam: tamoxifen; VAMP2: vesicle associated membrane protein 2; VCL: vinculin; wks: weeks.

The interplay between calcineurin inhibitors (CNIs) and WNK bodies

KS-WNK1 is a kidney-specific scaffolding isoform of the With-No-Lysine (WNK) kinase signaling pathway. It is the predominant WNK1 isoform in the kidney, lacks a functional kinase domain, and is highly expressed in the distal convoluted tubule (DCT) where it drives the formation of biomolecular condensates called WNK bodies. During hypokalemia, WNK bodies bring together KS-WNK1, L-WNK1, WNK4, and SPAK, facilitating activation of the kinase cascade. This leads to increased downstream phosphorylation of the SLC12 cotransporter NCC. Mechanisms governing the reversibility and size of WNK body condensate scaffolds are poorly understood. We propose that phosphatases regulate KS-WNK1 and WNK body formation--a likely candidate being the phosphatase calcineurin (PP3). Prior work suggests that calcineurin dephosphorylates and inhibits the WNK-SPAK-NCC pathway, whereas calcineurin inhibitors (CNIs) such as tacrolimus increase phosphorylation and activation of the pathway. Here, we tested the hypothesis that CNIs also increase the abundance of KS-WNK1, causing increased WNK body size and abundance, and activation of the WNK-SPAK-NCC pathway. To study the effect of endogenous WNK kinases on KS-WNK1 function, WT HEK293 cells or HEK293 L-WNK1 KO cells were transiently transfected with KS-WNK1-HA-mRuby, treated with tacrolimus and studied by immunofluorescence confocal microscopy and immunoblotting of supernatant and detergent-insoluble pellet fractions. In both WT and KO cells, tacrolimus significantly increased WNK body abundance and size. Consistent with its localization within WNK bodies, KS-WNK1 protein was enriched in the pellet fraction by 10-fold in both WT and KO cells. KS-WNK1 protein abundance was increased by tacrolimus in a dose- (0-400ng/ml) and time-dependent manner (0-72h), an effect that was independent of endogenous L-WNK1 expression. Consistent with previous studies, SPAK abundance and phosphorylation was increased with tacrolimus treatment in a dose and time-dependent manner. Interestingly, SPAK localization was shifted into the pellet fraction in WT cells expressing KS-WNK1, whereas it was localized in the supernatant in L-WNK1 KO cells. The tacrolimus-induced increase in KS-WNK1 and pSPAK correlated with increased SLC12 cotransporter phosphorylation in WT cells. These preliminary results suggest that tacrolimus promotes WNK body formation and activity via KS-WNK1, and that SPAK localization in HEK cell-reconstituted WNK bodies requires full length WNK1. There is growing evidence that biomolecular condensates concentrate biomolecules to optimize signaling pathways and enhance kinase specificity through spatiotemporal control. Here we propose a novel concept that WNK bodies are biomolecular condensates that are regulated by calcineurin inhibitors. NIH K08DK118211, U54DK137329, ASN Gottschalk Award, P30DK081943. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Serine Phosphorylation Marks Proteins for Degradation By the Mitochondrial Serine Protease Complex, Clpxp

ClpXP, a serine protease located in the mitochondrial matrix, regulates mitochondrial proteostasis by degrading damaged or aggregated proteins, including respiratory chain subunits. ClpXP is a bipartite protein complex comprised of the ClpX regulatory particle that caps each end of the ClpP degradation chamber. ClpX recognizes substrates, unfolds them, and feeds them into ClpP for proteolysis. Genetic or chemical inhibition of ClpXP leads to impaired oxidative phosphorylation function and leukemic cell death in vitro and in vivo. Importantly, however, the degradation marker of human ClpXP remains elusive. Since the bacterial homologue of ClpXP recognizes phosphorylated arginine (pArg)-tagged protein for degradation, we hypothesized that human ClpXP might also recognize phosphorylated amino acids as degrons. We first investigated whether phosphorylation influences substrate degradation by ClpXP using a model substrate, α-casein. Recombinant human ClpXP preferentially degraded phosphorylated α-casein compared to dephosphorylated α-casein. To further elucidate the effects of phosphorylation, we screened a panel of phosphorylated amino acids for their ability to impede ClpXP-mediated degradation of FITC-tagged α-casein. Phosphorylated serine (pSer) and phosphorylated threonine (pThr) free amino acids, or short peptides containing pSer or pThr, inhibited α-casein degradation by ClpXP in a dose-dependent manner. In contrast, phosphorylated pArg, and phosphorylated tyrosine (pTyr), as well as free unmodified Ser or Thr, did not impact ClpXP protease activity. Next, we applied a thermal shift assay to measure the binding capabilities of pSer and pThr to ClpX. We discovered that these phosphorylated amino acids, whether free or incorporated into short peptides, successfully associated with ClpX. In contrast, their dephosphorylated counterparts did not exhibit the same binding activity. Notably, pSer did not affect enzyme activity of the related LonP1 mitochondrial matrix serine protease, thereby demonstrating the specificity of pSer for ClpXP. Extending our studies to intact cells, we analyzed a proteomic dataset of post-translational modifications in Jurkat cells with and without treatment with bortezomib (1 µM), a proteasome inhibitor that inhibits ClpXP at this concentration. Notably, a global enrichment of phosphorylation was observed for mitochondrial proteins upon bortezomib treatment. We overlaid observed phosphorylated proteins with the ClpXP interactome as determined by BioID, and identified 9 phosphorylated mitochondrial proteins that also interact with ClpXP. Of these proteins, respiratory chain II complex subunit, SDHA, was the top hit, where serine phosphorylation was increased over 4 folds. Next we determined how depletion of ClpXP affected levels of phosphorylated substrate in intact cells. Knockdown of ClpX or ClpP in OCI-AML2 cells increased levels of serine phosphorylated SDHA (pSer-SDHA). We then added recombinant ClpXP protein to mitochondrial lysates and observed selective degradation of pSer-SDHA. To determine if pSer-SDHA are damaged proteins, we partitioned mitochondria into soluble and insoluble fractions using digitonin. pSer-SDHA was enriched in the detergent-insoluble fraction. In addition, we induced mitochondrial proteolytic stress by treating OCI-AML2 cells with antimycin to increase mitochondrial ROS or through heat shock by culturing cells at 42 oC. Both antimycin and heat shock increased pSer-SDHA in the detergent-insoluble fraction, further supporting pSer marking damaged proteins. Finally, we developed small molecule pSer mimics. We showed that these compounds bound ClpX in thermal shift assays. In addition, these compounds inhibited ClpXP protease activity in cell-free enzymatic assays. In summary, ClpXP recognizes serine phosphorylation as a degradation marker for damaged mitochondrial proteins. Small molecules that inhibit ClpX could be leads for novel anti-cancer agents.

Shift of the insoluble content of the proteome in the aging mouse brain.

During aging, the cellular response to unfolded proteins is believed to decline, resulting in diminished proteostasis. In model organisms, such as Caenorhabditis elegans, proteostatic decline with age has been linked to proteome solubility shifts and the onset of protein aggregation. However, this correlation has not been extensively characterized in aging mammals. To uncover age-dependent changes in the insoluble portion of a mammalian proteome, we analyzed the detergent-insoluble fraction of mouse brain tissue by mass spectrometry. We identified a group of 171 proteins, including the small heat shock protein α-crystallin, that become enriched in the detergent-insoluble fraction obtained from old mice. To enhance our ability to detect features associated with proteins in that fraction, we complemented our data with a meta-analysis of studies reporting the detergent-insoluble proteins in various mouse models of aging and neurodegeneration. Strikingly, insoluble proteins from young and old mice are distinct in several features in our study and across the collected literature data. In younger mice, proteins are more likely to be disordered, part of membraneless organelles, and involved in RNA binding. These traits become less prominent with age, as an increased number of structured proteins enter the pellet fraction. This analysis suggests that age-related changes to proteome organization lead a group of proteins with specific features to become detergent-insoluble. Importantly, these features are not consistent with those associated with proteins driving membraneless organelle formation. We see no evidence in our system of a general increase of condensate proteins in the detergent-insoluble fraction with age.

Degradability of Vicia ervilia Grain Using In Situ and CNCPS Methods, and Model-Based Analysis of Its Ruminal Degradation

Using nylon bag techniques, Cornell net carbohydrates and protein systems (CNCPS), and scanning electron microscopy, the authors examined the digestibility and structure of Vicia ervilia (ervil, bitter vetch) after steam flaking, roasting, and microwave processing. During the in situ technique, the samples were incubated at 0, 2, 4, 6, 8, 12, 16, 24, 36, and 48 h. For the description of the ruminal DM (dry matter) and CP (crude protein) degradation kinetics of treated and untreated Vicia ervilia, different models were selected as the best fit for the dry matter (DM) and crude protein (CP) degradation parameters of steam flaked samples. The results showed that both the steam flaking and microwave treatment samples contained high levels of non-protein nitrogen and buffer soluble protein, respectively. In comparison with steam flaking and microwave treatment, roasting decreased and increased the buffer soluble protein and neutral detergent insoluble protein, respectively. The control treatments showed the highest levels of neutral detergent soluble protein and the lowest levels of acid detergent soluble protein. Moreover, steam flaking and roasting decreased and increased the amount of acid detergent insoluble protein, respectively. When using dry heat (microwave and roasting), the acid detergent soluble and insoluble protein fractions were increased. As a result of this experiment, the nitrogen fractions were altered using heat processing. Hence, protein fermented feed and ruminal fermentation conditions can be expressed using these results.

Disruption of Proteostasis by Natural Products and Synthetic Compounds That Induce Pervasive Unfolding of Proteins: Therapeutic Implications.

Exposure of many cancer cells, including multiple myeloma cells, to cytotoxic concentrations of natural products celastrol and withaferin A or synthetic compounds of the IHSF series resulted in denaturation of a luciferase reporter protein. Proteomic analysis of detergent-insoluble extract fractions from HeLa-derived cells revealed that withaferin A, IHSF058 and IHSF115 caused denaturation of 915, 722 and 991 of 5132 detected cellular proteins, respectively, of which 440 were targeted by all three compounds. Western blots showed that important fractions of these proteins, in some cases approaching half of total protein amounts, unfolded. Relatively indiscriminate covalent modification of target proteins was observed; 1178 different proteins were modified by IHSF058. Further illustrating the depth of the induced proteostasis crisis, only 13% of these proteins detectably aggregated, and 79% of the proteins that aggregated were not targets of covalent modification. Numerous proteostasis network components were modified and/or found in aggregates. Proteostasis disruption caused by the study compounds may be more profound than that mediated by proteasome inhibitors. The compounds act by a different mechanism that may be less susceptible to resistance development. Multiple myeloma cells were particularly sensitive to the compounds. Development of an additional proteostasis-disrupting therapy of multiple myeloma is suggested.

Alzheimer's disease like neuropathology in Down syndrome cortical organoids.

Introduction: Down syndrome (DS) is a genetic disorder with an extra copy of chromosome 21 and DS remains one of the most common causes of intellectual disabilities in humans. All DS patients have Alzheimer's disease (AD)-like neuropathological changes including accumulation of plaques and tangles by their 40s, much earlier than the onset of such neuropathological changes in AD patients. Due to the lack of human samples and appropriate techniques, our understanding of DS neuropathology during brain development or before the clinical onset of the disease remains largely unexplored at the cellular and molecular levels. Methods: We used induced pluripotent stem cell (iPSC) and iPSC-derived 3D cortical organoids to model Alzheimer's disease in Down syndrome and explore the earliest cellular and molecular changes during DS fetal brain development. Results: We report that DS iPSCs have a decreased growth rate than control iPSCs due to a decreased cell proliferation. DS iPSC-derived cortical organoids have a much higher immunoreactivity of amyloid beta (Aß) antibodies and a significantly higher amount of amyloid plaques than control organoids. Although Elisa results did not detect a difference of Aß40 and Aß42 level between the two groups, the ratio of Aß42/Aß40 in the detergent-insoluble fraction of DS organoids was significantly higher than control organoids. Furthermore, an increased Tau phosphorylation (pTau S396) in DS organoids was confirmed by immunostaining and Western blot. Elisa data demonstrated that the ratio of insoluble Tau/total Tau in DS organoids was significantly higher than control organoids. Conclusion: DS iPSC-derived cortical organoids mimic AD-like pathophysiologyical phenotype in vitro, including abnormal Aß and insoluble Tau accumulation. The molecular neuropathologic signature of AD is present in DS much earlier than predicted, even in early fetal brain development, illustrating the notion that brain organoids maybe a good model to study early neurodegenerative conditions.

Glutamine induces remodeling of tight junctions in Caco-2 colorectal cancer cell

Malignant cells often exhibit significant metabolic alterations, including the utilization of different nutrients to meet energetic and biosynthetic demands. Recent studies have shown that glutamine can support primary colorectal tumor growth and also serve as an alternate energy source during distant metastasis under glucose-limited conditions. However, the overall effects of glutamine on cancer cell physiology are not completely understood. In this study, we investigated how glutamine impacts epithelial integrity in colorectal cancer cells under glucose deprivation. Human colorectal cancer (Caco-2) cells were grown to confluency in transwells and cultured in glucose/pyruvate-free DMEM with various glutamine concentrations (0–50 mM). Cell viability was assessed, and monolayer integrity was examined in terms of transepithelial resistance (TER) and paracellular permeability. Tight junction (TJ) component proteins were examined by immunofluorescence staining and Western blotting. A dose-dependent decrease in TER was observed in Caco-2 cells, but paracellular permeability was not affected after 24 h incubation with glutamine. At the same time, the TJ proteins, zonula occludens (ZO)-1 and Claudin-1, showed lateral undulations and punctate staining patterns accompanied by enlargement of cellular and nuclear sizes. Furthermore, decreased protein levels of ZO-1, but not claudin-1, were found in detergent-insoluble cellular fractions. Notably, the decreased TER and alterations in TJ structure were not associated with cell viability changes. Moreover, the addition of glutamate, which is produced by the first step of glutamine catabolism, had no impact on TER. Our results suggested that the enteral glutamine may play an important role in the regulation of TJ dynamics in colorectal cancer cells.

Small Heat Shock Proteins Collaborate with FAIM to Prevent Accumulation of Misfolded Protein Aggregates.

Cells and tissues are continuously subject to environmental insults, such as heat shock and oxidative stress, which cause the accumulation of cytotoxic, aggregated proteins. We previously found that Fas Apoptosis Inhibitory Molecule (FAIM) protects cells from stress-induced cell death by preventing abnormal generation of protein aggregates similar to the effect of small heat shock proteins (HSPs). Protein aggregates are often associated with neurodegenerative diseases, including Alzheimer’s disease (AD). In this study, we sought to determine how FAIM protein dynamics change during cellular stress and how FAIM prevents the formation of amyloid-β aggregates/fibrils, one of the pathological hallmarks of AD. Here, we found that the majority of FAIM protein shifts to the detergent-insoluble fraction in response to cellular stress. A similar shift to the insoluble fraction was also observed in small heat shock protein (sHSP) family molecules, such as HSP27, after stress. We further demonstrate that FAIM is recruited to sHSP-containing complexes after cellular stress induction. These data suggest that FAIM might prevent protein aggregation in concert with sHSPs. In fact, we observed the additional effect of FAIM and HSP27 on the prevention of protein aggregates using an in vitro amyloid-β aggregation model system. Our work provides new insights into the interrelationships among FAIM, sHSPs, and amyloid-β aggregation.

Multi-platform quantitation of alpha-synuclein human brain proteoforms suggests disease-specific biochemical profiles of synucleinopathies

Based on immunostainings and biochemical analyses, certain post-translationally modified alpha-synuclein (aSyn) variants, including C-terminally truncated (CTT) and Serine-129 phosphorylated (pSer129) aSyn, are proposed to be involved in the pathogenesis of synucleinopathies such as Parkinson’s disease with (PDD) and without dementia (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). However, quantitative information about aSyn proteoforms in the human brain in physiological and different pathological conditions is still limited. To address this, we generated sequential biochemical extracts of the substantia nigra, putamen and hippocampus from 28 donors diagnosed and neuropathologically-confirmed with different synucleinopathies (PD/PDD/DLB/MSA), as well as Alzheimer’s disease, progressive supranuclear palsy, and aged normal subjects. The tissue extracts were used to build a reverse phase array including 65 aSyn antibodies for detection. In this multiplex approach, we observed increased immunoreactivity in donors with synucleinopathies compared to controls in detergent-insoluble fractions, mainly for antibodies against CT aSyn and pSer129 aSyn. In addition, despite of the restricted sample size, clustering analysis suggested disease-specific immunoreactivity signatures in patient groups with different synucleinopathies. We aimed to validate and quantify these findings using newly developed immunoassays towards total, 119 and 122 CTT, and pSer129 aSyn. In line with previous studies, we found that synucleinopathies shared an enrichment of post-translationally modified aSyn in detergent-insoluble fractions compared to the other analyzed groups. Our measurements allowed for a quantitative separation of PDD/DLB patients from other synucleinopathies based on higher detergent-insoluble pSer129 aSyn concentrations in the hippocampus. In addition, we found that MSA stood out due to enrichment of CTT and pSer129 aSyn also in the detergent-soluble fraction of the SN and putamen. Together, our results achieved by multiplexed and quantitative immunoassay-based approaches in human brain extracts of a limited sample set point to disease-specific biochemical aSyn proteoform profiles in distinct neurodegenerative disorders.

DOCK4 Regulation of Rho GTPases Mediates Pulmonary Vascular Barrier Function.

The vascular endothelium maintains tissue-fluid homeostasis by controlling the passage of large molecules and fluid between the blood and interstitial space. The interaction of catenins and the actin cytoskeleton with VE-cadherin (vascular endothelial cadherin) is the primary mechanism for stabilizing AJs (adherens junctions), thereby preventing lung vascular barrier disruption. Members of the Rho (Ras homology) family of GTPases and conventional GEFs (guanine exchange factors) of these GTPases have been demonstrated to play important roles in regulating endothelial permeability. Here, we evaluated the role of DOCK4 (dedicator of cytokinesis 4)-an unconventional Rho family GTPase GEF in vascular function. We generated mice deficient in DOCK4' used DOCK4 silencing and reconstitution approaches in human pulmonary artery endothelial cells' used assays to evaluate protein localization, endothelial cell permeability, and small GTPase activation. Our data show that DOCK4-deficient mice are viable. However, these mice have hemorrhage selectively in the lung, incomplete smooth muscle cell coverage in pulmonary vessels, increased basal microvascular permeability, and impaired response to S1P (sphingosine-1-phosphate)-induced reversal of thrombin-induced permeability. Consistent with this, DOCK4 rapidly translocates to the cell periphery and associates with the detergent-insoluble fraction following S1P treatment, and its absence prevents S1P-induced Rac-1 activation and enhancement of barrier function. Moreover, DOCK4-silenced pulmonary artery endothelial cells exhibit enhanced basal permeability in vitro that is associated with enhanced Rho GTPase activation. Our findings indicate that DOCK4 maintains AJs necessary for lung vascular barrier function by establishing the normal balance between RhoA (Ras homolog family member A) and Rac-1-mediated actin cytoskeleton remodeling, a previously unappreciated function for the atypical GEF family of molecules. Our studies also identify S1P as a potential upstream regulator of DOCK4 activity.

A Matrigel-based 3D construct of SH-SY5Y cells models the α-synuclein pathologies of Parkinson's disease.

ABSTRACTParkinson's disease (PD) is associated with α-synuclein-based Lewy body pathology, which has been difficult to observe in conventional two-dimensional (2D) cell culture and even in animal models. We herein aimed to develop a three-dimensional (3D) cellular model of PD to recapitulate the α-synuclein pathologies. All-trans-retinoic acid-differentiated human SH-SY5Y cells and Matrigel were optimized for 3D construction. The 3D cultured cells displayed higher tyrosine hydroxylase expression than 2D cells and improved dopaminergic-like phenotypes, as suggested by RNA-sequencing analyses. Multiple forms of α-synuclein, including monomer, and low- and high-molecular mass oligomers, were differentially present in the 2D and 3D cells, but mostly remained unchanged upon N-methyl-4-phenyl pyridine or rotenone treatment. Phosphorylated α-synuclein was accumulated, and detergent-insoluble α-synuclein fraction was observed, in the neurotoxin-treated 3D cells. Importantly, Lewy body-like inclusions were captured in the 3D system, including proteinase K-resistant α-synuclein aggregates, ubiquitin aggregation, and β-amyloid and β-sheet protein deposition. The study provides a unique and convenient 3D model of PD that recapitulates critical α-synuclein pathologies and should be useful in multiple PD-associated applications.

Apolipoprotein E Proteinopathy Is a Major Dementia-Associated Pathologic Biomarker in Individuals with or without the APOE Epsilon 4 Allele

The amygdala is vulnerable to multiple or "mixed" mis-aggregated proteins associated with neurodegenerative conditions that can manifest clinically with amnestic dementia; the amygdala region is often affected even at earliest disease stages. With the original intent of identifying novel dementia-associated proteins, the detergent-insoluble proteome was characterized from the amygdalae of 40 participants from the University of Kentucky Alzheimer's Disease Center autopsy cohort. These individuals encompassed a spectrum of clinical conditions (cognitively normal to severe amnestic dementia). Polypeptides from the detergent-insoluble fraction were interrogated using liquid chromatography-electrospray ionization-tandem mass spectrometry. As anticipated, portions of peptides previously associated with neurologic diseases were enriched from subjects with dementia. Among all detected peptides, Apolipoprotein E (ApoE) stood out: even more than the expected Tau, APP/Aβ, and α-Synuclein peptides, ApoE peptides were strongly enriched in dementia cases, including from individuals lacking the APOE ε4 genotype. The amount of ApoE protein detected in detergent-insoluble fractions was robustly associated with levels of complement proteins C3 and C4. Immunohistochemical staining of APOE ε3/ε3 subjects' amygdalae confirmed ApoE co-localization with C4 in amyloid plaques. Thus, analyses of human amygdala proteomics indicate that rather than being only an "upstream" genetic risk factor, ApoE is an aberrantly aggregated protein in its own right, and show that the ApoE protein may play active disease-driving mechanistic roles in persons lacking the APOE ε4 allele.

Alpha adaptins show isoform-specific association with neurofibrillary tangles in Alzheimer's disease.

The heterotetrameric assembly protein complex 2 (AP-2) is a central hub for clathrin-dependent endocytosis. The AP-2 α-adaptin subunit has two major isoforms, encoded by two separate genes: AP2A1 and AP2A2. Endocytosis has been implicated in the pathogenesis of neurodegenerative disease, and recent studies linked α-adaptins (gene variants, splicing defects and altered expression) with late-onset Alzheimer's disease (LOAD) risk. Here, we used multiple antibodies to investigate α-adaptin isoforms and their localization in human brains. The specificities of 10 different α-adaptin antibodies were evaluated using immunoblots after human AP2A1 and AP2A2 plasmid transfection in cultured cells. Additional immunoblot analyses were then performed on protein homogenates from control and LOAD subjects. Formalin-fixed, paraffin-embedded brain sections from control and LOAD subjects were immunohistochemically stained, and immunofluorescence experiments were performed for quantitation of colocalisation with digital image analysis. Eight of the 10 evaluated antibodies recognised transfected α-adaptin proteins on immunoblots. The α-adaptin subspecies were relatively uniformly expressed in five different human brain regions. The α-adaptins were present in the detergent-insoluble fraction from cognitively impaired, but less so in control, brains. Immunohistochemical analyses showed colocalisation of AP2A1 with tau pathology in LOAD brains. By contrast, AP2A2 colocalised with microglial cells. These observations provide evidence of isoform-specific changes of α-adaptins in the brains of LOAD subjects. Antibodies that were verified to recognise AP2A1, but not AP2A2, labelled neurofibrillary tangles of LOAD patients. The findings extend our understanding of AP-2 proteins in the human brain in healthy and diseased states.

Abstract 14114: Role of Toxic Protein Sequestration by Aggregates in Cardiac Myocytes in Ischemic Cardiomyopathy

Introduction: Proteotoxic aggregation causes cardiomyopathy (CMP) associated with rare genetic mutations. The role of protein aggregates in ischemic cardiomyopathy is unclear. Hypothesis: Cardiac myocyte protein aggregation is deleterious in cardiomyopathy. Methods: Human (ischemic) and murine (ischemic and proteotoxic) CMP models were profiled by immunohistochemistry (IHC) and proteomic characterization of the detergent-insoluble fraction. Aggregates were evaluated by immunocytochemistry in p62 deficient neonatal mouse cardiac myocytes (NMCMs) with adenoviral transduction of mutant alpha-B crystallin (CRYABR120G). Mice with cardiac myocyte selective p62 deficiency (p62ckO) were generated and characterized (echocardiography, myocardial structure, autophagic flux, mitophagy, and biochemical analyses). p62cKO and floxed controls were subjected to closed-chest ischemia (90 minutes) or sham followed by reperfusion (IR) and evaluated 4 weeks later. Cardiac selective (AAV9-cTnT) reconstitution of p62, its aggregation-deficient K7R mutant or GFP, was performed. Wild-type mice transduced with a fungal disaggregase (AAV9-Hsp104 or control) were modeled for IR as above. Results: Protein aggregation increases in ischemic CMP, but not controls, with increased insoluble CRYAB with phosphorylation at serine 59 (pS59CRYAB). p62-deficient NMCMs are unable to form aggregates following CRYABR120G transduction, indicating the necessity of p62 in protein aggregation. p62cKO mice show preserved baseline cardiac structure/function, without significantly decreased mitophagy or autophagic flux. Compared to controls, p62cKO mice (post-IR) show infarct expansion and impaired LV remodeling at 4 weeks, associated with decreased sequestration of pS59CRYAB in insoluble fraction. Reconstitution with AAV9 p62, but not aggregation-deficient K7R, shows partial functional rescue after IR. Finally, disruption of protein aggregation by AAV9 Hsp104 (but not controls) results in decreased sequestration of pS59CRYAB, and decreased function post-IR. Conclusions: Contrary to the hypothesis, preventing or disrupting protein aggregation, associated with decreased pS59CRYAB sequestration, is maladaptive in ischemic cardiomyopathy.

Citrullination of Amyloid-β Peptides in Alzheimer's Disease.

Protein citrullination (deimination of arginine residue) is a well-known biomarker of inflammation. Elevated protein citrullination has been shown to colocalize with extracellular amyloid plaques in postmortem AD patient brains. Amyloid-β (Aβ) peptides which aggregate and accumulate in the plaques of Alzheimer's disease (AD) have sequential N-terminal truncations and multiple post-translational modifications (PTM) such as isomerization, pyroglutamate formation, phosphorylation, nitration, and dityrosine cross-linking. However, no conclusive biochemical evidence exists whether citrullinated Aβ is present in AD brains. In this study, using high-resolution mass spectrometry, we have identified citrullination of Aβ in sporadic and familial AD brains by characterizing the tandem mass spectra of endogenous N-truncated citrullinated Aβ peptides. Our quantitative estimations demonstrate that ∼ 35% of pyroglutamate3-Aβ pool was citrullinated in plaques in the sporadic AD temporal cortex and ∼ 22% in the detergent-insoluble frontal cortex fractions. Similarly, hypercitrullinated pyroglutamate3-Aβ (∼ 30%) was observed in both the detergent-soluble as well as insoluble Aβ pool in familial AD cases. Our results indicate that a common mechanism for citrullination of Aβ exists in both the sporadic and familial AD. We establish that citrullination of Aβ is a remarkably common PTM, closely associated with pyroglutamate3-Aβ formation and its accumulation in AD. This may have implications for Aβ toxicity, autoantigenicity of Aβ, and may be relevant for the design of diagnostic assays and therapeutic targeting.

Abstract P170: Targeting Angiotensin Ii-induced Endoplasmic Reticulum Stress In Vsmcs To Reduce Pathological Vascular Amyloid Burden And Remodeling

Hypertension is a complex disorder and risk factor for cardiovascular disease, ultimately contributing to premature death. The societal burden of hypertension is enormous, therefore better understanding of molecular mechanisms underlying hypertension and associated pathological vascular remodeling is needed for mechanism-targeted therapy development. Endoplasmic reticulum (ER) stress occurs in cells under increased protein synthesis, Ca 2+ flux, or ROS generating environments- all of which are induced by Angiotensin II signaling in vascular cells. ER stress leads to the accumulation of misfolded proteins, and mediates cell death, fibrotic, and hypertrophic responses. The aim of this study was to target ER stress via chemical chaperone 3-hydroxy-2-naphthoic acid (3HNA) or genetic overexpression of chaperone glucose-regulated protein 78 (GRP78) to reduce protein aggregation and pathological response in VSMCs. Rat primary VSMCs were treated with 500 μM 3HNA or 30 MOI lacZ-GRP78 prior to stimulation with Ang II (100 nM). Protein synthesis assessed via puromycin incorporation revealed ER stress inhibition blocked Ang II associated protein synthesis in VSMCs, fold change relative to control of 1.7 (saline + Ang II) to 1.2 (3-HNA + Ang II) treated cells (P<0.05). GRP78 overexpression attenuated Ang II induction pre-amyloid oligomers (P<0.01) from 2.1 aggregates/cell to 0.9 aggregates/cell. Proteomic assessment of aggregates revealed Ang II induced crystallin AB (1.3 fold rel to control), HSP70 (1.716 fold rel to control) and annexin a2 (1.3 fold increase) enrichment in detergent insoluble fractions, which was attenuated by GRP78 overexpression (P=0.03, 0.03, and 0.024, respectively). In a 2 week Ang II infusion model, 3HNA was injected in C57/Bl6 mice. Ang II-induced medial thickness and cardiac vessel fibrosis was significantly reduced by 3-HNA (P<0.05). SM22α Cre GRP78 mice were generated and found protective against Ang II induced cardiac hypertrophy via HW/BW ratio (Cre- vs Cre+ Ang II P<0.05), and a trend in reduction of aortic medial thickness (P=0.098). Overall, GRP78 chaperone expression reduced protein aggregation and subsequent vascular remodeling induced by hypertensive stimulus and may elude novel therapeutic targets.

Deubiquitinase Inhibitor WP1130 Blocks FLT3-ITD to Induce Apoptosis in Leukemic Cells

FMS-like tyrosine kinase 3 with internal tandem duplication (FLT3-ITD) is the most frequent kinase mutation seen in acute myeloid leukemia (AML). The ITD mutation promotes ligand-independent constitutive activation of FLT3 kinase, resulting in altered downstream signal pathway activation to enhance cell proliferation and prevent cell death. AML with FLT3-ITD is associated with poor prognosis, which neither high-dose chemotherapy nor allogeneic hematopoietic stem cell transplantation can overcome. Several tyrosine kinase inhibitors targeting FLT3 have been developed and tested in clinical trials, but their efficacies are limited so far partly due to resistance to kinase inhibition. Therefore, development of novel treatment strategies with distinct mechanisms to block FLT3 is warranted.Deubiquitinating enzymes (DUBs) are proteases that remove ubiquitin from target proteins, resulting in changes in protein stability and signaling. WP1130 is an inhibitor for DUBs, which has been reported to inhibit BCR/ABL not through blocking the kinase activity but through promoting ubiquitination and translocation of BCR/ABL protein into perinuclear aggresomes. WP1130 also downregulates JAK2 tyrosine kinase in the same manner. However, the effect of WP1130 against FLT3-ITD has not been reported.Here we demonstrate that WP1130 exerts anti-leukemic activity against leukemic cell lines and primary AML cells harboring FLT3-ITD. WP1130 efficiently downregulated activated form of FLT3-ITD through translocation of this mutant into aggresomes and activated the mitochondria-mediated intrinsic apoptotic pathway at least partly through accumulation of intracellular reactive oxygen species (ROS).First, we examined the anti-proliferative effect of WP1130 and found that proliferation of the FLT3-ITD-harboring AML cell line MV4-11 was suppressed in a dose dependent manner with IC50 of 1.3 μM. It was notable that MV4-11 was more susceptible to WP1130 compared with K562 (IC50 = 3.3 μM) and HEL (IC50 = 3.4 μM), leukemic cell lines harboring BCR/ABL and JAK2-V617F aberrant kinases, respectively.Next, we assessed the effect of WP1130 against FLT3-ITD in MV4-11 cells. WP1130 rapidly downregulated FLT3-ITD to suppress downstream signaling pathways. The aggresomal translocation of FLT3-ITD was confirmed by confocal microscopy and western blot analysis of detergent-insoluble fraction of cellular proteins. Importantly, the downregulation was prominently observed for the activated form of FLT3-ITD autophosphorylated on Y591. Consistent with this, inhibition of FLT3 autophosphorylation by AC220 (quizartinib) resulted in attenuation of WP1130-induced FLT3-ITD downregulation.Importantly, WP1130 induced apoptosis in MV4-11 cells much more efficiently than in other leukemic cell lines that we examined, including K562 and HEL. WP1130 activated the mitochondria-mediated intrinsic apoptotic pathway as demonstrated by Bax conformation change and loss of mitochondrial membrane potential (MMP). Forced overexpression of Bcl-xL, a member of pro-survival Bcl-2 family proteins that preserve MMP, blocked WP1130-induced apoptosis. Moreover, the apoptosis was synergistically enhanced by inhibition of Bcl-xL using ABT-737 or navitoclax, further supporting the involvement of Bcl-xL in WP1130-induced apoptosis. Interestingly, the apoptosis induction was also enhanced by 17-AAG, an HSP90 inhibitor that we previously demonstrated to disrupt the chaperone association of HSP90 with FLT3-ITD resulting in enhanced ubiquitination of FLT3-ITD. FLT3-ITD down regulation and apoptosis induction by WP1130 were also confirmed using other cell lines expressing FLT3-ITD and primary cells derived from FLT3-ITD positive AML patients.Finally, since FLT3-ITD has been reported to increase intracellular ROS, we examined if ROS was involved in the anti-leukemic effect of WP1130. Treatment with WP1130 increased ROS levels in MV4-11 cells. Antioxidants N-acetyl-cysteine (NAC) and tert-butylhydroquinone (tBHQ) both attenuated WP1130-induced apoptosis, suggesting that intracellular ROS accumulation may play a role in induction of apoptosis.Further studies are in progress to elucidate the exact molecular mechanisms involved in WP1130-mediated effects and to develop novel strategies to enhance its anti-leukemic effects in synergistic manners. DisclosuresNo relevant conflicts of interest to declare.

Immunoprecipitation Strategies to Isolate RIPK1/RIPK3 Complexes in Mouse Macrophages.

A large protein complex, containing RIPK1, RIPK3, and caspase-8 and known as Complex II, has emerged as one of the key mediators of cell death downstream from a range of innate immune triggers. This regulatory mechanism plays a prominent role in macrophages, where Complex II has been linked to apoptosis, pyroptosis, and necroptosis as well as the enhancement of inflammatory gene expression. Although core components of this complex are fairly well understood, more subtle proteomic changes that determine the direction of a response once the complex is assembled remain much less clear. In addition, Complex II components undergo a wealth of post-translational changes that modify the functions of the complex components. This necessitates development of robust and efficient methods of isolating Complex II for further interrogation of its composition and the post-translational modifications of its components. This article describes several methods that we have developed for Complex II isolation, which can be used to obtain complementary information about this signaling mechanism. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Isolation of Complex II in necroptotic and pyroptotic macrophages using FADD immunoprecipitation Basic Protocol 2: Isolation of the complexes formed by the conditionally expressed 3XFLAG-RIPK1 protein Alternate Protocol: Alternative methods of immunoprecipitation of RIPK1 and other Complex-II-related factors Support Protocol: Generation of stable macrophage cell lines using lentiviral expression Basic Protocol 3: Use of proximity labeling to identify necrosome components in the detergent-insoluble fraction of the cell lysates.