Cell Proteostasis Research Articles

The HSP90 Inhibitor Pimitespib Targets Regulatory T Cells in the Tumor Microenvironment.

Regulatory T (Treg) cells play key roles in cancer immunity by suppressing a range of antitumor immune responses and contributing to resistance to programmed death (PD)-1 blockade therapy. Given their critical roles in self-tolerance, local control of immunosuppression by Treg cells, such as in the tumor microenvironment (TME), has been intensively studied. Inhibition of heat shock protein 90 (HSP90), a chaperone with vital roles in regulating proteostasis in cancer cells, impedes cancer progression by interrupting oncogenic signaling pathways and potentially modulating antitumor immunity, but we have very little mechanistic insight into these immune modulatory effects. Here, we show that the number of Treg cells are selectively reduced by the HSP90 inhibitor pimitespib in animal models and patients with gastric cancer in a clinical trial (EPOC1704). Pimitespib reduced the highly immunosuppressive human FOXP3high effector Treg cells by inhibiting their proliferation and decreasing their expression of effector molecules, which improved the priming and activation of antigen-specific CD8+ T cells. Mechanistic studies revealed that pimitespib selectively degraded STAT5, a key transducer of the IL-2 signaling pathway, which is essential for Treg cell development and maintenance, and consequently compromised FOXP3 expression, leading to selective impairment of immunosuppression in the TME by Treg cells. Thus, pimitespib treatment combined with PD-1 blockade exhibited a far stronger antitumor effect than either treatment alone in animal models. Through these data, we propose that HSP90 inhibition is a promising therapeutic option for Treg cell-targeted cancer immunotherapy.

Carmaphycin B-Based Proteasome Inhibitors to Treat Human African Trypanosomiasis: Structure-Activity Relationship and In Vivo Efficacy.

The proteasome is essential for eukaryotic cell proteostasis, and inhibitors of the 20S proteasome are progressing preclinically and clinically as antiparasitics. We screenedTrypanosoma brucei, the causative agent of human and animal African trypanosomiasis, in vitro with a set of 27 carmaphycin B analogs, irreversible epoxyketone inhibitors that were originally developed to inhibit thePlasmodium falciparum20S (Pf20S). The structure-activity relationship was distinct from that of the human c20S antitarget by the acceptance of d-amino acids at the P3 position of the peptidyl backbone to yield compounds with greatly decreased toxicity to human cells. For the three most selective compounds, binding to the Tb20S β5 catalytic subunit was confirmed by competition with a fluorescent activity-based probe. For one compound, J-80, with its P3 d-configuration, the differential binding to the parasite's β5 subunit was supported by both covalent and noncovalent docking analysis. Further, J-80 was equipotent against both Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense in vitro. In a mouse model of Stage 1 T. brucei infection, a single intraperitoneal (i.p.) dose of 40 mg/kg J-80 halted the growth of the parasite, and when given at 50 mg/kg i.p. twice daily for 5 days, parasitemia was decreased to below the detectable limit, with parasite recrudescence 48 h after the last dose. The in vivo proof of principle demonstrated by a potent, selective, and irreversible inhibitor of Tb20S reveals an alternative path to the development of kinetoplastid proteasome inhibitors that differs from the current focus on allosteric reversible inhibitors.

The Proteostasis Network is a Therapeutic Target in Acute Myeloid Leukemia.

Oncogenic growth places great strain and dependence on the proteostasis network. This has made proteostasis pathways attractive therapeutic targets in cancer, but efforts to drug these pathways have yielded disappointing clinical outcomes. One exception is proteasome inhibitors, which are approved for frontline treatment of multiple myeloma. However, proteasome inhibitors are largely ineffective for treatment of other cancers, including acute myeloid leukemia (AML), although reasons for these differences are unknown. Here, we determined that proteasome inhibitors are ineffective in AML due to inability to disrupt proteostasis. In response to proteasome inhibition, AML cells activated HSF1 and autophagy, two key stem cell proteostasis pathways, to prevent unfolded protein accumulation. Inactivation of HSF1 sensitized human AML cells to proteasome inhibition, marked by unfolded protein accumulation, activation of the PERK-mediated integrated stress response, severe reductions in protein synthesis, proliferation and cell survival, and significant slowing of disease progression and extension of survival in vivo . Similarly, combined autophagy and proteasome inhibition suppressed proliferation, synergistically killed AML cells, and significantly reduced AML burden and extended survival in vivo . Furthermore, autophagy and proteasome inhibition preferentially suppressed protein synthesis and induced apoptosis in primary patient AML cells, including AML stem/progenitor cells, without severely affecting normal hematopoietic stem/progenitor cells. Combined autophagy and proteasome inhibition also activated the integrated stress response, but surprisingly this occurred in a PKR-dependent manner. These studies unravel how proteostasis pathways are co-opted to promote AML growth, progression and drug resistance, and reveal that disabling the proteostasis network is a promising strategy to therapeutically target AML.

Sod1-deficient cells are impaired in formation of the modified nucleosides mcm5s2U and yW in tRNA.

Uridine residues present at the wobble position of eukaryotic cytosolic tRNAs often carry a 5-carbamoylmethyl (ncm5), 5-methoxycarbonylmethyl (mcm5), or 5-methoxycarbonylhydroxymethyl (mchm5) side-chain. The presence of these side-chains allows proper pairing with cognate codons and they are particularly important in tRNA species where the U34 residue is also modified with a 2-thio (s2) group. The first step in synthesis of the ncm5, mcm5, and mchm5 side-chains is dependent on the six-subunit Elongator complex, whereas the thiolation of the 2-position is catalyzed by the Ncs6/Ncs2 complex. In both yeast and metazoans, allelic variants of Elongator subunit genes show genetic interactions with mutant alleles of SOD1, which encodes the cytosolic Cu,Zn-superoxide dismutase. However, the cause of these genetic interactions remains unclear. Here, we show that yeast sod1 null mutants are impaired in the formation of 2-thio-modified U34 residues. In addition, the lack of Sod1 induces a defect in the biosynthesis of wybutosine, which is a modified nucleoside found at position 37 of tRNAPhe Our results suggest that these tRNA modification defects are caused by superoxide-induced inhibition of the iron-sulfur cluster-containing Ncs6/Ncs2 and Tyw1 enzymes. Since mutations in Elongator subunit genes generate strong negative genetic interactions with mutant ncs6 and ncs2 alleles, our findings at least partially explain why the activity of Elongator can modulate the phenotypic consequences of SOD1/sod1 alleles. Collectively, our results imply that tRNA hypomodification may contribute to impaired proteostasis in Sod1-deficient cells.

Exploring the impact of lipid nanoparticles on protein stability and cellular proteostasis

Lipid nanoparticles (LNPs) have become pivotal in advancing modern medicine, from mRNA-based vaccines to gene editing with CRISPR-Cas9 systems. Though LNPs based therapeutics offer promising drug delivery with satisfactory clinical safety profiles, concerns are raised regarding their potential nanotoxicity. Here, we explore the impacts of LNPs on protein stability in buffer and cellular protein homeostasis (proteostasis) in HepG2 cells. First, we show that LNPs of different polyethylene glycol (PEG) molar ratios to total lipid ratio boost protein aggregation propensity by reducing protein stability in cell lysate and blood plasma. Second, in HepG2 liver cells, these LNPs induce global proteome aggregation, as imaged by a cellular protein aggregation fluorescent dye (AggStain). Such LNPs induced proteome aggregation is accompanied by decrease in cellular micro-environmental polarity as quantified by a solvatochromic protein aggregation sensor (AggRetina). The observed local polarity fluctuations may be caused by the hydrophobic contents of LNPs that promote cellular proteome aggregation. Finally, we exploit RNA sequencing analysis (RNA-Seq) to reveal activation of unfolded protein response (UPR) pathway and other proteostasis genes upon LNPs treatment. Together, these findings highlight that LNPs may induce subtle proteome stress by compromising protein stability and proteostasis even without obvious damage to cell viability.

LSD Modulates Proteins Involved in Cell Proteostasis, Energy Metabolism and Neuroplasticity in Human Cerebral Organoids.

Proteomic analysis of human cerebral organoids may reveal how psychedelics regulate biological processes, shedding light on drug-induced changes in the brain. This study elucidates the proteomic alterations induced by lysergic acid diethylamide (LSD) in human cerebral organoids. By employing high-resolution mass spectrometry-based proteomics, we quantitatively analyzed the differential abundance of proteins in cerebral organoids exposed to LSD. Our findings indicate changes in proteostasis, energy metabolism, and neuroplasticity-related pathways. Specifically, LSD exposure led to alterations in protein synthesis, folding, autophagy, and proteasomal degradation, suggesting a complex interplay in the regulation of neural cell function. Additionally, we observed modulation in glycolysis and oxidative phosphorylation, crucial for cellular energy management and synaptic function. In support of the proteomic data, complementary experiments demonstrated LSD's potential to enhance neurite outgrowth in vitro, confirming its impact on neuroplasticity. Collectively, our results provide a comprehensive insight into the molecular mechanisms through which LSD may affect neuroplasticity and potentially contribute to therapeutic effects for neuropsychiatric disorders.

Peroxiredoxin 4 deficiency induces accelerated ovarian aging through destroyed proteostasis in granulosa cells

Ovarian aging, a complex and challenging concern within the realm of reproductive medicine, is associated with reduced fertility, menopausal symptoms and long-term health risks. Our previous investigation revealed a correlation between Peroxiredoxin 4 (PRDX4) and human ovarian aging. The purpose of this research was to substantiate the protective role of PRDX4 against ovarian aging and elucidate the underlying molecular mechanism in mice. In this study, a Prdx4−/− mouse model was established and it was observed that the deficiency of PRDX4 led to only an accelerated decline of ovarian function in comparison to wild-type (WT) mice. The impaired ovarian function observed in this study can be attributed to an imbalance in protein homeostasis, an exacerbation of endoplasmic reticulum stress (ER stress), and ultimately an increase in apoptosis of granulosa cells. Furthermore, our research reveals a noteworthy decline in the expression of Follicle-stimulating hormone receptor (FSHR) in aging Prdx4−/− mice, especially the functional trimer, due to impaired disulfide bond formation. Contrarily, the overexpression of PRDX4 facilitated the maintenance of protein homeostasis, mitigated ER stress, and consequently elevated E2 levels in a simulated KGN cell aging model. Additionally, the overexpression of PRDX4 restored the expression of the correct spatial conformation of FSHR, the functional trimer. In summary, our research reveals the significant contribution of PRDX4 in delaying ovarian aging, presenting a novel and promising therapeutic target for ovarian aging from the perspective of endoplasmic reticulum protein homeostasis.

Exercise mitigates reductive stress-induced cardiac remodeling in mice

The endoplasmic reticulum (ER) regulates protein folding and maintains proteostasis in cells. We observed that the ER transcriptome is impaired during chronic reductive stress (RS) in cardiomyocytes. Here, we hypothesized that a prolonged moderate treadmill exercise mitigates the RS-induced ER dysfunction and cardiac remodeling in cardiac-specific constitutively active Nrf2 mice (CaNrf2-TG). RNA sequencing showed notable alterations in the ER transcriptome of TG hearts at 4, 12, and 24 weeks (16, 28, and 35 genes, respectively). Notably, the downregulation of ER genes was significant at 12 weeks, and further pronounced at 24 weeks, at which the cardiac pathology is evident. We also observed increased levels of ubiquitinated proteins in CaNrf2-TG hearts across all ages, along with VCP, a marker of ERAD function, at 24 weeks. These findings indicate that constitutive Nrf2 activation and RS impair protein-folding activity and augments ERAD function over time. Exercise intervention for 20 weeks (beginning at 6 weeks of age), reduced cardiomyocyte hypertrophy (from 448 μm2 to 280 μm2) in TG mice, through adaptive remodeling, and preserved the cardiac function. However, while exercise did not influence antioxidants or ER stress protein levels, it significantly improved ERAD function and autophagy flux (LC-I to LC-II) in the TG-EXE hearts. Collectively, our findings underscore the prophylactic potential of exercise in mitigating RS-associated pathology, highlighting its essential role in maintaining cellular proteostasis through ER-independent mechanisms.

The gluconeogenesis enzyme PCK2 has a non-enzymatic role in proteostasis in endothelial cells

Endothelial cells (ECs) are highly glycolytic, but whether they generate glycolytic intermediates via gluconeogenesis (GNG) in glucose-deprived conditions remains unknown. Here, we report that glucose-deprived ECs upregulate the GNG enzyme PCK2 and rely on a PCK2-dependent truncated GNG, whereby lactate and glutamine are used for the synthesis of lower glycolytic intermediates that enter the serine and glycerophospholipid biosynthesis pathways, which can play key roles in redox homeostasis and phospholipid synthesis, respectively. Unexpectedly, however, even in normal glucose conditions, and independent of its enzymatic activity, PCK2 silencing perturbs proteostasis, beyond its traditional GNG role. Indeed, PCK2-silenced ECs have an impaired unfolded protein response, leading to accumulation of misfolded proteins, which due to defective proteasomes and impaired autophagy, results in the accumulation of protein aggregates in lysosomes and EC demise. Ultimately, loss of PCK2 in ECs impaired vessel sprouting. This study identifies a role for PCK2 in proteostasis beyond GNG.

The role of Nrf2 signaling pathways in nerve damage repair.

The protein, Nuclear factor-E2-related factor 2 (Nrf2), is a transitory protein that acts as a transcription factor and is involved in the regulation of many cytoprotective genes linked to xenobiotic metabolism and antioxidant responses. Based on the existing clinical and experimental data, it can be inferred that neurodegenerative diseases are characterized by an excessive presence of markers of oxidative stress (OS) and a reduced presence of antioxidant defense systems in both the brain and peripheral tissues. The presence of imbalances in the homeostasis between oxidants and antioxidants has been recognized as a substantial factor in the pathogenesis of neurodegenerative disorders. The dysregulations include several cellular processes such as mitochondrial failure, protein misfolding, and neuroinflammation. These dysregulations all contribute to the disruption of proteostasis in neuronal cells, leading to their eventual mortality. A noteworthy component of Nrf2, as shown by recent research undertaken over the last decade, is to its role in the development of resistance to OS. Nrf2 plays a pivotal role in regulating systems that defend against OS. Extant research offers substantiation for the protective and defensive roles of Nrf2 in the context of neurodegenerative diseases. The purpose of this study is to provide a comprehensive analysis of the influence of Nrf2 on OS and its function in regulating antioxidant defense systems within the realm of neurodegenerative diseases. Furthermore, we evaluate the most recent academic inquiries and empirical evidence about the beneficial and potential role of certain Nrf2 activator compounds within the realm of therapeutic interventions.

The role of labile iron on brain proteostasis; could it be an early event of neurodegenerative disease?

Iron deposits in the brain are a natural consequence of aging. Iron accumulation, especially in the form of labile iron, can trigger a cascade of adverse effects, eventually leading to neurodegeneration and cognitive decline. Aging also increases the dysfunction of cellular proteostasis. The question of whether iron alters proteostasis is now being pondered. Herein, we investigated the effect of ferric citrate, considered as labile iron, on various aspects of proteostasis of neuronal cell lines, and also established an animal model having a labile iron diet in order to evaluate proteostasis alteration in the brain along with behavioral effects. According to an in vitro study, labile iron was found to activate lysosome formation but inhibits lysosomal clearance function. Furthermore, the presence of labile iron can alter autophagic flux and can also induce the accumulation of protein aggregates. RNA-sequencing analysis further reveals the upregulation of various terms related to proteostasis along with neurodegenerative disease-related terms. According to an in vivo study, a labile iron-rich diet does not induce iron overload conditions and was not detrimental to the behavior of male Wistar rats. However, an iron-rich diet can promote iron accumulation in a region-dependent manner. By staining for autophagic markers and misfolding proteins in the cerebral cortex and hippocampus, an iron-rich diet was actually found to alter autophagy and induce an accumulation of misfolding proteins. These findings emphasize the importance of labile iron on brain cell proteostasis, which could be implicated in developing of neurological diseases.

In the fed state, autophagy plays a crucial role in assisting the insect vector Rhodnius prolixus mobilize TAG reserves under forced flight activity.

Autophagy is a cellular degradation pathway mediated by highly conserved autophagy-related genes (Atgs). In our previous work, we showed that inhibiting autophagy under starvation conditions leads to significant physiological changes in the insect vector of Chagas disease Rhodnius prolixus; these changes include triacylglycerol (TAG) retention in the fat body, reduced survival and impaired locomotion and flight capabilities. Herein, because it is known that autophagy can be modulated in response to various stimuli, we further investigated the role of autophagy in the fed state, following blood feeding. Interestingly, the primary indicator for the presence of autophagosomes, the lipidated form of Atg8 (Atg8-II), displayed 20%-50% higher autophagic activation in the first 2weeks after feeding compared to the third week when digestion was complete. Despite the elevated detection of autophagosomes, RNAi-mediated suppression of RpAtg6 and RpAtg8 did not cause substantial changes in TAG or protein levels in the fat body or the flight muscle during blood digestion. We also found that knockdown of RpAtg6 and RpAtg8 led to modest modulations in the gene expression of essential enzymes involved in lipid metabolism and did not significantly stimulate the expression of the chaperones BiP and PDI, which are the main effectors of the unfolded protein response. These findings indicate that impaired autophagy leads to slight disturbances in lipid metabolism and general cell proteostasis. However, the ability of insects to fly during forced flight until exhaustion was reduced by 60% after knockdown of RpAtg6 and RpAtg8. This change was accompanied by TAG and protein increases as well as decreased ATP levels in the fat body and flight muscle, indicating that autophagy during digestion, i.e., under fed conditions, is necessary to sustain high-performance activity.

Maximum entropy determination of mammalian proteome dynamics

Full understanding of proteostasis and energy utilization in cells will require knowledge of the fraction of cell proteins being degraded with different half-lives and their rates of synthesis. We therefore developed a method to determine such information that combines mathematical analysis of protein degradation kinetics obtained in pulse-chase experiments with Bayesian data fitting using the maximum entropy principle. This approach will enable rapid analyses of whole-cell protein dynamics in different cell types, physiological states, and neurodegenerative disease. Using it, we obtained surprising insights about protein stabilities in cultured cells normally and upon activation of proteolysis by mTOR inhibition and increasing cAMP or cGMP. It revealed that >90% of protein content in dividing mammalian cell lines is long-lived, with half-lives of 24 to 200 h, and therefore comprises much of the proteins in daughter cells. The well-studied short-lived proteins (half-lives < 10 h) together comprise <2% of cell protein mass, but surprisingly account for 10 to 20% of measurable newly synthesized protein mass. Evolution thus appears to have minimized intracellular proteolysis except to rapidly eliminate misfolded and regulatory proteins.

Biomarker-based human and animal sperm phenotyping: the good, the bad and the ugly†.

Conventional, brightfield-microscopic semen analysis provides important baseline information about sperm quality of an individual; however, it falls short of identifying subtle subcellular and molecular defects in cohorts of "bad," defective human and animal spermatozoa with seemingly normal phenotypes. To bridge this gap, it is desirable to increase the precision of andrological evaluation in humans and livestock animals by pursuing advanced biomarker-based imaging methods. This review, spiced up with occasional classic movie references but seriously scholastic at the same time, focuses mainly on the biomarkers of altered male germ cell proteostasis resulting in post-testicular carryovers of proteins associated with ubiquitin-proteasome system. Also addressed are sperm redox homeostasis, epididymal sperm maturation, sperm-seminal plasma interactions, and sperm surface glycosylation. Zinc ion homeostasis-associated biomarkers and sperm-borne components, including the elements of neurodegenerative pathways such as Huntington and Alzheimer disease, are discussed. Such spectrum of biomarkers, imaged by highly specific vital fluorescent molecular probes, lectins, and antibodies, reveals both obvious and subtle defects of sperm chromatin, deoxyribonucleic acid, and accessory structures of the sperm head and tail. Introduction of next-generation image-based flow cytometry into research and clinical andrology will soon enable the incorporation of machine and deep learning algorithms with the end point of developing simple, label-free methods for clinical diagnostics and high-throughput phenotyping of spermatozoa in humans and economically important livestock animals.

Building an Understanding of Proteostasis in Reproductive Cells: The Impact of Reactive Carbonyl Species on Protein Fate.

Significance: Stringent regulation of protein homeostasis pathways, under both physiological and pathological conditions, is necessary for the maintenance of proteome fidelity and optimal cell functioning. However, when challenged by endogenous or exogenous stressors, these proteostasis pathways can become dysregulated with detrimental consequences for protein fate, cell survival, and overall organism health. Most notably, there are numerous somatic pathologies associated with a loss of proteostatic regulation, including neurodegenerative disorders, type 2 diabetes, and some cancers. Recent Advances: Lipid oxidation-derived reactive carbonyl species (RCS), such as 4-hydroxynonenal (4HNE) and malondialdehyde, are relatively underappreciated purveyors of proteostatic dysregulation, which elicit their effects via the nonenzymatic post-translational modification of proteins. Emerging evidence suggests that a subset of germline proteins can serve as substrates for 4HNE modification. Among these, prevalent targets include succinate dehydrogenase, heat shock protein A2 and A-kinase anchor protein 4, all of which are intrinsically associated with fertility. Critical Issues: Despite growing knowledge in this field, the RCS adductomes of spermatozoa and oocytes are yet to be comprehensively investigated. Furthermore, the manner by which RCS-mediated adduction impacts protein fate and drives cellular responses, such as protein aggregation, requires further examination in the germline. Given that RCS-protein adduction has been attributed a role in infertility, there has been sparked research investment into strategies to prevent lipid peroxidation in germ cells. Future Directions: An increased depth of knowledge regarding the mechanisms and substrates of RCS-mediated protein modification in reproductive cells may reveal important targets for the development of novel therapies to improve fertility and pregnancy outcomes for future generations.

Abstract 2104: Targeting stem cell proteostasis pathways sensitizes acute myeloid leukemia to proteasome inhibitors

Abstract Proteasome inhibitors are highly effective for treating multiple myeloma, but exhibit little efficacy in other blood cancers. In this study, we set out to determine why acute myeloid leukemia (AML) is largely refractory to proteasome inhibition and if we could sensitize human AML to this class of drugs. Efficacy of proteasome inhibitors in myeloma partly depends on disruption of protein homeostasis (proteostasis). This is driven by an accumulation of misfolded proteins that activate the unfolded protein response (UPR) and integrated stress response (ISR). Due to their highly secretory nature, myeloma cells exhibit relatively elevated baseline endoplasmic reticulum stress, and are thus sensitive to proteasome inhibition as they readily activate a terminal UPR. However, we found that proteasome inhibition fails to significantly disrupt proteostasis in human AML cells. We recently discovered that hematopoietic stem cells utilize distinct pathways to regulate proteostasis, and we hypothesized that AML cells co-opt stem cell proteostasis pathways to cope with proteasome inhibition and preserve proteostasis. To test this, we first performed RNA-sequencing which revealed that AML cells respond to proteasome inhibition by activating two distinct proteostasis pathways normally utilized by hematopoietic stem cells: autophagy and the heat shock response. Concurrent proteasome and autophagy inhibition significantly increased unfolded protein content in multiple human AML cell lines. This proteostasis disruption was sufficient to induce a terminal ISR marked by activation of the eIF2α-ATF4-CHOP signaling axis that severely attenuated protein synthesis, reduced proliferation, and induced widespread apoptosis in human AML cell lines and primary AML patient samples. In contrast to AML cells, dual proteasome and autophagy inhibition was well tolerated by healthy human CD34+ hematopoietic stem and progenitor cells, suggesting a tractable therapeutic window for treating AML. Indeed, dual proteasome and autophagy inhibition was tolerated in vivo, and decreased leukemia burden in mice xenografted with human AML cell lines. Similar to autophagy inhibition, disabling the heat shock response by deleting Heat shock factor 1 (HSF1) sensitized human AML cell lines to proteasome inhibitors. Treatment of HSF1-deficient AML cells with a proteasome inhibitior induced a significant increase in unfolded protein, activated the ISR, reduced growth and protein synthesis, and led to severe apoptosis. Concurrent HSF1 deletion and proteasome inhibition dramatically reduced AML disease burden in vivo and extended median survival from 40 to 140 days. Overall, this study revealed that AML cells hijack stem cell proteostasis pathways to promote growth, survival, and therapeutic resistance. Targeting the unique configuration of the proteostasis network is uncovering new therapeutic strategies to eliminate AML. Citation Format: Kentson Lam, Yoon Joon Kim, Carlo M. Ong, Fanny J. Zhou, Andrea Z. Liu, Bernadette A. Chua, Jie-Hua Zhou, Edward D. Ball, Robert A. Signer. Targeting stem cell proteostasis pathways sensitizes acute myeloid leukemia to proteasome inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2104.

Novel mechanism of drug resistance triggered by tumor-associated macrophages through Heat Shock Factor-1 activation

Macrophages constitute a major part of tumor microenvironment, and most of existing data demonstrate their ruling role in the development of anti-drug resistance of cancer cell. One of the most powerful protection system is based on heat shock proteins whose synthesis is triggered by activated Heat Shock Factor-1 (HSF1); the inhibition of the HSF1 with CL-43 sensitized A549 lung cancer cells to the anti-cancer effect of etoposide. Notably, analyzing A549 tumor xenografts in mice we observed nest-like pattern of co-localization of A549 cells demonstrating enhanced expression of HSF1 with macrophages, and decided to check whether the above arrangement has a functional value for both cell types. It was found that the incubation of A549 or DLD1 colon cancer cells with either human monocytes or THP1 monocyte-like cells activated HSF1 and increased resistance to etoposide. Importantly, the same effect was shown when primary cultures of colon tumors were incubated with THP1 cells or with human monocytes. To prove that HSF1 is implicated in enhanced resistance caused by monocytic cells, we generated an A549 cell subline devoid of HSF1 which did not respond to incubation with THP1 cells. The pharmacological inhibition of HSF1 with CL-43 also abolished the effect of THP1 cells on primary tumor cells, highlighting a new target of tumor-associated macrophages in a cell proteostasis mechanism.

Deciphering ApoE Genotype-Driven Proteomic and Lipidomic Alterations in Alzheimer's Disease Across Distinct Brain Regions.

Alzheimer's disease (AD) is a neurodegenerative disease with a complex etiology influenced by confounding factors such as genetic polymorphisms, age, sex, and race. Traditionally, AD research has not prioritized these influences, resulting in dramatically skewed cohorts such as three times the number of Apolipoprotein E (APOE) ε4-allele carriers in AD relative to healthy cohorts. Thus, the resulting molecular changes in AD have previously been complicated by the influence of apolipoprotein E disparities. To explore how apolipoprotein E polymorphism influences AD progression, 62 post-mortem patients consisting of 33 AD and 29 controls (Ctrl) were studied to balance the number of ε4-allele carriers and facilitate a molecular comparison of the apolipoprotein E genotype. Lipid and protein perturbations were assessed across AD diagnosed brains compared to Ctrl brains, ε4 allele carriers (APOE4+ for those carrying 1 or 2 ε4s and APOE4- for non-ε4 carriers), and differences in ε3ε3 and ε3ε4 Ctrl brains across two brain regions (frontal cortex (FCX) and cerebellum (CBM)). The region-specific influences of apolipoprotein E on AD mechanisms showcased mitochondrial dysfunction and cell proteostasis at the core of AD pathophysiology in the post-mortem brains, indicating these two processes may be influenced by genotypic differences and brain morphology.

Proteostasis in neurodegenerative diseases.

Proteostasis is essential for normal function of proteins and vital for cellular health and survival. Proteostasis encompasses all stages in the "life" of a protein, that is, from translation to functional performance and, ultimately, to degradation. Proteins need native conformations for function and in the presence of multiple types of stress, their misfolding and aggregation can occur. A coordinated network of proteins is at the core of proteostasis in cells. Among these, chaperones are required for maintaining the integrity of protein conformations by preventing misfolding and aggregation and guide those with abnormal conformation to degradation. The ubiquitin-proteasome system (UPS) and autophagy are major cellular pathways for degrading proteins. Although failure or decreased functioning of components of this network can lead to proteotoxicity and disease, like neuron degenerative diseases, underlying factors are not completely understood. Accumulating misfolded and aggregated proteins are considered major pathomechanisms of neurodegeneration. In this chapter, we have described the components of three major branches required for proteostasis-chaperones, UPS and autophagy, the mechanistic basis of their function, and their potential for protection against various neurodegenerative conditions, like Alzheimer's, Parkinson's, and Huntington's disease. The modulation of various proteostasis network proteins, like chaperones, E3 ubiquitin ligases, proteasome, and autophagy-associated proteins as therapeutic targets by small molecules as well as new and unconventional approaches, shows promise.

A cell‐autonomous role of DNAJC5 in microglial‐mediated proteostasis in Alzheimer’s disease

AbstractBackgroundDNAJC5 encodes co‐chaperone cysteine‐string protein alpha (CSPα), a synaptic and endolysosomal protein whose mutations cause adult‐onset neuronal ceroid lipofuscinosis (ANCL). Characterized by endolysosomal dysfunction, microgliosis, synaptic loss, and protein aggregation, ANCL shares pathological features with Alzheimer’s disease (AD). The endolysosomal role of CSPα in non‐neuronal cell types has been understudied. Here, we aimed to address this gap and hypothesized that CSPα plays a yet undefined microglial role in AD.MethodAβ levels and plaques in human tissue were assessed through ELISA and immunohistochemistry, respectively. Bulk‐RNAseq data from two independent cohorts of AD and ANCL patients with controls (GSE5281; GSE15222) were analyzed. To assess cell‐autonomous expression, single‐nuclei RNAseq (snRNA‐seq) was mined from the parietal cortex of AD patients with APP and PSEN1 mutations (n = 16) along with sporadic AD (n = 31), non‐AD (n = 9), presymptomatic (n = 3), and control patients (n = 8). An independent cohort (GSE138852) was analyzed for rigor. Using Clue.io, candidate therapeutics projected to reverse the observed transcriptomic profile from DNAJC5 mutants in myeloid cells were identified. A cohort from iPSC‐derived microglia (GSE178317) was cross‐analyzed with the ANCL RNAseq.ResultIn tissue from ANCL patients, soluble or insoluble Aβ levels were nearly nonexistent and synaptic protein levels were reduced, but intraneuronal Aβ accumulation was increased, suggesting altered APP processing. In two independent RNAseq cohorts (GSE5281; GSE15222), DNAJC5 was reduced in AD patients versus controls. SnRNA‐seq from parietal cortex revealed higher DNAJC5 transcript levels in microglia versus neurons. RNAseq obtained from ANCL patients was cross‐referenced with dataset obtained from iPSC‐derived microglia, revealing 35 differentially expressed genes associated with proliferative microglia and 32 genes associated with the interferon/chemokine response. Analysis of ANCL transcriptomics in myeloid cell lines using Clue.io indicate HSP inhibitors (BIIB‐021, geldanamycin), an opioid receptor antagonist (BNTX), and a proteasome inhibitor (bortezomib) suggesting convergence in proteostasis in microglial cells. ANCL‐patient transcriptomics displayed reduced TSPAN14, which facilitates ADAM10 maturation, and elevated APH1B and GSAP, enzymes that promote γ‐secretase activity suggesting a shift towards amyloidogenic pathway.ConclusionDNAJC5 is implicated in AD pathogenesis modulating APP processing and amyloidogenesis. Furthermore, endolysosomal DNAJC5 might play a novel cell‐autonomous role in microglia under altered proteostasis.