Endoplasmic Reticulum Accumulation Research Articles

Mutant dominant-negative rhodopsin∆I256 causes protein aggregates degraded via ERAD and prevents normal rhodopsin from proper membrane trafficking

Dominant mutations in the rhodopsin gene (Rho) contribute to 25% of autosomal dominant retinitis pigmentosa (adRP), characterized by photoreceptor loss and progressive blindness. One such mutation, Rho∆I256, carries a 3-bp deletion, resulting in the loss of one of two isoleucines at codons 255 and 256. Our investigation, using recombinant expression in HEK293 and COS-7 cells, revealed that Rho∆I256, akin to the known adRP mutation RhoP23H, induces the formation of rhodopsin protein (RHO) aggregates at the perinuclear region. Co-expression of Rho∆I256 or RhoP23H with wild-type RhoWT, mimicking the heterozygous genotype of adRP patients, demonstrated the dominant-negative effect, as all isoforms were retained in perinuclear aggregates, impeding membrane trafficking. In retinal explants from WT mice, mislocalization of labeled adRP isoforms at the outer nuclear layer was observed. Further analysis revealed that RHO∆I256 aggregates are retained at the endoplasmic reticulum (ER), undergo ER-associated degradation (ERAD), and colocalize with the AAA-ATPase escort chaperone valosin-containing protein (VCP). These aggregates are polyubiquitinated and partially colocalized with the 20S proteasome subunit beta-5 (PSMB5). Pharmacological inhibition of proteasome- or VCP activity increased RHO∆I256 aggregate size. In summary, RHO∆I256 exhibits dominant pathogenicity by sequestering normal RHOWT in ER aggregates, preventing its membrane trafficking and following the ERAD degradation.

Combinatorial selective ER-phagy remodels the ER during neurogenesis

The endoplasmic reticulum (ER) employs a diverse proteome landscape to orchestrate many cellular functions, ranging from protein and lipid synthesis to calcium ion flux and inter-organelle communication. A case in point concerns the process of neurogenesis, where a refined tubular ER network is assembled via ER shaping proteins into the newly formed neuronal projections to create highly polarized dendrites and axons. Previous studies have suggested a role for autophagy in ER remodelling, as autophagy-deficient neurons in vivo display axonal ER accumulation within synaptic boutons, and the membrane-embedded ER-phagy receptor FAM134B has been genetically linked with human sensory and autonomic neuropathy. However, our understanding of the mechanisms underlying selective removal of the ER and the role of individual ER-phagy receptors is limited. Here we combine a genetically tractable induced neuron (iNeuron) system for monitoring ER remodelling during in vitro differentiation with proteomic and computational tools to create a quantitative landscape of ER proteome remodelling via selective autophagy. Through analysis of single and combinatorial ER-phagy receptor mutants, we delineate the extent to which each receptor contributes to both the magnitude and selectivity of ER protein clearance. We define specific subsets of ER membrane or lumenal proteins as preferred clients for distinct receptors. Using spatial sensors and flux reporters, we demonstrate receptor-specific autophagic capture of ER in axons, and directly visualize tubular ER membranes within autophagosomes in neuronal projections by cryo-electron tomography. This molecular inventory of ER proteome remodelling and versatile genetic toolkit provide a quantitative framework for understanding the contributions of individual ER-phagy receptors for reshaping ER during cell state transitions.

Dynamic changes in endoplasmic reticulum morphology and its contact with the plasma membrane in motor neurons in response to nerve injury.

The endoplasmic reticulum (ER) extends throughout a cell and plays a critical role in maintaining cellular homeostasis. Changes in ER shape could provide a clue to explore the mechanisms that underlie the fate determination of neurons after axon injury because the ER drastically changes its morphology under neuronal stress to maintain cellular homeostasis and recover from damage. Because of their tiny structures and richness in the soma, the detailed morphology of the ER and its dynamics have not been well analysed. In this study, the focused ion beam/scanning electron microscopy (FIB/SEM) analysis was performed to explore the ultra-structures of the ER in the somata of motor neuron with axon regenerative injury models. In normal motor neurons, ER in the somata is abundantly localised near the perinucleus and represents lamella-like structures. After injury, analysis of the ER volume and ER branching points indicated a collapse of the normal distribution and a transformation from lamella-like structures to mesh-like structures. Furthermore, accompanied by ER accumulation near the plasma membrane (PM), the contact between the ER and PM (ER-PM contacts) significantly increased after injury. The accumulation of extended-synaptotagmin 1 (E-Syt1), a tethering protein of the ER and PM that regulates Ca2+-dependent lipid transfer, was also identified by immunohistochemistry and quantitative Real-time PCR after injury. These morphological alterations of ER and the increase in ER-PM contacts may be crucial events that occur in motor neurons as a resilient response for the survival after axonal injury.

DDRGK1-mediated ER-phagy attenuates acute kidney injury through ER-stress and apoptosis

Acute kidney injury (AKI) constitutes a prevalent clinical syndrome characterized by elevated morbidity and mortality rates, emerging as a significant public health issue. This study investigates the interplay between endoplasmic reticulum (ER) stress, unfolded protein response (UPR), and ER-associated degradation (ER-phagy) in the pathogenesis of AKI. We employed four distinct murine models of AKI—induced by contrast media, ischemia–reperfusion injury, cisplatin, and folic acid—to elucidate the relationship between ER-phagy, ER stress, and apoptosis. Our findings reveal a marked decrease in ER-phagy coinciding with an accumulation of damaged ER, elevated ER stress, and increased apoptosis across all AKI models. Importantly, overexpression of DDRGK1 in HK-2 cells enhanced ER-phagy levels, ameliorating contrast-induced ER stress and apoptosis. These findings unveil a novel protective mechanism in AKI, wherein DDRGK1–UFL1-mediated ER-phagy mitigates ER stress and apoptosis in renal tubular epithelial cells. Our results thereby contribute to understanding the molecular underpinnings of AKI and offer potential therapeutic targets for its treatment.

Overproduction of mycotoxin biosynthetic enzymes triggers Fusarium toxisome-shaped structure formation via endoplasmic reticulum remodeling.

Mycotoxin deoxynivalenol (DON) produced by the Fusarium graminearum complex is highly toxic to animal and human health. During DON synthesis, the endoplasmic reticulum (ER) of F. graminearum is intensively reorganized, from thin reticular structure to thickened spherical and crescent structure, which was referred to as "DON toxisome". However, the underlying mechanism of how the ER is reorganized into toxisome remains unknown. In this study, we discovered that overproduction of ER-localized DON biosynthetic enzyme Tri4 or Tri1, or intrinsic ER-resident membrane proteins FgHmr1 and FgCnx was sufficient to induce toxisome-shaped structure (TSS) formation under non-toxin-inducing conditions. Moreover, heterologous overexpression of Tri1 and Tri4 proteins in non-DON-producing fungi F. oxysporum f. sp. lycopersici and F. fujikuroi also led to TSS formation. In addition, we found that the high osmolarity glycerol (HOG), but not the unfolded protein response (UPR) signaling pathway was involved in the assembly of ER into TSS. By using toxisome as a biomarker, we screened and identified a novel chemical which exhibited high inhibitory activity against toxisome formation and DON biosynthesis, and inhibited Fusarium growth species-specifically. Taken together, this study demonstrated that the essence of ER remodeling into toxisome structure is a response to the overproduction of ER-localized DON biosynthetic enzymes, providing a novel pathway for management of mycotoxin contamination.

#72 : Embryo Quality and Pregnancy Outcome from Oocytes with Smooth Endoplasmic Reticulum (SER) Aggregates

Background and Aims: Calcium stored and released by the Smooth Endoplasmic Reticulum - SER which has an important role in oocyte maturation, fertilization, and embryo development. The presence of SER aggregate result in the disturbance of calcium stores and its occillation which in turn affect fertilization, embryo develompent, and increasing risk of abnormal cromoseme outcome. Alpha-ESHRE Consensus (2011), strongly recomended to not perform insemination on any oocytes with SER. In the other hand, some previous studies show that healthy babies had been born from oocytes presenting this dymorphism. The aim of this study was to evaluate embryo quality and pregnancy outcome patients derived from oocytes with SER. Method: This study was a crosssectional analysis from 2017-2019 of 223 MII oocytes with SER from 82 women underwent Intra cytoplasmic sperm injection - ICSI. Prior to ICSI, each oocyte was evaluated for the presence of SER Aggregates. The embryological outcome measures were the fertilization rate, embryo quality day 3 and day 5, and pregnancy rates. Results: This study result shown that from 223 oocytes with SER, 89.5% oocytes can be fertilized. Day 3 embryo 66.6% ± 0.000 qualified as good embryo, but only 29.1% ± 0.000 on day 5 embryo. The Pregnancy rate was 30.0%, with 2 miscarriages. It is important to give a well information to the patients on the risk of birth malformations as well as whether a decreased chance of pregnancy exists. Conclusion: Oocyte with SER still has the ability to develop and has the potential to support pregnancy.

Phytaspase Is Capable of Detaching the Endoplasmic Reticulum Retrieval Signal from Tobacco Calreticulin-3.

Soluble chaperones residing in the endoplasmic reticulum (ER) play vitally important roles in folding and quality control of newly synthesized proteins that transiently pass through the ER en route to their final destinations. These soluble residents of the ER are themselves endowed with an ER retrieval signal that enables the cell to bring the escaped residents back from the Golgi. Here, by using purified proteins, we showed that Nicotiana tabacum phytaspase, a plant aspartate-specific protease, introduces two breaks at the C-terminus of the N. tabacum ER resident calreticulin-3. These cleavages resulted in removal of either a dipeptide or a hexapeptide from the C-terminus of calreticulin-3 encompassing part or all of the ER retrieval signal. Consistently, expression of the calreticulin-3 derivative mimicking the phytaspase cleavage product in Nicotiana benthamiana cells demonstrated loss of the ER accumulation of the protein. Notably, upon its escape from the ER, calreticulin-3 was further processed by an unknown protease(s) to generate the free N-terminal (N) domain of calreticulin-3, which was ultimately secreted into the apoplast. Our study thus identified a specific proteolytic enzyme capable of precise detachment of the ER retrieval signal from a plant ER resident protein, with implications for the further fate of the escaped resident.

Dysregulation of the endoplasmic reticulum blocks recruitment of centrosome-associated proteins resulting in mitotic failure.

The endoplasmic reticulum (ER) undergoes a remarkable transition in morphology during cell division to aid in the proper portioning of the ER. However, whether changes in ER behaviors modulate mitotic events is less clear. Like many animal embryos, the early Drosophila embryo undergoes rapid cleavage cycles in a lipid-rich environment. Here, we show that mitotic spindle formation, centrosomal maturation, and ER condensation occur with similar time frames in the early syncytium. In a screen for Rab family GTPases that display dynamic function at these stages, we identified Rab1. Rab1 disruption led to an enhanced buildup of ER at the spindle poles and produced an intriguing 'mini-spindle' phenotype. ER accumulation around the mitotic space negatively correlates with spindle length/intensity. Importantly, centrosomal maturation is defective in these embryos, as mitotic recruitment of key centrosomal proteins is weakened after Rab1 disruption. Finally, division failures and ER overaccumulation is rescued by Dynein inhibition, demonstrating that Dynein is essential for ER spindle recruitment. These results reveal that ER levels must be carefully tuned during mitotic processes to ensure proper assembly of the division machinery.

Rationally Engineered hCES2A Near-Infrared Fluorogenic Substrate for Functional Imaging and High-Throughput Inhibitor Screening.

Human carboxylesterase 2A (hCES2A) is an important endoplasmic reticulum (ER)-resident enzyme that is responsible for the hydrolytic metabolism or activation of numerous ester-bearing drugs and environmental toxins. The previously reported hCES2A fluorogenic substrates suffer from limited emission wavelength, low specificity, and poor localization accuracy, thereby greatly limiting the in situ functional imaging of hCES2A and drug discovery. Herein, a rational ligand design strategy was adopted to construct a highly specific near-infrared (NIR) substrate for hCES2A. Following scaffold screening and recognition group optimization, HTCF was identified as a desirable NIR fluorophore with excellent photophysical properties and high ER accumulation ability, while several HTCF esters held a high potential to be good hCES2A substrates. Further investigations revealed that TP-HTCF (the tert-pentyl ester of HTCF) was an ideal substrate with ultrahigh sensitivity, excellent specificity, and a substantial signal-to-noise ratio. Upon the addition of hCES2A, TP-HTCF could be rapidly hydrolyzed to release HTCF, a chemically stable product that emitted bright fluorescent signals at around 670 nm. A TP-HTCF-based biochemical assay was then established for the high-throughput screening of potent and cell-active hCES2A inhibitors from an in-house compound library. Furthermore, TP-HTCF displayed high imaging resolution for imaging hCES2A in living cells as well as mouse liver slices and tumor-xenograft mice. Collectively, this study demonstrates a rational strategy for developing highly specific fluorogenic substrates for an ER-resident target enzyme, while TP-HTCF can act as a practical tool for sensing hCES2A in living systems.

Major histocompatibility complex class I assembly within endolysosomal pathways.

Major histocompatibility complex classI (MHC classI) molecules facilitate subcellular immune surveillance by presenting peptides on the cell surface. MHC classI assembly with peptides generally happens in the endoplasmic reticulum (ER). Peptides are processed in the cytosol, transported into the ER, and assembled with MHC classI heavy and light chains. However, as many pathogens reside within multiple subcellular organelles, peptide sampling across non-cytosolic compartments is also important. MHC classI molecules internalize from the cell surface into endosomes and constitutively traffic between endosomes and the cell surface. Within endosomes, MHC classI molecules assemble with both exogenous and endogenous antigens processed within these compartments. Human MHC classI polymorphisms, well known to affect ER assembly modes, also influence endosomal assembly outcomes, an area of current interest to the field.

Characterization of intracellular membrane structures derived from a massive expansion of endoplasmic reticulum (ER) membrane due to synthetic ER-membrane-resident polyproteins.

The endoplasmic reticulum (ER) is a dynamic organelle that is amenable to major restructuring. Introduction of recombinant ER-membrane-resident proteins that form homo oligomers is a known method of inducing ER proliferation: interaction of the proteins with each other alters the local structure of the ER network, leading to the formation large aggregations of expanded ER, sometimes leading to the formation of organized smooth endoplasmic reticulum (OSER). However, these membrane structures formed by ER proliferation are poorly characterized and this hampers their potential development for plant synthetic biology. Here, we characterize a range of ER-derived membranous compartments in tobacco and show how the nature of the polyproteins introduced into the ER membrane affect the morphology of the final compartment. We show that a cytosol-facing oligomerization domain is an essential component for compartment formation. Using fluorescence recovery after photobleaching, we demonstrate that although the compartment retains a connection to the ER, a diffusional barrier exists to both the ER and the cytosol associated with the compartment. Using quantitative image analysis, we also show that the presence of the compartment does not disrupt the rest of the ER network. Moreover, we demonstrate that it is possible to recruit a heterologous, bacterial enzyme to the compartment, and for the enzyme to accumulate to high levels. Finally, transgenic Arabidopsis constitutively expressing the compartment-forming polyproteins grew and developed normally under standard conditions.

The mechanisms to dispose of misfolded proteins in the endoplasmic reticulum of adipocytes

Endoplasmic reticulum (ER)-associated degradation (ERAD) and ER-phagy are two principal degradative mechanisms for ER proteins and aggregates, respectively; however, the crosstalk between these two pathways under physiological settings remains unexplored. Using adipocytes as a model system, here we report that SEL1L-HRD1 protein complex of ERAD degrades misfolded ER proteins and limits ER-phagy and that, only when SEL1L-HRD1 ERAD is impaired, the ER becomes fragmented and cleared by ER-phagy. When both are compromised, ER fragments containing misfolded proteins spatially coalesce into a distinct architecture termed Coalescence of ER Fragments (CERFs), consisted of lipoprotein lipase (LPL, a key lipolytic enzyme and an endogenous SEL1L-HRD1 substrate) and certain ER chaperones. CERFs enlarge and become increasingly insoluble with age. Finally, we reconstitute the CERFs through LPL and BiP phase separation in vitro, a process influenced by both redox environment and C-terminal tryptophan loop of LPL. Hence, our findings demonstrate a sequence of events centered around SEL1L-HRD1 ERAD to dispose of misfolded proteins in the ER of adipocytes, highlighting the profound cellular adaptability to misfolded proteins in the ER in vivo.

An ER-accumulated mutant of yeast Pma1 causes membrane-related stress to induce the unfolded protein response

Upon dysfunction of the endoplasmic reticulum (ER), namely ER stress, eukaryotic cells provoke the unfolded protein response (UPR), which is triggered by ER stress sensors including Ire1. While the ER luminal domain of Ire1 is known to directly recognize misfolded soluble proteins accumulated in the ER, the transmembrane domain of Ire1 is involved in its self-association and activation upon membrane lipid-related abnormalities, which are so-called lipid bilayer stress (LBS). Here we inquired how the ER accumulation of misfolded transmembrane proteins induces the UPR. In yeast Saccharomyces cerevisiae cells, a multi-transmembrane protein, Pma1, is not transported to the cell surface but aggregates on the ER membrane when carrying a point mutation (Pma1-2308). Here, we show that GFP-tagged Ire1 co-localized with the Pma1-2308-mCherry puncta. This co-localization and the UPR induced by Pma1-2308-mCherry were compromised by a point mutation in Ire1 that specifically impairs its activation upon LBS. We presume that Pma1-2308-mCherry locally affects the properties (probably the thickness) of the ER membrane at its aggregation sites, where Ire1 is then recruited, self-associated, and then activated.

Inhibition of the mevalonate pathway causes accumulation of CD14 in the endoplasmic reticulum of macrophages

Abstract Mevalonate kinase deficiency (MKD) is an autoinflammatory disease caused by mutations in the mevalonate kinase enzyme, which acts early in the mevalonate pathway to produce sterol and non-sterol isoprenoid compounds. Patients with MKD have episodes of inflammatory symptoms accompanied by increased plasma levels of pro-inflammatory cytokines. CD14 is a receptor that binds to lipopolysaccharide (LPS) and induces the release of cytokines. Isoprenoid depletion was previously shown to increase cellular and decrease extracellular levels of CD14 in macrophages after LPS stimulation. The objective of this study is to characterize the cellular localization of CD14 following isoprenoid depletion. RAW 264.7 macrophages were treated with lovastatin to deplete isoprenoids and CD14 localization was examined using immunofluorescence and western blotting after LPS stimulation. CD14 co-localized with the endoplasmic reticulum (ER) protein calnexin following lovastatin treatment. To quantitate ER CD14, cell lysates were digested with endoglycosidase (Endo) H, which removes high mannose glycans found on ER proteins, but not complex glycans added in the Golgi apparatus. Western blot results showed that the level of Endo H sensitive CD14 was higher in lovastatin as compared to carrier-treated cells. Co-incubation of lovastatin-treated cells with mevalonate or geranylgeranyl pyrophosphate (GGPP), but not cholesterol, prevented the ER accumulation of CD14 indicating that GGPP depletion may cause this effect. Understanding how mevalonate pathway products contribute to protein trafficking in macrophages will expand our understanding of inflammatory diseases.

Endo-lysosomal assembly variations among Human Leukocyte Antigen class I (HLA-I) allotypes

Abstract The extreme polymorphisms of HLA-I proteins enable the presentation of diverse peptides to cytotoxic T lymphocytes (CTL). The canonical endoplasmic reticulum (ER) HLA-I assembly pathway enables presentation of cytosolic peptides, but effective intracellular surveillance requires multi-compartmental antigen sampling. Endo-lysosomes are generally sites of HLA class II assembly, but human monocytes and monocyte-derived dendritic cells (moDCs) also contain significant reserves of endo-lysosomal HLA-I molecules. We hypothesized variable influences of HLA-I polymorphisms upon outcomes of endo-lysosomal trafficking, as the stabilities and peptide occupancies of cell surface HLA-I are variable. Consistent with this model, when the endo-lysosomal pH of moDCs is disrupted, HLA-B allotypes display varying propensities for reductions in surface expression, with HLA-B* 08:01 or HLA-B*35:01 being among the most resistant or sensitive respectively, among eight tested HLA-B allotypes. Perturbations of moDC endo-lysosomal pH result in redistribution of HLA-B*35:01, but not HLA-B* 08:01, to LAMP1 +compartments and increase HLA-B*35:01 peptide receptivity. These findings reveal the intersection of the vacuolar cross-presentation pathway with a constitutive assembly pathway for some HLA-B allotypes. Notably, cross-presentation of epitopes derived from two soluble antigens was also more efficient for B*35:01 compared to B*08:01, even when matched for T cell response sensitivity, and more affected by cathepsin inhibition. Thus, HLA-I polymorphisms dictate the degree of endo-lysosomal assembly, which can supplement ER assembly for constitutive HLA-I expression and increase the efficiency of cross-presentation. Supported by grants NIH R01 AI044115 T32GM008353 and T32AI007413

Does smooth endoplasmic reticulum aggregation in oocytes impact the chromosome aneuploidy of the subsequent embryos? A propensity score matching study

BackgroundThe appearance of smooth endoplasmic reticulum aggregation (SERa) is one of the most common dysmorphic phenotypes of oocytes, however, the impact of SERa occurrence on in vitro fertilization (IVF) outcomes is controversial. This study aimed to investigate the impact of SERa in oocytes on the aneuploidy of the subsequent embryos in IVF.MethodsIn this retrospective cohort study, a total of 114 intracytoplasmic sperm injection (ICSI) cycles with the appearance of SERa undergoing preimplantation genetic testing for aneuploidy (PGT-A) were enrolled, and among them there were 323 SERa(+) oocytes and 1253 sibling unaffected oocytes. The 907 PGT-A cycles without SERa during the same period were enrolled as controls. A propensity score matching of 1:1 ratio between these two groups resulted in 113 matched cycles. The outcome parameters between the SERa(+) cycles/oocytes and the controls were compared. IVF laboratory outcomes, PGT-A outcomes, and clinical and neonatal outcomes were the main outcomes.ResultsIncreased abnormal fertilization rate and reduced blastocyst formation rate can be observed in both SERa(+) cycles and oocytes, some other parameters on developmental potential, such as available embryo rate at Day 3 and available blastocyst rate, were also impaired in the case of SERa occurrences. Among the 910 blastocysts for PGT-A, the percentage of euploid embryos was similar between the matched cohorts, while an unpredicted increase of the proportions of euploid in the SERa(+) oocytes, compared to the SERa(-) oocytes. Moreover, there was no significance in terms of clinical and neonatal outcomes, such as implantation rate, biochemical pregnancy rate, clinical pregnancy rate, miscarriage rate, and live birth rate, regardless of the presence of SERa in cycles and oocytes.ConclusionsThe appearance of SERa within mature oocytes has no significant impact on the aneuploidy of subsequent blastocysts. It is recommended to utilize these oocytes, especially for those with few oocytes or advanced maternal age, which is likely to increase the cumulative pregnancy rate. This study may offer evidence to assist embryologists to make clinical decisions concerning SERa(+) oocytes more consciously and rationally.

Real value of the oocytes with smooth endoplasmic reticulum aggregates in in vitro fertilization/intracytoplasmic sperm injection cycle: a retrospective cohort study

Objective: The management of oocytes affected by smooth endoplasmic reticulum aggregates (SERa) remains debatable. To understand how to manage SERa+ oocytes and cycles, we performed a retrospective cohort study and analyzed the impact of SERa+ cycles and oocytes on clinical and neonatal outcomes. Methods: We included 4856 cycles (149 SERa+ and 4707 SERa−) from 4201 women (age: 21–42 years) who received in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) treatments at the Center of Reproductive Medicine, First Affiliated Hospital of Army Military University, from 2016 to 2019. SERa+ cycles had at least one SERa oocyte in the oocyte cohort. All 1722 oocytes in the SERa+ cycle were divided into SERa+ (405) and SERa− (1317) oocytes. Results: The rates of two pronuclei (2PN) and high-quality embryos were lower in SERa+ cycles than in SERa− cycles, regardless of IVF or ICSI (P <0.05). As the proportion of SERa+ oocytes increased in the SERa+ cycles, the rate of high-quality embryos declined gradually (P <0.05). Furthermore, the rate of 2PN in SERa+ oocytes was significantly lower than that in SERa− oocytes (P <0.05). Regardless of whether IVF or ICSI insemination was performed, no significant differences in terms of clinical pregnancy rate and spontaneous abortion rate were observed between SERa+ and SERa− cycles or between SERa+ and SERa− oocytes (P >0.05). Conclusion: Normal fertilization with SERa+ cycles and oocytes was substantially reduced, regardless of the insemination method. Embryos originating from SERa+ oocytes can be transferred when there are no other options, but fully informed consent and strict follow-up of fetal development are mandatory.

Endo-lysosomal assembly variations among human leukocyte antigen class I (HLA class I) allotypes.

The extreme polymorphisms of human leukocyte antigen class I (HLA class I) proteins enable the presentation of diverse peptides to cytotoxic T lymphocytes. The canonical endoplasmic reticulum (ER) HLA class I assembly pathway enables presentation of cytosolic peptides, but effective intracellular surveillance requires multi-compartmental antigen sampling. Endo-lysosomes are generally sites of HLA class II assembly, but human monocytes and monocyte-derived dendritic cells (moDCs) also contain significant reserves of endo-lysosomal HLA class I molecules. We hypothesized variable influences of HLA class I polymorphisms upon outcomes of endo-lysosomal trafficking, as the stabilities and peptide occupancies of cell surface HLA class I molecules are variable. Consistent with this model, when the endo-lysosomal pH of moDCs is disrupted, HLA-B allotypes display varying propensities for reductions in surface expression, with HLA-B*08:01 or HLA-B*35:01 being among the most resistant or sensitive, respectively, among eight tested HLA-B allotypes. Perturbations of moDC endo-lysosomal pH result in accumulation of HLA-B*35:01 in LAMP1+ compartments and increase HLA-B*35:01 peptide receptivity. These findings reveal the intersection of the vacuolar cross-presentation pathway with a constitutive assembly pathway for some HLA-B allotypes. Notably, cross-presentation of epitopes derived from two soluble antigens was also more efficient for B*35:01 compared to B*08:01, even when matched for T cell response sensitivity, and more affected by cathepsin inhibition. Thus, HLA class I polymorphisms dictate the degree of endo-lysosomal assembly, which can supplement ER assembly for constitutive HLA class I expression and increase the efficiency of cross-presentation.

Fundamental Role of Pentose Phosphate Pathway within the Endoplasmic Reticulum in Glutamine Addiction of Triple-Negative Breast Cancer Cells.

Cancer utilization of large glutamine equivalents contributes to diverging glucose-6-P flux toward the pentose phosphate shunt (PPP) to feed the building blocks and the antioxidant responses of rapidly proliferating cells. In addition to the well-acknowledged cytosolic pathway, cancer cells also run a largely independent PPP, triggered by hexose-6P-dehydrogenase within the endoplasmic reticulum (ER), whose activity is mandatory for the integrity of ER-mitochondria networking. To verify whether this reticular metabolism is dependent on glutamine levels, we complemented the metabolomic characterization of intermediates of the glucose metabolism and tricarboxylic acid cycle with the estimation of proliferating activity, energy metabolism, redox damage, and mitochondrial function in two breast cancer cell lines. ER-PPP activity and its determinants were estimated by the ER accumulation of glucose analogs. Glutamine shortage decreased the proliferation rate despite increased ATP and NADH levels. It depleted NADPH reductive power and increased malondialdehyde content despite a marked increase in glucose-6P-dehydrogenase. This paradox was explained by the deceleration of ER-PPP favored by the decrease in hexose-6P-dehydrogenase expression coupled with the opposite response of its competitor enzyme glucose-6P-phosphatase. The decreased ER-PPP activity eventually hampered mitochondrial function and calcium exchanges. These data configure the ER-PPP as a powerful, unrecognized regulator of cancer cell metabolism and proliferation.

Notch Missense Mutations in Drosophila Reveal Functions of Specific EGF-like Repeats in Notch Folding, Trafficking, and Signaling.

Notch signaling plays various roles in cell-fate specification through direct cell-cell interactions. Notch receptors are evolutionarily conserved transmembrane proteins with multiple epidermal growth factor (EGF)-like repeats. Drosophila Notch has 36 EGF-like repeats, and while some play a role in Notch signaling, the specific functions of most remain unclear. To investigate the role of each EGF-like repeat, we used 19 previously identified missense mutations of Notch with unique amino acid substitutions in various EGF-like repeats and a transmembrane domain; 17 of these were identified through a single genetic screen. We assessed these mutants' phenotypes in the nervous system and hindgut during embryogenesis, and found that 10 of the 19 Notch mutants had defects in both lateral inhibition and inductive Notch signaling, showing context dependency. Of these 10 mutants, six accumulated Notch in the endoplasmic reticulum (ER), and these six were located in EGF-like repeats 8-10 or 25. Mutations with cysteine substitutions were not always coupled with ER accumulation. This suggests that certain EGF-like repeats may be particularly susceptible to structural perturbation, resulting in a misfolded and inactive Notch product that accumulates in the ER. Thus, we propose that these EGF-like repeats may be integral to Notch folding.