Recruitment Of Receptors Research Articles

Selective Glycan Presentation in Liquid‐Ordered or ‐Disordered Membrane Phases and its Effect on Lectin Binding

AbstractGlycan‐protein interactions play a key role in various biological processes from fertilization to infections. Many of these interactions take place at the glycocalyx—a heavily glycosylated layer at the cell surface. Despite its significance, studying the glycocalyx remains challenging due to its complex, dynamic, and heterogeneous nature. This study introduces a glycocalyx model allowing for the first time to control spatial organization and heterogeneity of the glycan moieties. Glycan‐mimetics with lipid‐moieties that partition into either liquid‐ordered (Lo, lipid rafts) or liquid‐disordered (Ld) phases of giant unilamellar vesicles (GUVs), which serve as simplified cell membrane models mimicking lipid rafts, are developed. This phase‐specific allocation allows controlled placement of glycan motifs in distinct membrane environments, creating heteromultivalent systems that replicate the natural glycocalyx's complexity. We show that phase localization of glycan mimetics significantly influences recruitment of protein receptors to the membrane. Glycan‐conjugates in the ordered phase demonstrate enhanced lectin binding, supporting the idea that raft‐like domains facilitate stronger receptor interactions. This study provides a platform for systematically investigating spatial and dynamic presentation of glycans in biological systems and presents the first experimental evidence that glycan accumulation in lipid rafts enhances receptor binding affinity, offering deeper insights into the glycocalyx‘s functional mechanisms.

Selective Glycan Presentation in Liquid-Ordered or -Disordered Membrane Phases and its Effect on Lectin Binding.

Glycan-protein interactions play a key role in various biological processes from fertilization to infections. Many of these interactions take place at the glycocalyx-a heavily glycosylated layer at the cell surface. Despite its significance, studying the glycocalyx remains challenging due to its complex, dynamic, and heterogeneous nature. This study introduces a glycocalyx model allowing for the first time to control spatial organization and heterogeneity of the glycan moieties. Glycan-mimetics with lipid-moieties that partition into either liquid-ordered (Lo, lipid rafts) or liquid-disordered (Ld) phases of giant unilamellar vesicles (GUVs), which serve as simplified cell membrane models mimicking lipid rafts, are developed. This phase-specific allocation allows controlled placement of glycan motifs in distinct membrane environments, creating heteromultivalent systems that replicate the natural glycocalyx's complexity. We show that phase localization of glycan mimetics significantly influences recruitment of protein receptors to the membrane. Glycan-conjugates in the ordered phase demonstrate enhanced lectin binding, supporting the idea that raft-like domains facilitate stronger receptor interactions. This study provides a platform for systematically investigating spatial and dynamic presentation of glycans in biological systems and presents the first experimental evidence that glycan accumulation in lipid rafts enhances receptor binding affinity, offering deeper insights into the glycocalyx's functional mechanisms.

Regulation of BCR-dependent germinal center B-cell formation by HGAL and insight into its emerging myeloid ortholog, C1ORF150.

The specificity of cytokine and immunoreceptor signaling frequently depends upon receptor recruitment of select adaptor proteins and specifically engaged effectors. This review focuses on the orthologous adaptor proteins, HGAL and C1ORF150, and aims to provide insight into their respective modulation of lymphoid and myeloid cell signaling, formation, and function. HGAL acts predominantly within germinal center B cells as an important BCR signal transducer. Effects on BCR signalosome assembly involve HGAL's localization to the plasma membrane via its lipidation, initial interactions with SYK, the pY-phosphorylation of HGAL including its recruitment of GRB2, and HGAL engagement of PDZ-RhoGEF and RhoA signaling. At ligated BCRs, this includes HGAL(-GRB2) stimulation of SYK kinase, attenuation of calcium flux-dependent and NF-κB expression, promotion of cSMAC formation, and cytoskeletal remodeling associated with HGAL-attenuated cell migration. HGAL and partnered effectors also impact on DLBCL pathogenesis, and studies are summarized on HGAL's actions (using DLBCL and Burkitt lymphoma B cells) including cell migration effects, HGAL modulation of cytoskeletal components, and insightful HGAL transgenic mouse and xenograft models. For C1ORF150, its HGAL-homologous subdomains are considered, together with studies that demonstrate C1OR150's FcϵRI- and KIT-mediated expression and phosphorylation in primary human mast cells. Intriguingly, recent GWAS studies have identified a C1ORF150 in-frame splice variant that is strongly associated with urticaria. Candidate mechanisms via which the encoded "C1ORF150-Δexon2" isoform affects mast cell degranulation are considered, including FcϵR1 and/or KIT receptor connections, and candidate "myristoylation switch" mechanisms.

Estrogen Receptor Beta Agonist Influences Presynaptic NMDA Receptor Distribution in the Paraventricular Hypothalamic Nucleus Following Hypertension in a Mouse Model of Perimenopause.

Women become susceptible to hypertension as they transition to menopause (i.e., perimenopause); however, the underlying mechanisms are unclear. Animal studies using an accelerated ovarian failure (AOF) model of peri-menopause (peri-AOF) demonstrate that peri-AOF hypertension is associated with increased postsynaptic NMDA receptor plasticity in the paraventricular hypothalamic nucleus (PVN), a brain area critical for blood pressure regulation. However, recent evidence indicates that presynaptic NMDA receptors also play a role in neural plasticity. Here, using immuno-electron microscopy, we examine the influence of peri-AOF hypertension on the subcellular distribution of the essential NMDA GluN1 receptor subunit in PVN axon terminals in peri-AOF and in male mice. Hypertension was produced by 14-day slow-pressor angiotensin II (AngII) infusion. The involvement of estrogen signaling was investigated by co-administering an estrogen receptor beta (ERß) agonist. Although AngII induced hypertension in both peri-AOF and male mice, peri-AOF females showed higher cytoplasmic GluN1 levels. In peri-AOF females, activation of ERß blocked hypertension and increased plasmalemmal GluN1 in axon terminals. In contrast, stimulation of ERß did not inhibit hypertension or influence presynaptic GluN1 localization in males. These results indicate that sex-dependent recruitment of presynaptic NMDA receptors in the PVN is influenced by ERß signaling in mice during early ovarian failure.

Chronic Lithium Treatment Alters NMDA and AMPA Receptor Synaptic Availability and Dendritic Spine Organization in the Rat Hippocampus.

The mechanisms underlying the action of lithium (LiCl) in bipolar disorder (BD) are still far from being completely understood. Previous evidence has revealed that BD is characterized by glutamate hyperexcitability, suggesting that LiCl may act, at least partially, by toning down glutamatergic signaling abnormalities. In this study, taking advantage of western blot and confocal microscopy, we used a combination of integrative molecular and morphological approaches in rats exposed to repeated administration of LiCl at a therapeutic dose (between 0.6 and 1.2 mmol/l) and sacrificed at two different time points, i.e., 24 hours and 7 days after the last exposure. We report that repeated LiCl treatment activates multiple, parallel, but also converging forms of compensatory neuroplasticity related to glutamatergic signaling. More specifically, LiCl promoted a wave of neuroplasticity in the hippocampus, involving the synaptic recruitment of GluN2A-containing NMDA receptors, GluA1-containing AMPA receptors, and the neurotrophin BDNF that are indicative of a more plastic spine. The latter is evidenced by morphological analyses showing changes in dendritic spine morphology, such as increased length and head diameter of such spines. These changes may counteract the potentially negative extra-synaptic movements of GluN2B-containing NMDA receptors as well as the increase in the formation of GluA2-lacking Ca2+-permeable AMPA receptors. Our findings highlight a previously unknown cohesive picture of the glutamatergic implications of LiCl action that persist long after the end of its administration, revealing for the first time a profound and persistent reorganization of the glutamatergic postsynaptic density receptor composition and structure.

Olfactory receptor coexpression and co-option in the dengue mosquito.

The olfactory sensory neurons of vinegar flies and mice tend to express a single ligand-specific receptor. While this 'one neuron-one receptor' motif has long been expected to apply broadly across insects, recent evidence suggests it may not extend to mosquitoes. We sequenced and analyzed the transcriptomes of 46,000 neurons from antennae of the dengue mosquito Aedes aegypti to resolve all olfactory, thermosensory, and hygrosensory neuron subtypes and identify the receptors expressed therein. We find that half of all olfactory subtypes coexpress multiple receptors. However, coexpression occurs almost exclusively among genes from the same family-among odorant receptors (ORs) or among ionotropic receptors (IRs). Coexpression of ORs with IRs is exceedingly rare. Many coexpressed receptors are recent duplicates. In other cases, the recruitment or co-option of single receptors by multiple neuron subtypes has placed these genes together in the same cells with distant paralogs. Close examination of data from Drosophila reveal rare cases of both phenomena, indicating that the olfactory systems of these two species are not fundamentally different, but instead fall at different locations along a continuum likely to encompass diverse insects.

A biallelic mutation in CACNA2D2 associated with developmental and epileptic encephalopathy affects calcium channel-dependent as well as synaptic functions of α2δ-2.

α2δ proteins serve as auxiliary subunits of voltage-gated calcium channels and regulate channel membrane expression and current properties. Besides their channel function, α2δ proteins regulate synapse formation, differentiation, and synaptic wiring. Considering these important functions, it is not surprising that CACNA2D1-4, the genes encoding for α2δ-1 to -4 isoforms, have been implicated in neurological, neurodevelopmental, and neuropsychiatric disorders. Mutations in CACNA2D2 have been associated with developmental and epileptic encephalopathy (DEE) and cerebellar atrophy. In our present study, we performed a detailed functional characterization of the p.R593P mutation in α2δ-2, a homozygous mutation previously identified in two siblings with DEE. Importantly, we analyzed both calcium channel-dependent as well as synaptic functions of α2δ-2. Our data show that the corresponding p.R596P mutation in mouse α2δ-2 drastically decreases membrane expression and synaptic targeting of α2δ-2. This defect correlates with altered biophysical properties of postsynaptic CaV1.3 channel but has no effect on presynaptic CaV2.1 channels upon heterologous expression in tsA201 cells. However, homologous expression of α2δ-2_R596P in primary cultures of hippocampal neurons affects the ability of α2δ-2 to induce a statistically significant increase in the presynaptic abundance of endogenous CaV2.1 channels and presynaptic calcium transients. Moreover, our data demonstrate that in addition to lowering membrane expression, the p.R596P mutation reduces the trans-synaptic recruitment of GABAA receptors and presynaptic synapsin clustering in glutamatergic synapses. Lastly, the α2δ-2_R596P reduces the amplitudes of glutamatergic miniature postsynaptic currents in transduced hippocampal neurons. Taken together, our data strongly link the human biallelic p.R593P mutation to the underlying severe neurodevelopmental disorder and highlight the importance of studying α2δ mutations not only in the context of channelopathies but also synaptopathies.

Interleukin-17F suppressed colon cancer by enhancing caspase 4 mediated pyroptosis of endothelial cells

The combination of anti-angiogenic treatment and immunotherapy presents a promising strategy against colon cancer. Interleukin-17F (IL-17F) emerges as a critical immune cell cytokine expressed in colonic epithelial cells, demonstrating potential in inhibiting angiogenesis. In order to clarify the roles of IL-17F in the colon cancer microenvironment and elucidate its mechanism, we established a mouse colon carcinoma cell line CT26 overexpressing IL-17F and transplanted it subcutaneously into syngeneic BALB/c mice. We also analyzed induced colon tumor in IL-17F knockout and wild type mice. Our results demonstrated that IL-17F could suppress colon tumor growth in vivo with inhibited angiogenesis and enhanced recruitment of cysteine-cysteine motif chemokine receptor 6 (CCR6) positive immune cells. Additionally, IL-17F suppressed the tube formation, cell growth and migration of endothelial cells EOMA in vitro. Comprehensive bioinformatics analysis of transcriptome profiles between EOMA cells and those treated with three different concentrations of IL-17F identified 109 differentially expressed genes. Notably, a potential new target, Caspase 4, showed increased expressions after IL-17F treatment in endothelial cells. Further molecular validation revealed a novel downstream signaling for IL-17F: IL-17F enhanced Caspase 4/GSDMD signaling of endothelial cells, CT26 cells and CT26 transplanted tumors, while IL-17F knockout colon tumors exhibited decreased Caspase 4/GSDMD signaling. The heightened expression of the GSDMD N-terminus, coupled with increased cellular propidium iodide (PI) uptake and lactate dehydrogenase (LDH) release, revealed that IL-17F promoted pyroptosis of endothelial cells. Altogether, IL-17F could modulate the colon tumor microenvironment with inhibited angiogenesis, underscoring its potential as a therapeutic target for colon cancer.

Discovery of Monovalent Direct Degraders of BRD4 that Act via the Recruitment of DCAF11.

Targeted protein degradation (TPD) using the ubiquitin proteasome system (UPS) is a rapidly growing drug discovery modality to eliminate pathogenic proteins. Strategies for TPD have focused on heterobifunctional degraders that often suffer from poor drug-like properties, and molecular glues that rely on serendipitous discovery. Monovalent "direct" degraders represent an alternative approach, in which small molecules bind to a target protein and induce degradation of that protein through the recruitment of an E3 ligase complex. Using an ultra-high throughput cell-based screening platform, degraders of the bromodomain extraterminal protein BRD4 were identified and optimized to yield a lead compound, PLX-3618. In this paper, we demonstrate that PLX-3618 elicited UPS-mediated selective degradation of BRD4, resulting in potent antitumor activity in vitro and in vivo. Characterization of the degradation mechanism identified DCAF11 as the E3 ligase required for PLX-3618-mediated degradation of BRD4. Protein-protein interaction studies verified a BRD4:PLX-3618:DCAF11 ternary complex, and mutational studies provided further insights into the DCAF11-mediated degradation mechanism. Collectively, these results demonstrate the discovery and characterization of a novel small molecule that selectively degrades BRD4 through the recruitment of the E3 substrate receptor, DCAF11, and promotes potent antitumor activity in vivo.

Agonistic anti-DCIR antibody inhibits ITAM-mediated inflammatory signaling and promotes immune resolution.

DC inhibitory receptor (DCIR) is a C-type lectin receptor selectively expressed on myeloid cells, including monocytes, macrophages, DCs, and neutrophils. Its role in immune regulation has been implicated in murine models and human genome-wide association studies, suggesting defective DCIR function associates with increased susceptibility to autoimmune diseases such as rheumatoid arthritis, lupus, and Sjögren's syndrome. However, little is known about the mechanisms underlying DCIR activation to dampen inflammation. Here, we developed anti-DCIR agonistic antibodies that promote phosphorylation on DCIR's immunoreceptor tyrosine-based inhibitory motifs and recruitment of SH2 containing protein tyrosine phosphatase-2 for reducing inflammation. We also explored the inflammation resolution by depleting DCIR+ cells with antibodies. Utilizing a human DCIR-knock-in mouse model, we validated the antiinflammatory properties of the agonistic anti-DCIR antibody in experimental peritonitis and colitis. These findings provide critical evidence for targeting DCIR to develop transformative therapies for inflammatory diseases.

Recognizing the opponent: The consolidation of long-term social memory in zebrafish males

Recognizing and remembering another individual in a social context could be beneficial for individual fitness. Especially in agonistic encounters, remembering an opponent and the previous fight could allow for avoiding new conflicts. Considering this, we hypothesized that this type of social interaction forms a long-term recognition memory lasting several days. It has been shown that a second encounter 24 h later between the same pair of zebrafish males is resolved with lower levels of aggression. Here, we evaluated if this behavioral change could last for longer intervals and a putative mechanism associated with memory storage: the recruitment of NMDA receptors. We found that if a pair of zebrafish males fight and fight again 48 or 72 h later, they resolve the second encounter with lower levels of aggression. However, if opponents were exposed to MK-801 (NMDA receptor antagonist) immediately after the first encounter, they solved the second one with the same levels of aggression: that is, no reduction in aggressive behaviors was observed. These amnesic effect suggest the formation of a long-term social memory related to recognizing a particular opponent and/or the outcome and features of a previous fight.

Neto proteins differentially modulate the gating properties of Drosophila NMJ glutamate receptors.

The formation of functional synapses requires co-assembly of ion channels with their accessory proteins which controls where, when, and how neurotransmitter receptors function. The auxiliary protein Neto modulates the function of kainate-type glutamate receptors in vertebrates as well as at the Drosophila neuromuscular junction (NMJ), a glutamatergic synapse widely used for genetic studies on synapse development. We previously reported that Neto is essential for the synaptic recruitment and function of glutamate receptors. Here, using outside-out patch-clamp recordings and fast ligand application, we examine for the first time the biophysical properties of recombinant Drosophila NMJ receptors expressed in HEK293T cells and compare them with native receptor complexes of genetically controlled composition. The two Neto isoforms, Neto-α and Neto-β, differentially modulate the gating properties of NMJ receptors. Surprisingly, we found that deactivation is extremely fast and that the decay of synaptic currents resembles the rate of iGluR desensitization. The functional analyses of recombinant iGluRs that we report here should greatly facilitate the interpretation of compound in vivo phenotypes of mutant animals.

WHAMM functions in kidney reabsorption and polymerizes actin to promote autophagosomal membrane closure and cargo sequestration.

The actin cytoskeleton is essential for many functions of eukaryotic cells, but the factors that nucleate actin assembly are not well understood at the organismal level or in the context of disease. To explore the function of the actin nucleation factor WHAMM in mice, we examined how Whamm inactivation impacts kidney physiology and cellular proteostasis. We show that male WHAMM knockout mice excrete elevated levels of albumin, glucose, phosphate, and amino acids, and display structural abnormalities of the kidney proximal tubule, suggesting that WHAMM activity is important for nutrient reabsorption. In kidney tissue, the loss of WHAMM results in the accumulation of the lipidated autophagosomal membrane protein LC3, indicating an alteration in autophagy. In mouse fibroblasts and human proximal tubule cells, WHAMM and its binding partner the Arp2/3 complex control autophagic membrane closure and cargo receptor recruitment. These results reveal a role for WHAMM-mediated actin assembly in maintaining kidney function and promoting proper autophagosome membrane remodeling.

Activity-dependent mitochondrial ROS signaling regulates recruitment of glutamate receptors to synapses.

Our understanding of mitochondrial signaling in the nervous system has been limited by the technical challenge of analyzing mitochondrial function in vivo. In the transparent genetic model Caenorhabditis elegans, we were able to manipulate and measure mitochondrial reactive oxygen species (mitoROS) signaling of individual mitochondria as well as neuronal activity of single neurons in vivo. Using this approach, we provide evidence supporting a novel role for mitoROS signaling in dendrites of excitatory glutamatergic C. elegans interneurons. Specifically, we show that following neuronal activity, dendritic mitochondria take up calcium (Ca2+) via the mitochondrial Ca2+ uniporter (MCU-1) that results in an upregulation of mitoROS production. We also observed that mitochondria are positioned in close proximity to synaptic clusters of GLR-1, the C. elegans ortholog of the AMPA subtype of glutamate receptors that mediate neuronal excitation. We show that synaptic recruitment of GLR-1 is upregulated when MCU-1 function is pharmacologically or genetically impaired but is downregulated by mitoROS signaling. Thus, signaling from postsynaptic mitochondria may regulate excitatory synapse function to maintain neuronal homeostasis by preventing excitotoxicity and energy depletion.

Alpha1- and Beta-norepinephrinergic receptors of dorsomedial and ventromedial hypothalamic nuclei modulate panic attack-like defensive behaviour elicited by diencephalic GABAergic neurotransmission disinhibition

Gamma-aminobutyric acid (GABA) disinhibition in medial hypothalamus (MH) nuclei of rats elicits some defensive reactions that are considered panic attack-like behaviours. Recent evidence showed that the norepinephrine-mediated system modulates fear-related defensive behaviours organised by MH neurons at least in part via noradrenergic receptors recruitment on midbrain tegmentum. However, it is unknown whether noradrenergic receptors of the MH also modulate the panic attack-like reactions. The aim of this work was to investigate the distribution of noradrenergic receptors in MH, and the effects of either α1-, α2- or β-noradrenergic receptors blockade in the MH on defensive behaviours elaborated by hypothalamic nuclei. Defensive behaviours were evaluated after the microinjection of the selective GABAA receptor antagonist bicuculline into the MH that was preceded by microinjection of either WB4101, RX821002, propranolol (α1-, α2- and β-noradrenergic receptor selective antagonists, respectively), or physiological saline into the MH of male Wistar rats. The α1-, α2- and β-noradrenergic receptors were found in neuronal perikarya of all MH nuclei, and the α2-noradrenergic receptor were also found on glial cells mainly situated in the ventrolateral division of the ventromedial hypothalamic nucleus. The α1- and β-noradrenergic receptors blockade in the MH decreased defensive attention and escape reactions elicited by the intra-MH microinjections of bicuculline. These findings suggest that, despite the profuse distributions of α1-, α2- and β-noradrenergic receptors in the MH, both α1- and β-noradrenergic receptor- rather than α2-noradrenergic receptor-signalling in MH are critical for the neuromodulation of panic-like behaviour.

Developmental Windows for Effects of Choline and Folate on Excitatory and Inhibitory Neurotransmission During Human Gestation.

Choline and folate are critical nutrients for fetal brain development, but the timing of their influence during gestation has not been previously characterized. At different periods during gestation, choline stimulation of α7-nicotinic receptors facilitates conversion of γ-aminobutyric acid (GABA) receptors from excitatory to inhibitory and recruitment of GluR1-R2 receptors for faster excitatory responses to glutamate. The outcome of the fetal development of inhibition and excitation was assessed in 159 newborns by P50 cerebral auditory-evoked responses. Paired stimuli, S1, S2, were presented 500 msec apart. Higher P50 amplitude in response to S1 (P50S1microV) assesses excitation, and lower P50S2microV assesses inhibition in this paired-stimulus paradigm. Development of inhibition was related solely to maternal choline plasma concentration and folate supplementation at 16 weeks' gestation. Development of excitation was related only to maternal choline at 28 weeks. Higher maternal choline concentrations later in gestation did not compensate for earlier lower concentrations. At 4 years of age, increased behavior problems on the Child Behavior Checklist 1½-5yrs were related to both newborn inhibition and excitation. Incomplete development of inhibition and excitation associated with lower choline and folate during relatively brief periods of gestation thus has enduring effects on child development.

Assessment of the effectiveness of the peptide inhibitor homologous to the transforming growth factor β cytokine blocking the TGFβRI/TGFβRII receptor complex-pilot study.

A key player in the fibrotic process is the transforming growth factor β (TGF-β) which enhances extracellular matrix production by increasing the transcription of matrix proteins. The cytokine TGF-β first binds to the TGFβRII receptor (dimer), resulting in the recruitment of the TGFβRI receptor (dimer). The complex thus formed leads to the phosphorylation of the kinase domain of TGFβRI, which in turn results in activation of the Smad pathway. This is therefore a targeted pathway for research into the application of peptide inhibitors in blocking the TGF-β-Smad signaling pathway. The aim of this study was to design a peptide inhibitor (homologous to the cytokine TGF-β) which, after binding to the TGFβRI/TGFβRII receptor, would block the cytokine binding and thus prevent the formation of an activating complex. Preliminary work on the design and synthesis of inhibitors for TGFβRI/TGFβRII has allowed us to identify and describe five key regions of the TGF-β-TGFβRI/TGFβRII interface. The following five peptide inhibitors were synthesized for Region 1: 1.1 ALDAAYCFR, 1.2 LDAAYCFRN, 1.3 DAAYCFRNV, 1.4 AAYCFRNVQ, 1.5 AYCFRNVQD. The expression of the SEAP reporter gene, Smad2, Smad3, Smad4, and JNK1 gene was measured using quantitative real-time polymerase chain reaction. For Region 1 peptide inhibitors tested for TGFβRI/TGFβRII, reduced SEAP (reporter gene) expression was observed in cells of the MFB-F11 line, which suggests inhibited the formation of cytokine-receptor complexes. For IP1_2, 1_3 and 1_5 Region 1 peptides tested for TGFβRI/TGFβRII, reduced cytokine-receptor signal by adding newly designed inhibitors. The study revealed an impact of these peptide inhibitors on the reduction of mRNA expression of Smad2, Smad3, Smad4 and JNK1 genes.

Control of CCR5 Cell-Surface Targeting by the PRAF2 Gatekeeper.

The cell-surface targeting of neo-synthesized G protein-coupled receptors (GPCRs) involves the recruitment of receptors into COPII vesicles budding at endoplasmic reticulum exit sites (ERESs). This process is regulated for some GPCRs by escort proteins, which facilitate their export, or by gatekeepers that retain the receptors in the ER. PRAF2, an ER-resident four trans- membrane domain protein with cytoplasmic extremities, operates as a gatekeeper for the GB1 protomer of the heterodimeric GABAB receptor, interacting with a tandem di-leucine/RXR retention motif in the carboxyterminal tail of GB1. PRAF2 was also reported to interact in a two-hybrid screen with a peptide corresponding to the carboxyterminal tail of the chemokine receptor CCR5 despite the absence of RXR motifs in its sequence. Using a bioluminescence resonance energy transfer (BRET)-based subcellular localization system, we found that PRAF2 inhibits, in a concentration-dependent manner, the plasma membrane export of CCR5. BRET-based proximity assays and Co-IP experiments demonstrated that PRAF2/CCR5 interaction does not require the presence of a receptor carboxyterminal tail and involves instead the transmembrane domains of both proteins. The mutation of the potential di-leucine/RXR motif contained in the third intracellular loop of CCR5 does not affect PRAF2-mediated retention. It instead impairs the cell-surface export of CCR5 by inhibiting CCR5's interaction with its private escort protein, CD4. PRAF2 and CD4 thus display opposite roles on the cell-surface export of CCR5, with PRAF2 inhibiting and CD4 promoting this process, likely operating at the level of CCR5 recruitment into COPII vesicles, which leave the ER.

Amino acid substitutions in norovirus VP1 dictate host dissemination via variations in cellular attachment.

All viruses initiate infection by utilizing receptors to attach to target host cells. These virus-receptor interactions can therefore dictate viral replication and pathogenesis. Understanding the nature of virus-receptor interactions could also be important for the development of novel therapies. Noroviruses are non-enveloped icosahedral viruses of medical importance. They are a common cause of acute gastroenteritis with no approved vaccine or therapy and are a tractable model for studying fundamental virus biology. In this study, we utilized the murine norovirus model system to show that variation in a single amino acid of the major capsid protein alone can affect viral infectivity through improved attachment to suspension cells. Modulating plasma membrane mobility reduced infectivity, suggesting an importance of membrane mobility for receptor recruitment and/or receptor conformation. Furthermore, different substitutions at this site altered viral tissue distribution in a murine model, illustrating how in-host capsid evolution could influence viral infectivity and/or immune evasion.

Effects of junk-food on food-motivated behavior and nucleus accumbens glutamate plasticity; insights into the mechanism of calcium-permeable AMPA receptor recruitment

In rats, eating obesogenic diets increases calcium-permeable AMPA receptor (CP-AMPAR) transmission in the nucleus accumbens (NAc) core, and enhances food-motivated behavior. Interestingly, these diet-induced alterations in NAc transmission are pronounced and sustained in obesity-prone (OP) male rats and absent in obesity-resistant (OR) populations. However, effects of diet manipulation on food motivation, and the mechanisms underlying this NAc plasticity in OPs is unknown. Using male selectively-bred OP and OR rats, we assessed food-motivated behavior following ad lib access to chow (CH), junk-food (JF), or 10d of JF followed by a return to chow diet (JF-Dep). Motivation for food was greater in OP than OR rats, as expected. However, JF-Dep only produced enhancements in food-seeking in OP groups, while continuous JF access reduced food-seeking in both OPs and ORs. Additionally, optogenetic, chemogenetic, and pharmacological approaches were used to examine NAc CP-AMPAR recruitment following diet manipulation and ex vivo treatment of brain slices. Reducing excitatory transmission in the NAc was sufficient to recruit CP-AMPARs to synapses in OPs, but not ORs. In OPs, JF-induced increases in CP-AMPARs occurred in mPFC-, but not BLA-to-NAc inputs. Together results show that diet differentially affects behavioral and neural plasticity in obesity susceptible populations. We also identify conditions for acute recruitment of NAc CP-AMPARs; these results suggest that synaptic scaling mechanisms contribute to NAc CP-AMPAR recruitment. Overall, this work helps elucidate how diet interacts with obesity susceptibility to influence food-motivated behavior and extends our fundamental understanding of NAc CP-AMPAR recruitment.