Small-molecule-induced polymerization triggers degradation of BCL6.

Effective and sustained inhibition of non-enzymatic oncogenic driver proteins is a major pharmacological challenge. The clinical success of thalidomide analogues demonstrates the therapeutic efficacy of drug-induced degradation of transcription factors and other cancer targets1-3, but a substantial subset of proteins are resistant to targeted degradation using existing approaches4,5. Here we report an alternative mechanism of targeted protein degradation, in which a small molecule induces the highly specific, reversible polymerization of a target protein, followed by its sequestration into cellular foci and subsequent degradation. BI-3802 is a small molecule that binds to the Broad-complex, Tramtrack and Bric-à-brac (BTB) domain of the oncogenic transcription factor B cell lymphoma 6 (BCL6) and leads to the proteasomal degradation of BCL66. We use cryo-electron microscopy to reveal how the solvent-exposed moiety of a BCL6-binding molecule contributes to a composite ligand-protein surface that engages BCL6 homodimers to form a supramolecular structure. Drug-induced formation of BCL6 filaments facilitates ubiquitination by the SIAH1 E3 ubiquitin ligase. Our findings demonstrate that a small molecule such as BI-3802 can induce polymerization coupled to highly specific protein degradation, which in the case of BCL6 leads to increased pharmacological activity compared to the effects induced by other BCL6 inhibitors. These findings open new avenues for the development of therapeutic agents and synthetic biology.

Noble gas dative bonding with coinage metal carbene complexes: A theoretical study.

The structure and stability of noble gas (Ng) bound [NHCM]+ complexes (M = Cu, Ag, and Au) were investigated using Quantum chemical calculations. Dissociation energies, enthalpy, and free energy changes were computed to comprehend the stability of these Ng-bonded complexes. The nature of interactions associated to the bonding between metal and noble gas atoms was studied through the computation of electron density-based descriptors. Detailed electronic structure study revealed electron donation from the noble gas atoms towards the metal center, resulting in the formation of dative bonds.

Coordination of noble gases by alkali-metal-like superatom cation Be3 B.

Quantum chemical calculations were carried out to investigate the noble gas binding ability of Be3 B+ cluster. Calculations reveal that heavier noble gas atoms (ArXe) form stable complexes with this cluster. Detailed bonding analyses reveal that the noble gas atoms act as donor fragment in the formation of Ng → Be donor-acceptor bonds. Three noble gas atoms can consecutively form bonds with the Be atom of the Be3 B+ cluster.

Beryllium bonding with noble gas atoms.

Quantum chemical calculations were carried out to investigate the nature of the bonding between a neutral Be3 ring and noble gas atom. Electronic structure calculation for these complexes was carried out at different computational levels in association with natural bond orbital, quantum theory of atoms in molecules, electron localization function, symmetry adapted perturbation theory, and molecular electrostatic potential surface analysis of Be3 complexes. The Be atoms in the Be3 moiety are chemically bonded to one another, with the BeBe bond dissociation energy being ~125 kJ mol-1 . The Be3 ring interacts with the noble gases through non-covalent interactions. The binding energies of the noble gas atoms with the Be3 ring increases with increase in their atomic number. The non-covalent interaction index, density overlap region indicator and independent gradient model analyses reveal the presence of non-covalent inter-fragment interactions in the complexes. Energy decomposition analysis reveals that dispersion plays the major role towards stabilizing these systems.

Solvent Effect on the Photoinduced Intramolecular Charge-Transfer and Redox Potentials of Three Difuranones.

We report solvent-dependent excited state properties of three difuranone derivatives with a quinoidal backbone by steady-state and lifetime fluorescence measurements and theoretical calculations. Remarkable bathochromic shifts in fluorescence with diminished intensity indicate the occurrence of strong intramolecular charge-transfer transitions in high polar solvents. Cyclic voltammetric redox potentials reveal an interesting variation of biradical characters of the compounds with increasing solvent polarity. Solvent polarity also significantly modulates the energy levels of the charge-transfer (CT) states, as observed from the combined analyses of redox potentials and photophysical data via the Rehm-Weller equation. When high polar solvents favor forward CT by a more exoergic driving force and stabilize the charge-separated states, the reverse CT process diminishes. Estimated free energies of activation for CT suggest that high polar solvents lessen the activation barrier. Calculated excited state energies of the compounds at the CAM-B3LYP/6-31+G* level fulfill the primary conditions required for singlet fission, a process that can substantially increase the efficiency of solar cells, and the crystal packing for compound 1 also reveals a favorable geometry for singlet fission.

Harlequin syndrome.

A 56-year-old man presented to the clinic with episodic headaches for several years which had been worsening over a few months prior to the presentation. He described headache as sharp, stabbing pain around the left eye associated with nausea, vomiting, photophobia, and phonophobia lasting for hours associated with flushing on the left side of the face. The picture of his face during these episodes showed flushing of the left side of the face, ptosis of the right eyelid, and miosis (panel A). Flushing in his face would resolve with the abortion of the headache. At the time of presentation to the clinic, his neurological exam was only significant for mild left eye ptosis and miosis (panels B and C). Extensive workup including MRI brain, cervical spine, thoracic spine, lumbar spine, CTA head and neck, and CT maxillofacial was unremarkable. He had tried several medications in the past including valproic acid, nortriptyline, and verapamil without significant benefit. He was started on erenumab for migraine prophylaxis and was given sumatriptan for abortive therapy following which his headaches improved. The patient was diagnosed with idiopathic left Horner's syndrome and his migraines with autonomic dysfunction would present with unilateral flushing opposite to the site of Horner's presenting as Harlequin syndrome [1, 2].

A prospective comparative study of mini-PCNL using Trilogy™ or thulium fibre laser with suction.

INTRODUCTION: There has been recent introduction of novel lithotripters and high-power lasers for stone disintegration. With miniaturization of PCNL, there is need of effective disintegration and faster stone-clearance. This study aimed to evaluate efficiency of Trilogy™ and Thulium fibre laser (TFL) in mini-percutaneous nephrolithotomy (mini-PCNL). METHODS: This is prospective study comparing efficiency and outcomes of Trilogy™ and TFL in mini-PCNL between January 2019 and February 2020. Primary objective was to compare stone fragmentation rates, with secondary objectives beings stone-free rates and complications. RESULTS: There were 60 mini-PCNL with suction using either Trilogy™ or TFL energy source. Mean stone size and density were 27.60 ± 10.17 mm, 22.04 ± 9.69 mm (p = 0.05) and 1172.9 ± 313.5HU, 1308.9 ± 333.9HU (p = 0.10) for Trilogy™ and TFL, respectively. Using 3D doctor imaging software from CT images, mean stone volumes were 3718.9 ± 3038.7mm3 for Trilogy™ and 3425.9 ± 3096.1mm3 for TFL(p = 0.77). Using probe-activation time or lasing time, stone-fragmentation rate was 5.98 ± 4.25mm3/sec for Trilogy™ and 3.95 ± 1.00mm3/sec for TFL(p = 0.015). Treatment time (puncture to complete clearance) was 32.48 ± 15.39 min for Trilogy™ and 28.63 ± 18.56 min for TFL(p = 0.38). Haemoglobin drop was 1.19 ± 0.76gm/dl for Trilogy™ and 0.99 ± 0.74gm/dl for TFL (p = 0.30). Trilogy™ arm had 96.6% complete clearance and TFL had 76.6% in TFL at 48 h. One patient in Trilogy™ arm required auxiliary RIRS for residual stone. Both arms had complete stone clearance at 1 month follow-up. Trilogy™ arm had 3 Clavien-Dindo grade-II complications while TFL had 2 Clavien-Dindo grade-II complications (UTI requiring antibiotics). There was no blood transfusion in either of arm. CONCLUSION: Trilogy™ had significantly better stone fragmentation rate than TFL in managing renal stones. However, stone-free rates and complications were comparable for Trilogy™ and TFL.

Autoimmune encephalitis and seizures, cerebrospinal fluid, imaging, and EEG findings: a case series.

Antibody-mediated encephalitides constitute a group of inflammatory brain diseases characterized by prominent neuropsychiatric symptoms and are associated with antibodies against neuronal cell-surface proteins, ion channels, or receptors. The diagnosis and management of autoimmune encephalitis include evaluation of the clinical presentation, brain imaging, cerebrospinal fluid (CSF) findings, antibody detection, and electroencephalography (EEG) findings. This is a retrospective study of adults 18 years or older with autoimmune encephalitis due to antibodies against membrane surface antigens as well as anti-glutamic acid decarboxylase (anti-GAD) antibodies. The electronic medical record was reviewed for demographic data, clinical data, laboratory results, EEG, and imaging findings. Antibody screening was requested for 341 patients between May 2014 and December 2019. Antibody screening was positive in 37 patients presenting with seizures and/or encephalopathy. Of these, 10 patients tested positive for antibodies against neuronal surface antigens or anti-GAD antibodies-2 patients had anti-GAD antibody encephalitis, 5 had anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis, and 3 had anti-leucine-rich glioma-inactivated 1 (anti-LGI1) encephalitis. Demographics, clinical presentation, EEG, imaging, and CSF findings are reported. Autoimmune encephalitides are a diverse group of disorders with a few common clinical features and MRI findings. MRI, EEG, and CSF findings can be normal or show nonspecific findings in autoimmune encephalitis. Therefore, early diagnosis of these disorders requires a high level of suspicion to avoid delaying the diagnosis. Carefully looking for diagnostic clinical features (e.g., faciobrachial dystonic seizures in anti-LGI1 encephalitis), significant findings in MRI (e.g., limbic encephalitis), and some EEG patterns (e.g., extreme delta brush and generalized rhythmic delta activity in anti-NMDAR encephalitis) may help in early diagnosis.

The post-synaptic scaffolding protein tamalin regulates ligand-mediated trafficking of metabotropic glutamate receptors.

Group I metabotropic glutamate receptors (mGluRs) play important roles in various neuronal functions and have also been implicated in multiple neuropsychiatric disorders like fragile X syndrome, autism, and others. mGluR trafficking not only plays important roles in controlling the spatiotemporal localization of these receptors in the cell but also regulates the activity of these receptors. Despite this obvious significance, the cellular machineries that control the trafficking of group I metabotropic glutamate receptors in the central nervous system have not been studied in detail. The post-synaptic scaffolding protein tamalin has been shown to interact with group I mGluRs and also with many other proteins involved in protein trafficking in neurons. Using a molecular replacement approach in mouse hippocampal neurons, we show here that tamalin plays a critical role in the ligand-dependent internalization of mGluR1 and mGluR5, members of the group I mGluR family. Specifically, knockdown of endogenous tamalin inhibited the ligand-dependent internalization of these two receptors. Both N-terminal and C-terminal regions of tamalin played critical roles in mGluR1 endocytosis. Furthermore, we found that tamalin regulates mGluR1 internalization by interacting with S-SCAM, a protein that has been implicated in vesicular trafficking. Finally, we demonstrate that tamalin plays a critical role in mGluR-mediated internalization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, a process believed to be the cellular correlate for mGluR-dependent synaptic plasticity. Taken together, these findings reveal a mechanistic role of tamalin in the trafficking of group I mGluRs and suggest its physiological implications in the brain.

Hybrid cytokine IL233 renders protection in murine acute graft vs host disease (aGVHD).

Previously, we generated IL233, a hybrid cytokine composed of interleukin (IL)-2 and IL-33, with better therapeutic potential than either cytokine in multiple inflammatory diseases, in part through promoting T-regulatory cells (Tregs). Here we test the potential of IL233 pretreatment in a murine model of excessive Th1 activation, the parent-into-F1 model of acute GVHD (aGVHD). Five days of IL233 pretreatment of the recipients blocked or delayed the aGVHD-linked loss of B cells as seen in either the peripheral blood (day-11) or lymph nodes (day-14). IL233 pretreatment also prevented the expansion of donor CD8 T-cells in blood and LN at day-14 and significantly reduced day-14 serum IFNγ and TNFα compared to saline treated GVHD mice although, the level of Tregs did not statistically differ between saline and IL233-treated mice. Overall, the current study provides support for the use of IL233 as a therapeutic option in excessive Th1/CD8-driven conditions.

Patterns of substrate affinity, competition, and degradation kinetics underlie biological activity of thalidomide analogs.

Pharmacologic agents that modulate ubiquitin ligase activity to induce protein degradation are a major new class of therapeutic agents, active in a number of hematologic malignancies. However, we currently have a limited understanding of the determinants of activity of these agents and how resistance develops. We developed and used a novel quantitative, targeted mass spectrometry (MS) assay to determine the relative activities, kinetics, and cell-type specificity of thalidomide and 4 analogs, all but 1 of which are in clinical use or clinical trials for hematologic malignancies. Thalidomide analogs bind the CRL4CRBN ubiquitin ligase and induce degradation of particular proteins, but each of the molecules studied has distinct patterns of substrate specificity that likely underlie the clinical activity and toxicities of each drug. Our results demonstrate that the activity of molecules that induce protein degradation depends on the strength of ligase-substrate interaction in the presence of drug, the levels of the ubiquitin ligase, and the expression level of competing substrates. These findings highlight a novel mechanism of resistance to this class of drugs mediated by competition between substrates for access to a limiting pool of the ubiquitin ligase. We demonstrate that increased expression of a nonessential substrate can lead to decreased degradation of other substrates that are critical for antineoplastic activity of the drug, resulting in drug resistance. These studies provide general rules that govern drug-dependent substrate degradation and key differences between thalidomide analog activity in vitro and in vivo.

Isolated primary neurolymphomatosis with cranial multineuritis: a case presentation.

BACKGROUND: Isolated primary neurolymphomatosis (NL) of cranial multineuritis is a very rare condition that refers to the lymphomatous invasion of cranial nerves only. There are sparse cases of isolated cranial nerves NL reported worldwide. CASE PRESENTATION: We present magnetic resonance imaging (MRI) and cerebrospinal fluid (CSF) findings of a 63-year-old female patient suffering from isolated neurolymphomatosis of cranial multineuritis with a wide constellation of syndromes including binocular diplopia, left facial paralysis and pain, syncope episodes, and progressive dysphagia. A contrasted MRI brain showed multiple cranial nerves enhancement. Extensive workup for infectious, autoimmune, neoplastic, paraneoplastic, or inflammatory etiologies had been unrevealing except CSF cytology revealed large atypical monotypic B cells that were suspicious for non-Hodgkin lymphoma on the third large volume tap. The decision of biopsy was deferred after the risks and benefits discussion. Following the four cycles of empiric methotrexate-based induction chemotherapy, the patient's symptoms resolved, and a complete radiographic response was achieved without whole-brain radiation or autologous hematopoietic cell transplantation. In the latest follow-up, she is independent with her daily activities and remains in clinical and radiographic remission more than 3 years since initial chemotherapy. CONCLUSION: Isolated NL of cranial nerves can present diagnostic and management pitfalls for the neurologist, neurosurgeons, and oncologists. Since current diagnostic modalities have modest sensitivity and a pathological diagnosis is often difficult, empiric treatment once other possibilities are ruled out can carry a good prognosis.

Spectrum of neuropsychiatric manifestations in COVID-19.

Corona Virus Disease 2019 (COVID-19) emerged in December 2019 from Wuhan, China. It typically presents with mild upper respiratory tract infection symptoms and may have life threatening complications, including acute respiratory distress syndrome, acute stroke, myocardial infarction, kidney failure, shock, and even death. Coronavirus infections are known to have neuroinvasive potential with consequent neuropsychiatric manifestations. We analyzed COVID-19 adult patients in the TriNetX database, which is a global health collaborative clinical research platform collecting real-time electronic medical records data from a network of health care organizations (HCOs) from January 20, 2020 to June 10th, 2020. 40,469 patients were diagnosed with COVID-19 among whom 9086 (22.5%) patients had neuropsychiatric manifestations. The most common neurologic manifestations included headache (3.7%) and sleep disorders (3.4%), Encephalopathy (2.3%), Stroke and transient ischemic attack (TIA) (1.0%) and 0.6% had seizures. Most common psychiatric manifestations included anxiety and other related disorders (4.6%), mood disorders (3.8%), while 0.2% patients had suicidal ideation. Early recognition and prompt management of neuropsychiatric manifestations in these patients have a potential to decrease overall morbidity and mortality.

EMS Lithoclast Trilogy™: an effective single-probe dual-energy lithotripter for mini and standard PCNL.

PURPOSE: Several energy sources are available to clear stones during PCNL. Required improvements are faster stone clearance, optimized suction and ease of use while maintaining high patient safety standard. EMS LithoClast® Trilogy, is the first device combining electromagnetic impactor with ultrasonic energy and suction, all-in-one probe. Animal studies and in vitro phantom stone studies have proven safety and efficacy of this device. We aim to study safety and clinical efficacy of Trilogy in our patients. METHODS: 31 patients with renal stones were included. Amplatz sheath sizes/Trilogy probe size was 22-28 Fr/10.2 Fr for standard PNL (n = 20) and 15 Fr./5.7 Fr for mini PNL access (n = 11). Analysis was done with respect to demography, stone characteristics, operation duration (total time and lithotripter activation time), post op Hb drop, clearance rate and adverse events. Stone area/volume was calculated based on CT using 3D doctor. Efficacy was determined by stone volume clearance rate (mm3/min). RESULTS: Male:female ratio was 6:5 and 16:4 for mini (MPNL)/standard PNL (SPNL). Stone densities were 1229 ± 206 vs. 1168 ± 344 HU (MPNL vs. SPNL). Mean stone volumes were 3776.1 ± 2132 mm3 for MPNL and 7096 ± 6441 for SPNL. Mean stone volume clearance ratios were 370.5 ± 171 mm3/min and 590.7 ± 250mm3/min for MPNL and SPNL, respectively. Hb drop was 1.24 ± 0.64 g/dL (MPNL) and 1.23 ± 0.89gm/dL (SPNL). Total procedure time/lithotripter activation time was 53.4 ± 23.8/14.7 ± 12.4 min for MPNL and 65.2 ± 23.5/12.0 ± 8.9 for SPNL. Immediate post-operative/1 month stone clearance rates were 93%/96% with one clinically insignificant residual fragment (< 3 mm) and no necessity for auxiliary procedures. No device failure occured and three Clavien grade I and one grade II complications were observed. CONCLUSION: Swiss LithoClast® Trilogy provides fast stone clearance in standard/mini-PCNL procedures. Ease of use, high tissue safety and optimized suction that avoids fragment blockings are other key features.

A Critical Role for Sorting Nexin 1 in the Trafficking of Metabotropic Glutamate Receptors.

Group I metabotropic glutamate receptors (mGluRs) function as modulators of neuronal physiology and they have also been implicated in various neuropsychiatric disorders. Trafficking of mGluRs plays important roles in controlling the precise localization of these receptors at specific region of the cell, as well as it regulates the activity of these receptors. Despite this obvious significance, we know very little about the cellular machineries that control the trafficking of these receptors in the CNS. Sorting nexin 1 (SNX1) has been shown to regulate the endosomal sorting of few cell surface receptors either to lysosomes where they are downregulated or back to the cell surface. Using "molecular replacement" approach in hippocampal neurons derived from mice of both sexes, we show here that SNX1 plays critical role in the trafficking of mGluR1, a member of the group I mGluR family. Overexpression of dominant-negative SNX1 or knockdown of endogenous SNX1 resulted in the rapid recycling of the receptor. Importantly, recycling via the rapid recycling route, did not allow the resensitization of the receptors. Our data suggest that both, N-terminal and C-terminal region of SNX1 play critical role in the normal trafficking of the receptor. In addition, we also show here that SNX1 regulates the trafficking of mGluR1 through the interaction with Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate), a protein that has been implicated in both signaling and vesicular trafficking. Thus, these studies reveal a mechanistic role of SNX1 in the trafficking of group I mGluRs and its physiological implications.SIGNIFICANCE STATEMENT Group I mGluRs are activated by the neurotransmitter glutamate in the CNS, and play various important roles in the brain. Similar to many other receptors, trafficking plays crucial roles in controlling the precise localization as well as activity of these receptors. Despite this obvious significance very little is known about the cellular machineries that control the trafficking of these receptors. We demonstrate here, that SNX1 plays a critical role in the trafficking of mGluR1, a member of the group I mGluR family. SNX1-mediated trafficking is critical for the resensitization of the receptor. SNX1 controls the trafficking of the receptor through the interaction with another protein, Hrs. The results suggest a role for SNX1 in the regulation of group I mGluRs.

Dodecafluoropentane Emulsion in Acute Ischemic Stroke: A Phase Ib/II Randomized and Controlled Dose-Escalation Trial.

PURPOSE: This randomized, placebo-controlled, double-blind, dose-escalation acute ischemic stroke trial was designed to demonstrate maximum tolerated dose, characterize adverse events (AEs), and explore clinical outcomes when intravenous dodecafluoropentane emulsion (DDFPe) was used as neuroprotection. METHODS: Acute ischemic stroke patients (n = 24) with National Institutes of Health Stroke Scale (NIHSS) score of 2-20 were randomized to either 3 doses of intravenous DDFPe or placebo, 1 every 90 minutes, starting within 12 hours of symptom onset. Doses were given without affecting standard stroke care. Each of the 3 dose cohorts included 8 patients, with 2 receiving placebo and 6 receiving DDFPe. Primary outcomes were serious adverse events (SAEs), AEs, NIHSS score, and modified Rankin Score (mRS). RESULTS: No dose-limiting toxicities were encountered, and no maximum tolerated dose was defined. One unrelated delayed death occurred in a DDFPe patient, and another occurred in the placebo group. Group SAEs and AEs were similar in incidence and severity. Early initiation of DDFPe treatment resulted in better NIHSS score response than late initiation (P = .03). Thirty- and 90-day mRS after high-dose therapy suggested clinical improvement (P = .01 and P = .03, respectively). However, the significance of differences in clinical outcomes was limited by small patient numbers and differences in stroke severity between cohorts. CONCLUSIONS: Intravenous DDFPe appears to be safe at all doses tested. Clinical improvements in NIHSS score and mRS were significant but compromised by small sample size.

Altered lipid metabolism in post-traumatic epileptic rat model: one proposed pathway.

Post-traumatic epilepsy (PTE) is a common long-term risk associated with traumatic brain injury (TBI). PTE rat model, proposed by Willmore et al., is a well known model that mimics human PTE. The present study explored the lipid metabolism in this PTE rat model by using in vitro, high-resolution NMR (nuclear magnetic resonance) spectroscopy and lipid staining based investigations. The level of gene expression, cytokines and enzyme activity was estimated. Level of TG (triglycerides), PL (phospholipids) and CHOL (cholesterol) was found to increase in brain tissue of PTE rats. This is an indication of the altered lipid metabolism in PTE rats. Level of lipid peroxidation and cytokines was enhanced in the brain tissue of PTE rats. A positive correlation was also observed in cytokines vs. lipid peroxidation. These results make available the evidence of the oxidative stress induced damage or destruction of the lipid components and also the cause of the inflammatory events in PTE rats. Antioxidant enzyme activity and respective gene expression were found to increase in brain tissue of PTE rats. A positive correlation was also observed in antioxidant enzyme's activity vs. respective enzyme gene expression and lipid peroxidation vs. activity of antioxidant enzymes. Such outcomes reflect the oxidative stress induced lipid damage responsible for production enhancement of antioxidant enzymes, which further responsible for enhancing the activity of antioxidant enzymes. A positive correlation was observed in lipid peroxidation vs. lipid components (TG, PL and CHOL) and provides the confirmatory verification of alteration in the level of lipid components. A negative correlation was observed in the level of cytokines and the quantity of TG. This showed that TG is consumed in the production of cytokines. MUA (Motor unit activity) is highly correlated with the level of LP and indicated that oxidative stress is responsible for the event of epileptogenesis. Positive correlation of MUA with RA (rearing activity) and MWM (Morris-water maze) showed that epileptogenesis also influences the memory of PTE rats. Overall results based analyses clearly indicate that the inflammatory activity and oxidative stress in brain tissue of PTE rats, which are responsible to establish a significant change in the lipid metabolism. This can be visualized through a well constructed possible pathway of altered lipid metabolism. This study will improve our understanding and approach in the field of epilepsy that need to be considered for the development of new drugs or therapy for patients with PTE. Representation of the proposed pathway of altered lipid metabolism in posttraumatic epileptic rats.

Revisiting the role of cholesterol in regulating the pore-formation mechanism of Vibrio cholerae cytolysin, a membrane-damaging ß-barrel pore-forming toxin.

Vibrio cholerae cytolysin (VCC) is a ß-barrel pore-forming toxin with potent membrane-damaging cell-killing activity. Previous studies employing the model membranes of lipid vesicles (liposomes) have shown that pore formation by VCC requires the presence of cholesterol in the liposome membranes. However, the exact role of cholesterol in the mode of action of VCC still remains unclear. Most importantly, implication of cholesterol, if any, in regulating the pore-formation mechanism of VCC in the biomembranes of eukaryotic cells remains unexplored. Here, we show that the presence of cholesterol promotes the interaction of VCC with the membrane lipid bilayer, when non-lipid-dependent interactions are absent. However, in the case of biomembranes of human erythrocytes, where accessory interactions are available, cholesterol appears to play a less critical role in the binding step. Nevertheless, in the absence of an optimal level of membrane cholesterol in the human erythrocytes, membrane-bound fraction of the toxin remains trapped in the form of abortive oligomeric assembly, devoid of functional pore-forming activity. Our study also shows that VCC exhibits a prominent propensity to associate with the cholesterol-rich membrane micro-domains of human erythrocytes. Interestingly, mutation of the cholesterol-binding ability of VCC does not block association with the cholesterol-rich membrane micro-domains on human erythrocytes. Based on these results, we propose that the specific cholesterol-binding ability of VCC does not appear to dictate its association with the cholesterol-rich micro-domains on human erythrocytes. Rather, targeting of VCC toward the membrane micro-domains of human erythrocytes possibly acts to facilitate the cholesterol-dependent pore-formation mechanism of the toxin.

A Critical Role for Ubiquitination in the Endocytosis of Glutamate Receptors.

Group I metabotropic glutamate receptors (mGluRs) play important roles in various neuronal processes and elicit changes in synaptic efficacy through AMPA receptor (AMPAR) endocytosis. Trafficking of mGluRs plays an important role in controlling the precise localization of these receptors at specific region of the cell; it also regulates the activity of these receptors. Despite this obvious significance, we know very little about the cellular mechanisms that control the trafficking of group I mGluRs. We show here that ligand-mediated internalization of group I mGluRs is ubiquitination-dependent. A lysine residue (Lys1112) at the C-terminal tail of mGluR1 (a member of the group I mGluR family) plays crucial role in this process. Our data suggest that Lys63-linked polyubiquitination is involved in the ligand-mediated endocytosis of mGluR1. We also show here that the mGluR1 internalization is dependent on a specific E3 ubiquitin ligase, Siah-1A. Furthermore, acute knockdown of Siah-1A enhances the mGluR-mediated AMPAR endocytosis. These studies reveal a novel function of ubiquitination in the regulation of group I mGluRs, as well as its role in mGluR-dependent AMPAR endocytosis.