ATG16L1 induces the formation of phagophore-like membrane cups.

The hallmark of non-selective autophagy is the formation of cup-shaped phagophores that capture bulk cytoplasm. The process is accompanied by the conjugation of LC3B to phagophores by an E3 ligase complex comprising ATG12-ATG5 and ATG16L1. Here we combined two complementary reconstitution approaches to reveal the function of LC3B and its ligase complex during phagophore expansion. We found that LC3B forms together with ATG12-ATG5-ATG16L1 a membrane coat that remodels flat membranes into cups that closely resemble phagophores. Mechanistically, we revealed that cup formation strictly depends on a close collaboration between LC3B and ATG16L1. Moreover, only LC3B, but no other member of the ATG8 protein family, promotes cup formation. ATG16L1 truncates that lacked the C-terminal membrane binding domain catalyzed LC3B lipidation but failed to assemble coats, did not promote cup formation and inhibited the biogenesis of non-selective autophagosomes. Our results thus demonstrate that ATG16L1 and LC3B induce and stabilize the characteristic cup-like shape of phagophores.

Membrane insertion mechanism of the caveola coat protein Cavin1.

Caveolae are small plasma membrane invaginations, important for control of membrane tension, signaling cascades, and lipid sorting. The caveola coat protein Cavin1 is essential for shaping such high curvature membrane structures. Yet, a mechanistic understanding of how Cavin1 assembles at the membrane interface is lacking. Here, we used model membranes combined with biophysical dissection and computational modeling to show that Cavin1 inserts into membranes. We establish that initial phosphatidylinositol (4, 5) bisphosphate [PI(4,5)P2]-dependent membrane adsorption of the trimeric helical region 1 (HR1) of Cavin1 mediates the subsequent partial separation and membrane insertion of the individual helices. Insertion kinetics of HR1 is further enhanced by the presence of flanking negatively charged disordered regions, which was found important for the coassembly of Cavin1 with Caveolin1 in living cells. We propose that this intricate mechanism potentiates membrane curvature generation and facilitates dynamic rounds of assembly and disassembly of Cavin1 at the membrane.

Efficacy and Safety of Acotiamide Versus Mosapride in Patients With Functional Dyspepsia Associated With Meal-Induced Postprandial Distress Syndrome: A Phase III Randomized Clinical Trial.

BACKGROUND:  Acotiamide is a novel prokinetic drug that acts by enhancing the release of acetylcholine and is used in the treatment of functional dyspepsia-postprandial distress syndrome (FD-PDS). Mosapride is indicated to FD-PDS as per the Rome III treatment guidelines. Mosapride 5 mg three times daily (TID) is approved by the Drugs Controller General of India (DCGI) for the treatment of FD-PDS. The objective of this study was to determine the efficacy and safety of Acotiamide in comparison with Mosapride on FD-PDS. METHODS: The 220 patients of either gender (aged 18-64 years) with active PDS included in the study were centrally randomized 1:1 to receive either 100 mg Acotiamide (test product) or 5 mg Mosapride (reference product) TID for four weeks. Responder rates for the overall treatment effect (OTE) at the end of four weeks were the primary efficacy endpoint. Secondary efficacy endpoints included the elimination rate of postprandial fullness, upper abdominal bloating, and early satiation. The study also evaluated the OTE at each week, individual symptom scores, and quality of life (QoL) assessed by the Short Form-Nepean Dyspepsia Index questionnaire (SF-NDI). The safety endpoints included assessments of treatment-emergent adverse events (TEAEs). RESULTS:  At the end of four weeks, the responders in the Acotiamide versus Mosapride group for OTE was 98% versus 93.27% in the per-protocol (PP) population. Among the intent to treat (ITT) population, the comparison of Acotiamide versus Mosapride stood at 95.15% versus 89.81%. Secondary efficacy endpoints were significantly improved with 100 mg TID Acotiamide, which was evident from the improvement in postprandial fullness (14.56%), upper abdominal bloating (15.53%), early satiation (10.68%), and QoL (13.7 ± 4.67). CONCLUSIONS:  Our study results demonstrated that Acotiamide is effective, safe, and well-tolerated and had significantly improved the QoL over a four-week treatment period in FD-PDS patients. The efficacy and safety profiles of Acotiamide were similar to Mosapride.

Membrane remodeling by SARS-CoV-2 - double-enveloped viral replication.

The ongoing pandemic of the new severe acute respiratory syndrome coronavirus (SARS-CoV-2) has caused more than one million deaths, overwhelmed many public health systems, and led to a worldwide economic recession. This has raised an unprecedented need to develop antiviral drugs and vaccines, which requires profound knowledge of the fundamental pathology of the virus, including its entry, replication, and release from host cells. The genome of coronaviruses comprises around 30 kb of positive single-stranded RNA, representing one of the largest RNA genomes of viruses. The 5' part of the genome encodes a large polyprotein, PP1ab, which gives rise to 16 non-structural proteins (nsp1- nsp16). Two proteases encoded in nsp3 and nsp5 cleave the polyprotein into individual proteins. Most nsps belong to the viral replicase complex that promotes replication of the viral genome and translation of structural proteins by producing subgenomic mRNAs. The replicase complexes are found on double-membrane vesicles (DMVs) that contain viral double-stranded RNA. Expression of a small subset of viral proteins, including nsp3 and nsp4, is sufficient to induce formation of these DMVs in human cells, suggesting that both proteins deform host membranes into such structures. We will discuss the formation of DMVs and provide an overview of other membrane remodeling processes that are induced by coronaviruses.

The Chlamydia effector CT622/TaiP targets a nonautophagy related function of ATG16L1.

The obligate intracellular bacteria Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted diseases, multiply in a vacuolar compartment, the inclusion. From this niche, they secrete "effector" proteins, that modify cellular activities to enable bacterial survival and proliferation. Here, we show that the host autophagy-related protein 16-1 (ATG16L1) restricts inclusion growth and that this effect is counteracted by the secretion of the bacterial effector CT622/TaiP (translocated ATG16L1 interacting protein). ATG16L1 is mostly known for its role in the lipidation of the human homologs of ATG8 (i.e., LC3 and homologs) on double membranes during autophagy as well as on single membranes during LC3-associated phagocytosis and other LC3-lipidation events. Unexpectedly, the LC3-lipidation-related functions of ATG16L1 are not required for restricting inclusion development. We show that the carboxyl-terminal domain of TaiP exposes a mimic of an eukaryotic ATG16L1-binding motif that binds to ATG16L1's WD40 domain. By doing so, TaiP prevents ATG16L1 interaction with the integral membrane protein TMEM59 and allows the rerouting of Rab6-positive compartments toward the inclusion. The discovery that one bacterial effector evolved to target ATG16L1's engagement in intracellular traffic rather than in LC3 lipidation brings this "secondary" activity of ATG16L1 in full light and emphasizes its importance for maintaining host cell homeostasis.

Human ubiquitin-like proteins as central coordinators in autophagy.

Autophagy is one of the most versatile recycling systems of eukaryotic cells. It degrades diverse cytoplasmic components such as organelles, protein aggregates, ribosomes and multi-enzyme complexes. Not surprisingly, any failure of autophagy or reduced activity of the pathway contributes to the onset of various pathologies, including neurodegeneration, cancer and metabolic disorders such as diabetes or immune diseases. Furthermore, autophagy contributes to the innate immune response and combats bacterial or viral pathogens. The hallmark of macroautophagy is the formation of a membrane sack that sequesters cytoplasmic cargo and delivers it to lysosomes for degradation. More than 40 autophagy-related (ATG) proteins have so far been identified. A unique protein-conjugation system represents one of the core components of this highly elaborate machinery. It conjugates six homologous ATG8 family proteins to the autophagic membrane. In this review, we summarize the current knowledge regarding the various functions of ATG8 proteins in autophagy and briefly discuss how physical approaches and in vitro reconstitution contributed in deciphering their function.

BIOAVAILABILITY OF BIOTRANSFORMED ZINC ENRICHED DAHI IN WISTAR RATS.

In the present study, we have investigated the bioavailability of biotransformed organic zinc enriched dahi in vivo. The results evidence that the rats fed with zinc enriched dahi (ZED and ZEP) significantly increased (p<0.001) body weight and food intake from zero to third weeks. Analysis of zinc by AAS in body parts of different rat groups indicated that zinc content was significantly higher (p<0.001) in serum, femur bone, liver and hair of rats fed ZED/ZEP. Basal diet and inorganic zinc sulphate fed rat group excreted a greater amount of zinc in faeces. The results of in-vivo studies indicated that the bioavailability of organic zinc through dahi/probiotic dahi is high compared to its inorganic form.

A retrospective analysis comparing APCEDEN® dendritic cell immunotherapy with best supportive care in refractory cancer.

AIM: A retrospective survival benefit analysis of APCEDEN®, APAC BIOTECH Pvt Ltd 69, Jacranda Marg, DLF PHASE II, Gurugram, Haryana, India, an autologous dendritic cell-based product for management of refractory solid malignancies, was performed in comparison with a control group. METHODS: Subjects (retrospective data) whose survival data, geographical region, age, gender, ECOG performance status and stage of disease that could be matched with the treatment group were considered for analysis. RESULTS: The analysis suggests a significant survival benefit of 199 days for the APCEDEN therapy treatment group when compared with the control group (356 vs 157 days). The event-free survival time of APCEDEN therapy was 439 days in patients who demonstrated an objective response at first evaluation as per immune-related response criteria. CONCLUSION: APCEDEN demonstrated highly convincing survival benefits when compared with the control group.

EHD2 restrains dynamics of caveolae by an ATP-dependent, membrane-bound, open conformation.

The EH-domain-containing protein 2 (EHD2) is a dynamin-related ATPase that confines caveolae to the cell surface by restricting the scission and subsequent endocytosis of these membrane pits. For this, EHD2 is thought to first bind to the membrane, then to oligomerize, and finally to detach, in a stringently regulated mechanistic cycle. It is still unclear how ATP is used in this process and whether membrane binding is coupled to conformational changes in the protein. Here, we show that the regulatory N-terminal residues and the EH domain keep the EHD2 dimer in an autoinhibited conformation in solution. By significantly advancing the use of infrared reflection-absorption spectroscopy, we demonstrate that EHD2 adopts an open conformation by tilting the helical domains upon membrane binding. We show that ATP binding enables partial insertion of EHD2 into the membrane, where G-domain-mediated oligomerization occurs. ATP hydrolysis is related to detachment of EHD2 from the membrane. Finally, we demonstrate that the regulation of EHD2 oligomerization in a membrane-bound state is crucial to restrict caveolae dynamics in cells.

Cavin3 interacts with cavin1 and caveolin1 to increase surface dynamics of caveolae.

Caveolae are invaginations of the cell surface thought to regulate membrane tension, signalling, adhesion and lipid homeostasis owing to their dynamic behaviour ranging from stable surface association to dynamic rounds of fission and fusion with the plasma membrane. The caveolae coat is generated by oligomerisation of the membrane protein caveolin and the family of cavin proteins. Here, we show that cavin3 (also known as PRKCDBP) is targeted to caveolae by cavin1 (also known as PTRF) where it interacts with the scaffolding domain of caveolin1 and promote caveolae dynamics. We found that the N-terminal region of cavin3 binds a trimer of the cavin1 N-terminus in competition with a homologous cavin2 (also known as SDPR) region, showing that the cavins form distinct subcomplexes through their N-terminal regions. Our data shows that cavin3 is enriched at deeply invaginated caveolae and that loss of cavin3 in cells results in an increase of stable caveolae and a decrease of caveolae that are only present at the membrane for a short time. We propose that cavin3 is recruited to the caveolae coat by cavin1 to interact with caveolin1 and regulate the duration time of caveolae at the plasma membrane.

Relating nucleotide-dependent conformational changes in free tubulin dimers to tubulin assembly.

The complex dynamic behavior of microtubules (MTs) is believed to be primarily due to the αß-tubulin dimer architecture and its intrinsic GTPase activity. Hence, a detailed knowledge of the conformational variations of isolated α-GTP-ß-GTP- and α-GTP-ß-GDP-tubulin dimers in solution and their implications to interdimer interactions and stability is directly relevant to understand the MT dynamics. An attempt has been made here by combining molecular dynamics (MD) simulations and protein-protein docking studies that unravels key structural features of tubulin dimer in different nucleotide states and correlates their association to tubulin assembly. Results from simulations suggest that tubulin dimers and oligomers attain curved conformations in both GTP and GDP states. Results also indicate that the tubulin C-terminal domain and the nucleotide state are closely linked. Protein-protein docking in combination with MD simulations suggest that the GTP-tubulin dimers engage in relatively stronger interdimer interactions even though the interdimer interfaces are bent in both GTP and GDP tubulin complexes, providing valuable insights on in vitro finding that GTP-tubulin is a better assembly candidate than GDP-tubulin during the MT nucleation and elongation processes.

Molecular interactions of the physiological anti-hypertensive peptide catestatin with the neuronal nicotinic acetylcholine receptor.

Catestatin (CST), a chromogranin-A-derived peptide, is a potent endogenous inhibitor of the neuronal nicotinic acetylcholine receptor (nAChR). It exerts an anti-hypertensive effect by acting as a 'physiological brake' on transmitter release into the circulation. However, the mechanism of interaction of CST with nAChR is only partially understood. To unravel molecular interactions of the wild-type human CST (CST-WT) as well as its naturally occurring variants (CST-364S and CST-370L, which have Gly→Ser and Pro→Leu substitutions, respectively) with the human α3ß4 nAChR, we generated a homology-modeled human α3ß4 nAChR structure and solution structures of CST peptides. Docking and molecular dynamics simulations showed that ~90% of interacting residues were within 15 N-terminal residues of CST peptides. The rank order of binding affinity of these peptides with nAChR was: CST-370L>CST-WT>CST-364S; the extent of occlusion of the receptor pore by these peptides was also in the same order. In corroboration with computational predictions, circular dichroism analysis revealed significant differences in global structures of CST peptides (e.g. the order of α-helical content was: CST-370L>CST-WT>CST-364S). Consistently, CST peptides blocked various stages of nAChR signal transduction, such as nicotine- or acetylcholine-evoked inward current, rise in intracellular Ca(2+) and catecholamine secretion in or from neuron-differentiated PC12 cells, in the same rank order. Taken together, this study shows molecular interactions between human CST peptides and human α3ß4 nAChR, and demonstrates that alterations in the CST secondary structure lead to the gain of potency for CST-370L and loss of potency for CST-364S. These findings have implications for understanding the nicotinic cholinergic signaling in humans.