Celebrating the Birthday of AMPA Receptor Nanodomains: Illuminating the Nanoscale Organization of Excitatory Synapses with 10 Nanocandles.

A decade ago, in 2013, and over the course of 4 summer months, three separate observations were reported that each shed light independently on a new molecular organization that fundamentally reshaped our perception of excitatory synaptic transmission (Fukata et al., 2013; MacGillavry et al., 2013; Nair et al., 2013). This discovery unveiled an intricate arrangement of AMPA-type glutamate receptors and their principal scaffolding protein PSD-95, at synapses. This breakthrough was made possible, thanks to advanced super-resolution imaging techniques. It fundamentally changed our understanding of excitatory synaptic architecture and paved the way for a brand-new area of research. In this Progressions article, the primary investigators of the nanoscale organization of synapses have come together to chronicle the tale of their discovery. We recount the initial inquiry that prompted our research, the preceding studies that inspired our work, the technical obstacles that were encountered, and the breakthroughs that were made in the subsequent decade in the realm of nanoscale synaptic transmission. We review the new discoveries made possible by the democratization of super-resolution imaging techniques in the field of excitatory synaptic physiology and architecture, first by the extension to other glutamate receptors and to presynaptic proteins and then by the notion of trans-synaptic organization. After describing the organizational modifications occurring in various pathologies, we discuss briefly the latest technical developments made possible by super-resolution imaging and emerging concepts in synaptic physiology.

Japanese encephalitis virus NS5 protein interacts with nucleolin to enhance the virus replication.

Japanese encephalitis virus (JEV) is an arthropod-borne, plus-strand flavivirus causing viral encephalitis in humans with a high case fatality rate. The JEV non-structural protein 5 (NS5) with the RNA-dependent RNA polymerase activity interacts with the viral and host proteins to constitute the replication complex. We have identified the multifunctional protein Nucleolin (NCL) as one of the several NS5-interacting host proteins. We demonstrate the interaction and colocalization of JEV NS5 with NCL in the virus-infected HeLa cells. The siRNA-mediated knockdown of NCL indicated that it was required for efficient viral replication. Importantly, JEV grew to higher titers in cells over-expressing exogenous NCL, demonstrating its pro-viral role. We demonstrated that NS5 interacted with the RRM and GAR domains of NCL. We show that the NCL-binding aptamer AS1411 containing the G-quadruplex (GQ) structure and the GQ ligand BRACO-19 caused significant inhibition of JEV replication. The antiviral effect of AS1411 and BRACO-19 could be overcome in HeLa cells by the overexpression of exogenous NCL. We demonstrated that the synthetic RNAs derived from the 3'-NCR of JEV genomic RNA containing the GQ sequence could bind NCL in vitro. The replication complex binding to the 3'-NCR is required for the viral RNA synthesis. It is likely that NCL present in the replication complex destabilizes the GQ structures in the genomic RNA, thus facilitating the movement of the replication complex resulting in efficient virus replication.IMPORTANCEJapanese encephalitis virus (JEV) is endemic in most parts of South-East Asia and the Western Pacific region, causing epidemics of encephalitis with a high case fatality rate. While a tissue culture-derived JEV vaccine is available, no antiviral therapy exists. The JEV NS5 protein has RNA-dependent RNA polymerase activity. Together with several host and viral proteins, it constitutes the replication complex necessary for virus replication. Understanding the interaction of NS5 with the host proteins could help design novel antivirals. We identified Nucleolin (NCL) as a crucial host protein interactor of JEV NS5 having a pro-viral role in virus replication. The NS5-interacting NCL binds to the G-quadruplex (GQ) structure sequence in the 3'-NCR of JEV RNA. This may smoothen the movement of the replication complex along the genomic RNA, thereby facilitating the virus replication. This study is the first report on how NCL, a host protein, helps in JEV replication through GQ-binding.

A broadly neutralizing monoclonal antibody overcomes the mutational landscape of emerging SARS-CoV-2 variants of concern.

The emergence of new variants of SARS-CoV-2 necessitates unremitting efforts to discover novel therapeutic monoclonal antibodies (mAbs). Here, we report an extremely potent mAb named P4A2 that can neutralize all the circulating variants of concern (VOCs) with high efficiency, including the highly transmissible Omicron. The crystal structure of the P4A2 Fab:RBD complex revealed that the residues of the RBD that interact with P4A2 are a part of the ACE2-receptor-binding motif and are not mutated in any of the VOCs. The pan coronavirus pseudotyped neutralization assay confirmed that the P4A2 mAb is specific for SARS-CoV-2 and its VOCs. Passive administration of P4A2 to K18-hACE2 transgenic mice conferred protection, both prophylactically and therapeutically, against challenge with VOCs. Overall, our data shows that, the P4A2 mAb has immense therapeutic potential to neutralize the current circulating VOCs. Due to the overlap between the P4A2 epitope and ACE2 binding site on spike-RBD, P4A2 may also be highly effective against a number of future variants.

Real-time heterogeneity of supramolecular assembly of amyloid precursor protein is modulated by an endocytic risk factor PICALM.

Recently, the localization of amyloid precursor protein (APP) into reversible nanoscale supramolecular assembly or "nanodomains" has been highlighted as crucial towards understanding the onset of the molecular pathology of Alzheimer's disease (AD). Surface expression of APP is regulated by proteins interacting with it, controlling its retention and lateral trafficking on the synaptic membrane. Here, we evaluated the involvement of a key risk factor for AD, PICALM, as a critical regulator of nanoscale dynamics of APP. Although it was enriched in the postsynaptic density, PICALM was also localized to the presynaptic active zone and the endocytic zone. PICALM colocalized with APP and formed nanodomains with distinct morphological properties in different subsynaptic regions. Next, we evaluated if this localization to subsynaptic compartments was regulated by the C-terminal sequences of APP, namely, the "Y682ENPTY687" domain. Towards this, we found that deletion of C-terminal regions of APP with partial or complete deletion of Y682ENPTY687, namely, APP-Δ9 and APP-Δ14, affected the lateral diffusion and nanoscale segregation of APP. Lateral diffusion of APP mutant APP-Δ14 sequence mimicked that of a detrimental Swedish mutant of APP, namely, APP-SWE, while APP-Δ9 diffused similar to wild-type APP. Interestingly, elevated expression of PICALM differentially altered the lateral diffusion of the APP C-terminal deletion mutants. These observations confirm that the C-terminal sequence of APP regulates its lateral diffusion and the formation of reversible nanoscale domains. Thus, when combined with autosomal dominant mutations, it generates distinct molecular patterns leading to onset of Alzheimer's disease (AD).

Neuronal PET tracers for Alzheimer's disease.

Advancements in brain imaging techniques have emerged as a significant tool in detecting Alzheimer's disease (AD) progression. The complicated cascade of AD progression can be detected using radio imaging, especially with Positron emission tomography (PET). The review focus on recently introduced investigational PET tracers targeting neurofibrillary tau aggregates found typically in AD. Herein, we also address the use of different PET tracers and the clinical implementation of established and newer generation tracers. This review also intends to discuss the importance of several PET radiotracers and challenges in PET imaging.

Society for Vascular Surgery appropriate use criteria for management of intermittent claudication.

The Society for Vascular Surgery appropriate use criteria (AUC) for the management of intermittent claudication were created using the RAND appropriateness method, a validated and standardized method that combines the best available evidence from medical literature with expert opinion, using a modified Delphi process. These criteria serve as a framework on which individualized patient and clinician shared decision-making can grow. These criteria are not absolute. AUC should not be interpreted as a requirement to administer treatments rated as appropriate (benefit outweighs risk). Nor should AUC be interpreted as a prohibition of treatments rated as inappropriate (risk outweighs benefit). Clinical situations will occur in which moderating factors, not included in these AUC, will shift the appropriateness level of a treatment for an individual patient. Proper implementation of AUC requires a description of those moderating patient factors. For scenarios with an indeterminate rating, clinician judgement combined with the best available evidence should determine the treatment strategy. These scenarios require mechanisms to track the treatment decisions and outcomes. AUC should be revisited periodically to ensure that they remain relevant. The panelists rated 2280 unique scenarios for the treatment of intermittent claudication (IC) in the aortoiliac, common femoral, and femoropopliteal segments in the round 2 rating. Of these, only nine (0.4%) showed a disagreement using the interpercentile range adjusted for symmetry formula, indicating an exceptionally high degree of consensus among the panelists. Post hoc, the term "inappropriate" was replaced with the phrase "risk outweighs benefit." The term "appropriate" was also replaced with "benefit outweighs risk." The key principles for the management of IC reflected within these AUC are as follows. First, exercise therapy is the preferred initial management strategy for all patients with IC. Second, for patients who have not completed exercise therapy, invasive therapy might provide net a benefit for selected patients with IC who are nonsmokers, are taking optimal medical therapy, are considered to have a low physiologic and technical risk, and who are experiencing severe lifestyle limitations and/or a short walking distance. Third, considering the long-term durability of the currently available technology, invasive interventions for femoropopliteal disease should be reserved for patients with severe lifestyle limitations and a short walking distance. Fourth, in the common femoral segment, open common femoral endarterectomy will provide greater net benefit than endovascular intervention for the treatment of IC. Finally, in the infrapopliteal segment, invasive intervention for the treatment of IC is of unclear benefit and could be harmful.

Vinculin-mediated axon growth requires interaction with actin but not talin in mouse neocortical neurons.

The actin-binding protein vinculin is a major constituent of focal adhesion, but its role in neuronal development is poorly understood. We found that vinculin deletion in mouse neocortical neurons attenuated axon growth both in vitro and in vivo. Using functional mutants, we found that expression of a constitutively active vinculin significantly enhanced axon growth while the head-neck domain had an inhibitory effect. Interestingly, we found that vinculin-talin interaction was dispensable for axon growth and neuronal migration. Strikingly, expression of the tail domain delayed migration, increased branching, and stunted axon. Inhibition of the Arp2/3 complex or abolishing the tail domain interaction with actin completely reversed the branching phenotype caused by tail domain expression without affecting axon length. Super-resolution microscopy showed increased mobility of actin in tail domain expressing neurons. Our results provide novel insights into the role of vinculin and its functional domains in regulating neuronal migration and axon growth.

Measuring mRNA translation in neuronal processes and somata by tRNA-FRET.

In neurons, the specific spatial and temporal localization of protein synthesis is of great importance for function and survival. Here, we visualized tRNA and protein synthesis events in fixed and live mouse primary cortical culture using fluorescently-labeled tRNAs. We were able to characterize the distribution and transport of tRNAs in different neuronal sub-compartments and to study their association with the ribosome. We found that tRNA mobility in neural processes is lower than in somata and corresponds to patterns of slow transport mechanisms, and that larger tRNA puncta co-localize with translational machinery components and are likely the functional fraction. Furthermore, chemical induction of long-term potentiation (LTP) in culture revealed up-regulation of mRNA translation with a similar effect in dendrites and somata, which appeared to be GluR-dependent 6 h post-activation. Importantly, measurement of protein synthesis in neurons with high resolutions offers new insights into neuronal function in health and disease states.

Effect of Early vs Standard Approach to Tracheostomy on Functional Outcome at 6 Months Among Patients With Severe Stroke Receiving Mechanical Ventilation: The SETPOINT2 Randomized Clinical Trial.

Importance: Many patients with severe stroke have impaired airway protective reflexes, resulting in prolonged invasive mechanical ventilation. Objective: To test whether early vs standard tracheostomy improved functional outcome among patients with stroke receiving mechanical ventilation. Design, Setting, and Participants: In this randomized clinical trial, 382 patients with severe acute ischemic or hemorrhagic stroke receiving invasive ventilation were randomly assigned (1:1) to early tracheostomy (≤5 days of intubation) or ongoing ventilator weaning with standard tracheostomy if needed from day 10. Patients were randomized between July 28, 2015, and January 24, 2020, at 26 US and German neurocritical care centers. The final date of follow-up was August 9, 2020. Interventions: Patients were assigned to an early tracheostomy strategy (n = 188) or to a standard tracheostomy (control group) strategy (n = 194). Main Outcomes and Measures: The primary outcome was functional outcome at 6 months, based on the modified Rankin Scale score (range, 0 [best] to 6 [worst]) dichotomized to a score of 0 (no disability) to 4 (moderately severe disability) vs 5 (severe disability) or 6 (death). Results: Among 382 patients randomized (median age, 59 years; 49.8% women), 366 (95.8%) completed the trial with available follow-up data on the primary outcome (177 patients [94.1%] in the early group; 189 patients [97.4%] in the standard group). A tracheostomy (predominantly percutaneously) was performed in 95.2% of the early tracheostomy group in a median of 4 days after intubation (IQR, 3-4 days) and in 67% of the control group in a median of 11 days after intubation (IQR, 10-12 days). The proportion without severe disability (modified Rankin Scale score, 0-4) at 6 months was not significantly different in the early tracheostomy vs the control group (43.5% vs 47.1%; difference, -3.6% [95% CI, -14.3% to 7.2%]; adjusted odds ratio, 0.93 [95% CI, 0.60-1.42]; P = .73). Of the serious adverse events, 5.0% (6 of 121 reported events) in the early tracheostomy group vs 3.4% (4 of 118 reported events) were related to tracheostomy. Conclusions and Relevance: Among patients with severe stroke receiving mechanical ventilation, a strategy of early tracheostomy, compared with a standard approach to tracheostomy, did not significantly improve the rate of survival without severe disability at 6 months. However, the wide confidence intervals around the effect estimate may include a clinically important difference, so a clinically relevant benefit or harm from a strategy of early tracheostomy cannot be excluded. Trial Registration: ClinicalTrials.gov Identifier: NCT02377167.

A polar filter in DNA polymerases prevents ribonucleotide incorporation.

The presence of ribonucleotides in DNA can lead to genomic instability and cellular lethality. To prevent adventitious rNTP incorporation, the majority of the DNA polymerases (dPols) possess a steric filter. The dPol named MsDpo4 (Mycobacterium smegmatis) naturally lacks this steric filter and hence is capable of rNTP addition. The introduction of the steric filter in MsDpo4 did not result in complete abrogation of the ability of this enzyme to incorporate ribonucleotides. In comparison, DNA polymerase IV (PolIV) from Escherichia coli exhibited stringent selection for deoxyribonucleotides. A comparison of MsDpo4 and PolIV led to the discovery of an additional polar filter responsible for sugar selectivity. Thr43 represents the filter in PolIV and this residue forms interactions with the incoming nucleotide to draw it closer to the enzyme surface. As a result, the 2'-OH in rNTPs will clash with the enzyme surface, and therefore ribonucleotides cannot be accommodated in the active site in a conformation compatible with productive catalysis. The substitution of the equivalent residue in MsDpo4-Cys47, with Thr led to a drastic reduction in the ability of the mycobacterial enzyme to incorporate rNTPs. Overall, our studies evince that the polar filter serves to prevent ribonucleotide incorporation by dPols.

Vitamin B12 may inhibit RNA-dependent-RNA polymerase activity of nsp12 from the SARS-CoV-2 virus.

SARS-CoV-2 is the causative agent for the ongoing COVID19 pandemic, and this virus belongs to the Coronaviridae family. Like other members of this family, the virus possesses a positive-sense single-stranded RNA genome. The genome encodes for the nsp12 protein, which houses the RNA-dependent-RNA polymerase (RdRP) activity responsible for the replication of the viral genome. A homology model of nsp12 was prepared using the structure of the SARS nsp12 (6NUR) as a model. The model was used to carry out in silico screening to identify molecules among natural products, or Food and Drug Administration-approved drugs that can potentially inhibit the activity of nsp12. This exercise showed that vitamin B12 (methylcobalamin) may bind to the active site of the nsp12 protein. A model of the nsp12 in complex with substrate RNA and incoming NTP showed that vitamin B12 binding site overlaps with that of the incoming nucleotide. A comparison of the calculated energies of binding for RNA plus NTP and methylcobalamin suggested that the vitamin may bind to the active site of nsp12 with significant affinity. It is, therefore, possible that methylcobalamin binding may prevent association with RNA and NTP and thus inhibit the RdRP activity of nsp12. Overall, our computational studies suggest that methylcobalamin form of vitamin B12 may serve as an effective inhibitor of the nsp12 protein.

mEosBrite Are Bright Variants of mEos3.2 Developed by Semirational Protein Engineering.

mEos3.2 is a photoconvertible fluorescence protein with comparatively low brightness, which limits its application in live Super resolution microscopy. To address this issue, we have used semi-rational protein engineering to develop mEosBrite, a new class of improved brightness variants. The improvement in the brightness was confirmed by expression in E.coli as well as mammalian cell lines. Furthermore, biophysical characterization suggests that all the three mEosBrite variant proteins display higher quantum yield, truly monomeric form, less cytotoxicity and lower protein aggregation as compared to the wild type mEos3.2 protein. Most importantly, because of their high photoconversion efficiency mEosBrite variants could be an excellent tool for single-molecule and intensity fluctuation based super-resolution microscopy.

Pyrophosphate hydrolysis is an intrinsic and critical step of the DNA synthesis reaction.

DNA synthesis by DNA polymerases (dPols) is central to duplication and maintenance of the genome in all living organisms. dPols catalyze the formation of a phosphodiester bond between the incoming deoxynucleoside triphosphate and the terminal primer nucleotide with the release of a pyrophosphate (PPi) group. It is believed that formation of the phosphodiester bond is an endergonic reaction and PPi has to be hydrolyzed by accompanying pyrophosphatase enzymes to ensure that the free energy change of the DNA synthesis reaction is negative and it can proceed in the forward direction. The fact that DNA synthesis proceeds in vitro in the absence of pyrophosphatases represents a long-standing conundrum regarding the thermodynamics of the DNA synthesis reaction. Using time-resolved crystallography, we show that hydrolysis of PPi is an intrinsic and critical step of the DNA synthesis reaction catalyzed by dPols. The hydrolysis of PPi occurs after the formation of the phosphodiester bond and ensures that the DNA synthesis reaction is energetically favorable without the need for additional enzymes. Also, we observe that DNA synthesis is a two Mg2+ ion assisted stepwise associative SN2 reaction. Overall, this study provides deep temporal insight regarding the primary enzymatic reaction responsible for genome duplication.

Mechanism of formation of a toroid around DNA by the mismatch sensor protein.

The DNA mismatch repair (MMR) pathway removes errors that appear during genome replication. MutS is the primary mismatch sensor and forms an asymmetric dimer that encircles DNA to bend it to scan for mismatches. The mechanism utilized to load DNA into the central tunnel was unknown and the origin of the force required to bend DNA was unclear. We show that, in absence of DNA, MutS forms a symmetric dimer wherein a gap exists between the monomers through which DNA can enter the central tunnel. The comparison with structures of MutS-DNA complexes suggests that the mismatch scanning monomer (Bm) will move by nearly 50 Å to associate with the other monomer (Am). Consequently, the N-terminal domains of both monomers will press onto DNA to bend it. The proposed mechanism of toroid formation evinces that the force required to bend DNA arises primarily due to the movement of Bm and hence, the MutS dimer acts like a pair of pliers to bend DNA. We also shed light on the allosteric mechanism that influences the expulsion of adenosine triphosphate from Am on DNA binding. Overall, this study provides mechanistic insight regarding the primary event in MMR i.e. the assembly of the MutS-DNA complex.

Outcomes of standard 12-core transrectal ultrasound-guided prostate biopsy in biopsy naive Indian men -single center experience.

INTRODUCTION: Prostate cancer is now the second-most common cancer in many parts of India. Despite being the second-largest population in the world, data regarding outcomes of biopsy in Indian men are limited. We report the correlation of biopsy finding with prostate-specific antigen (PSA) level in Indian men undergoing biopsy for either elevated PSA and/or abnormal digital rectal examination (DRE) findings. MATERIALS AND METHODS: We retrospectively analyzed data of 853 men who underwent TRUS-guided prostate biopsy in a single institution from January 2014 to October 2019. The biopsy was performed when serum PSA was more than 4.00 ng/mL and/or DRE findings were suspicious for malignancy. RESULTS: Overall cancer detection rate was 38.8%. Patients were classified in five groups based on PSA levels, irrespective of DRE findings (0-3.99 ng/mL, 4.00-9.99 ng/mL, 10.00-19.99 ng/mL, 20.00-39.99 ng/mL, and ≥40 ng/mL). Overall prostate cancer detection rates at corresponding at PSA levels were 3/23 (13%), 62/282 (21.9%), 86/226 (38.05%), 66/126 (52.3%), and 165/196 (84.18%), respectively. 331 (38.8%) patients of the total 853 had suspicious DRE, the cancer detection rate in corresponding PSA groups, based on DRE alone was 3/23 (13.04%), 23/42 (54.76%), 39/56 (69.64%), 43/52 (82.69%), and 157/160 (98.13%), respectively. CONCLUSION: The overall prostate cancer detection rate at our center was 38.8%, which is much higher as compared to other Indian data. Our study also emphasizes the role of DRE in Indian men presenting with elevated PSA.

Aß mediates F-actin disassembly in dendritic spines leading to cognitive deficits in Alzheimer's disease.

Dendritic spine loss is recognized as an early feature of Alzheimer's disease (AD), but the underlying mechanisms are poorly understood. Dendritic spine structure is defined by filamentous actin (F-actin) and we observed depolymerization of synaptosomal F-actin accompanied by increased globular-actin (G-actin) at as early as 1 month of age in a mouse model of AD (APPswe/PS1ΔE9, male mice). This led to recall deficit after contextual fear conditioning (cFC) at 2 months of age in APPswe/PS1ΔE9 male mice, which could be reversed by the actin-polymerizing agent jasplakinolide. Further, the F-actin-depolymerizing agent latrunculin induced recall deficit after cFC in WT mice, indicating the importance of maintaining F-/G-actin equilibrium for optimal behavioral response. Using direct stochastic optical reconstruction microscopy (dSTORM), we show that F-actin depolymerization in spines leads to a breakdown of the nano-organization of outwardly radiating F-actin rods in cortical neurons from APPswe/PS1ΔE9 mice. Our results demonstrate that synaptic dysfunction seen as F-actin disassembly occurs very early, before onset of pathological hallmarks in AD mice, and contributes to behavioral dysfunction, indicating that depolymerization of F-actin is causal and not consequent to decreased spine density. Further, we observed decreased synaptosomal F-actin levels in postmortem brain from mild cognitive impairment and AD patients compared with subjects with normal cognition. F-actin decrease correlated inversely with increasing AD pathology (Braak score, Aß load, and tangle density) and directly with performance in episodic and working memory tasks, suggesting its role in human disease pathogenesis and progression.SIGNIFICANCE STATEMENT Synaptic dysfunction underlies cognitive deficits in Alzheimer's disease (AD). The cytoskeletal protein actin plays a critical role in maintaining structure and function of synapses. Using cultured neurons and an AD mouse model, we show for the first time that filamentous actin (F-actin) is lost selectively from synapses early in the disease process, long before the onset of classical AD pathology. We also demonstrate that loss of synaptic F-actin contributes directly to memory deficits. Loss of synaptosomal F-actin in human postmortem tissue correlates directly with decreased performance in memory test and inversely with AD pathology. Our data highlight that synaptic cytoarchitectural changes occur early in AD and they may be targeted for the development of therapeutics.

Synthesis of N2-Deoxyguanosine Modified DNAs and the Studies on Their Translesion Synthesis by the E. coli DNA Polymerase IV.

We report the synthesis of N2-aryl (benzyl, naphthyl, anthracenyl, and pyrenyl)-deoxyguanosine (dG) modified phosphoramidite building blocks and the corresponding damaged DNAs. Primer extension studies using E. coli Pol IV, a translesion polymerase, demonstrate that translesion synthesis (TLS) across these N2-dG adducts is error free. However, the efficiency of TLS activity decreases with increase in the steric bulkiness of the adducts. Molecular dynamics simulations of damaged DNA-Pol IV complexes reveal the van der Waals interactions between key amino acid residues (Phe13, Ile31, Gly32, Gly33, Ser42, Pro73, Gly74, Phe76, and Tyr79) of the enzyme and adduct that help to accommodate the bulky damages in a hydrophobic pocket to facilitate TLS. Overall, the results presented here provide insights into the TLS across N2-aryl-dG damaged DNAs by Pol IV.

Structure-guided identification of function: role of Capsicum annuum vicilin during oxidative stress.

Proteins belonging to cupin superfamily are known to have critical and diverse physiological functions. However, 7S globulins family, which is also a part of cupin superfamily, were undermined as only seed storage proteins. Structure determination of native protein - Vic_CAPAN from Capsicum annuum - was carried out, and its physiological functions were explored after purifying the protein by ammonium sulfate precipitation followed by size exclusion chromatography. The crystal structure of vicilin determined at 2.16 Šresolution revealed two monomers per asymmetric unit which are juxtaposed orthogonal with each other. Vic_CAPAN consists predominately of ß-sheets that folds to form a ß-barrel structure commonly called cupin fold. Each monomer of Vic_CAPAN consists of two cupin fold domains, N-terminal and C-terminal, which accommodate two different ligands. A bound ligand was identified at the C-terminal cupin fold in the site presumably conserved for metabolites in the crystal structure. The ligand was confirmed to be salicylic acid through mass spectrometric analysis. A copper-binding site was further observed near the conserved ligand-binding pocket, suggesting possible superoxide dismutase activity of Vic_CAPAN which was subsequently confirmed biochemically. Vicilins from other sources did not exhibit this activity indicating functional specificity of Vic_CAPAN. Discovery of bound salicylic acid, which is a known regulator of antioxidant pathway, and revelation of superoxide dismutase activity suggest that Vic_CAPAN has an important role during oxidative stress. As salicylic acid changes the redox state of cell, it may act as a downstream signal for various pathways involved in plant biotic and abiotic stress rescue.

Dimerization through the RING-Finger Domain Attenuates Excision Activity of the piggyBac Transposase.

The movement of the piggyBac transposon is mediated through its cognate transposase. The piggyBac transposase binds to the terminal repeats present at the ends of the transposon. This is followed by excision of the transposon and release of the nucleoprotein complex. The complex translocates, followed by integration of the transposon at the target site. Here, we show that the RING-finger domain (RFD) present toward the C-terminus of the transposase is vital for dimerization of this enzyme. The deletion of the RFD or the last seven residues of the RFD results in a monomeric protein that binds the terminal end of the transposon with nearly the same affinity as wild type piggyBac transposase. Surprisingly, the monomeric constructs exhibit >2-fold enhancement in the excision activity of the enzyme. Overall, our studies suggest that dimerization attenuates the excision activity of the piggyBac transposase. This attribute of the piggyBac transposase may serve to prevent excessive transposition of the piggyBac transposon that might be catastrophic for the host cell.

Nanoscale segregation of actin nucleation and elongation factors determines dendritic spine protrusion.

Actin dynamics drive morphological remodeling of neuronal dendritic spines and changes in synaptic transmission. Yet, the spatiotemporal coordination of actin regulators in spines is unknown. Using single protein tracking and super-resolution imaging, we revealed the nanoscale organization and dynamics of branched F-actin regulators in spines. Branched F-actin nucleation occurs at the PSD vicinity, while elongation occurs at the tip of finger-like protrusions. This spatial segregation differs from lamellipodia where both branched F-actin nucleation and elongation occur at protrusion tips. The PSD is a persistent confinement zone for IRSp53 and the WAVE complex, an activator of the Arp2/3 complex. In contrast, filament elongators like VASP and formin-like protein-2 move outwards from the PSD with protrusion tips. Accordingly, Arp2/3 complexes associated with F-actin are immobile and surround the PSD. Arp2/3 and Rac1 GTPase converge to the PSD, respectively, by cytosolic and free-diffusion on the membrane. Enhanced Rac1 activation and Shank3 over-expression, both associated with spine enlargement, induce delocalization of the WAVE complex from the PSD. Thus, the specific localization of branched F-actin regulators in spines might be reorganized during spine morphological remodeling often associated with synaptic plasticity.