Jacob-induced transcriptional inactivation of CREB promotes Aß-induced synapse loss in Alzheimer's disease.

Synaptic dysfunction caused by soluble ß-amyloid peptide (Aß) is a hallmark of early-stage Alzheimer's disease (AD), and is tightly linked to cognitive decline. By yet unknown mechanisms, Aß suppresses the transcriptional activity of cAMP-responsive element-binding protein (CREB), a master regulator of cell survival and plasticity-related gene expression. Here, we report that Aß elicits nucleocytoplasmic trafficking of Jacob, a protein that connects a NMDA-receptor-derived signalosome to CREB, in AD patient brains and mouse hippocampal neurons. Aß-regulated trafficking of Jacob induces transcriptional inactivation of CREB leading to impairment and loss of synapses in mouse models of AD. The small chemical compound Nitarsone selectively hinders the assembly of a Jacob/LIM-only 4 (LMO4)/ Protein phosphatase 1 (PP1) signalosome and thereby restores CREB transcriptional activity. Nitarsone prevents impairment of synaptic plasticity as well as cognitive decline in mouse models of AD. Collectively, the data suggest targeting Jacob protein-induced CREB shutoff as a therapeutic avenue against early synaptic dysfunction in AD.

One-step purification of tag free and soluble lamin B1 from an E. coli bacterial expression system.

Lamin B1 is an intermediate filament protein that is a core component of the nuclear lamina. Structural studies and biochemical characterization of lamin B1 are severely hampered by the tendency of the protein to form inclusion bodies in E. coli bacterial expression systems. Therefore, the purity and consistency of the protein varies from batch to batch. In this work, we have purified a tag-free lamin B1 protein from a soluble fraction following bacterial expression. We also checked the functional properties of the purified as well as of the subsequently lyophilised protein. The current protocol helps to purify functional lamin B1 in a single step.

Uncovering Structural and Molecular Dynamics of ESAT-6:ß2M Interaction: Asp53 of Human ß2-Microglobulin Is Critical for the ESAT-6:ß2M Complexation.

ESAT-6 is a small secreted protein of Mycobacterium tuberculosis involved in the ESAT-6 secretion system (ESX-1)-mediated virulence and pathogenesis. The protein interacts with ß2M, causing downregulation of MHC class I Ag presentation, which could be one of the mechanisms by which it favors increased survival of the bacilli inside the host. In an earlier study, we have shown that the C-terminal region of ESAT-6 is crucial for its interaction with ß2M. However, the interface of ß2M involved in interaction with ESAT-6 and detailed physicochemical changes associated with ESAT-6:ß2M complexation are not fully defined. In this study, using computational and site-directed mutagenesis studies, we demonstrate the presence of strong noncovalent hydrophobic interactions between ESAT-6 and ß2M in addition to the vital hydrogen bonding between the aspartate residue (Asp53) of ß2M and methionine (Met93) of ESAT-6. Docking-based high-throughput virtual screening followed by 16-point screening on microscale thermophoresis resulted in the identification of two potent inhibitors (SM09 and SM15) that mask the critical Met93 residue of ESAT-6 that is required for ESAT-6:ß2M interaction and could rescue cell surface expression of ß2M and HLA in human macrophages as well as MHC class I Ag presentation suppressed by ESAT-6 in peritoneal macrophages isolated from C57BL/6 mice. Both SM09 and SM15 significantly inhibited intracellular survival of M. tuberculosis in human macrophages. Further, we characterized the physicochemical properties involved in the ESAT-6:ß2M complexation, which may help in understanding host-pathogen interactions.

Interface interactions between ßγ-crystallin domain and Ig-like domain render Ca2+ -binding site inoperative in abundant perithecial protein of Neurospora crassa.

We describe a set of proteins in which a ßγ-crystallin domain pairs with an Ig-like domain, and which are confined to microbes, like bacteria, slime molds and fungi. DdCAD-1 (Ca2+ -dependent cell adhesion molecule-1) and abundant perithecial protein (APP) represent this class of molecules. Using the crystal structure of APP-NTD (N-terminal domain of APP), we describe its mode of Ca2+ binding and provide a generalized theme for correct identification of the Ca2+ -binding site within this class of molecules. As a common feature, one of the two Ca2+ -binding sites is non-functional in the ßγ-crystallin domains of these proteins. While APP-NTD binds Ca2+ with a micromolar affinity which is comparable to DdCAD-1, APP surprisingly does not bind Ca2+ . Crystal structures of APP and Ca2+ -bound APP-NTD reveal that the interface interactions in APP render its Ca2+ -binding site inoperative. Thus, heterodomain association provides a novel mode of Ca2+ -binding regulation in APP. Breaking the interface interactions (mutating Asp30Ala, Leu132Ala and Ile135Ala) or separation from the Ig-like domain removes the constraints upon the required conformational transition and enables the ßγ-crystallin domain to bind Ca2+ . In mechanistic detail, our work demonstrates an interdomain interface adapted to distinct functional niches in APP and its homolog DdCAD-1.

Abundant Perithecial Protein (APP) from Neurospora is a primitive functional analog of ocular crystallins.

The eye arose during the Cambrian explosion from pre-existing proteins that would have been recruited for the formation of the specialized components of this organ, such as the transparent lens. Proteins suitable for the role of lens crystallins would need to possess unusual physical properties and the study of such earliest analogs of ocular crystallins would add to our understanding of the nature of recruitment of proteins as lens/corneal crystallins. We show that the Abundant Perithecial Protein (APP) of the fungi Neurospora and Sordaria fulfils the criteria for an early crystallin analog. The perithecia in these fungal species are phototropic, and APP accumulates at a high concentration in the neck of the pitcher-shaped perithecium. Spores are formed at the base of the perithecium, and light contributes to their maturation. The hydrodynamic properties of APP appear to exclude dimer formation or aggregation at high protein concentrations. APP is also deficient in Ca2+ binding, a property seen in its close homolog, the calcium-binding cell adhesion molecule (DdCAD-1) from Dictyostelium discoidum. Comparable to crystallins, APP occurs in high concentrations and seems to have dispensed with Ca2+ binding in exchange for greater stability. These crystallin-like attributes of APP lead us to demonstrate that it is a primitive form of ocular crystallins.

Posttranslational modification impact on the mechanism by which amyloid-ß induces synaptic dysfunction.

Oligomeric amyloid-ß (Aß) 1-42 disrupts synaptic function at an early stage of Alzheimer's disease (AD). Multiple posttranslational modifications of Aß have been identified, among which N-terminally truncated forms are the most abundant. It is not clear, however, whether modified species can induce synaptic dysfunction on their own and how altered biochemical properties can contribute to the synaptotoxic mechanisms. Here, we show that a prominent isoform, pyroglutamated Aß3(pE)-42, induces synaptic dysfunction to a similar extent like Aß1-42 but by clearly different mechanisms. In contrast to Aß1-42, Aß3(pE)-42 does not directly associate with synaptic membranes or the prion protein but is instead taken up by astrocytes and potently induces glial release of the proinflammatory cytokine TNFα. Moreover, Aß3(pE)-42-induced synaptic dysfunction is not related to NMDAR signalling and Aß3(pE)-42-induced impairment of synaptic plasticity cannot be rescued by D1-agonists. Collectively, the data point to a scenario where neuroinflammatory processes together with direct synaptotoxic effects are caused by posttranslational modification of soluble oligomeric Aß and contribute synergistically to the onset of synaptic dysfunction in AD.

Strategizing for the purification of a multiple Big domain-containing protein in native conformation is worth it!

The reliability and accuracy of conformational or functional studies of any novel multidomain protein rely on the quality of protein. The bottleneck in structural studies with the complete Big_2 domain containing proteins like LigA, LigB or MpIBP is usually their large molecular size owing to their multidomain (>10-12 domains) architectures. Interestingly, a soil bacterium Paenarthrobacter aurescens TC1, harbours a gene that encodes a protein comprising of four predicted Big_2 domains. We report here the expression and purification of this novel, multiple Big_2 domains containing protein, Arig of P. aurescens TC1. During overexpression, recombinant Arig formed inclusion bodies and hence was purified by on-column refolding. The refolded Arig revealed a ß-sheet conformation and a well-resolved near-UV CD spectra but did not exhibit a well-dispersed 2D [1H-15N]-HSQC NMR spectrum, as expected for a well-folded ß-sheet native conformation. We, therefore, further optimized Arig overexpression in the soluble fraction by including osmolytes. CD spectroscopic and 2D [1H-15N]-HSQC analyses consolidate that Arig purified alternatively has a well-folded native conformation. While we describe different strategies for purification of Arig, we also present the spectral properties of this novel all-ß-sheet protein.

A Transition Metal-Binding, Trimeric ßγ-Crystallin from Methane-Producing Thermophilic Archaea, Methanosaeta thermophila.

ßγ-Crystallins are important constituents of the vertebrate eye lens, whereas in microbes, they are prevalent as Ca2+-binding proteins. In archaea, ßγ-crystallins are conspicuously confined to two methanogens, viz., Methanosaeta and Methanosarcina. One of these, i.e., M-crystallin from Methanosarcina acetivorans, has been shown to be a typical Ca2+-binding ßγ-crystallin. Here, with the aid of a high-resolution crystal structure and isothermal titration calorimetry, we report that "Methallin", a ßγ-crystallin from Methanosaeta thermophila, is a trimeric, transition metal-binding protein. It binds Fe, Ni, Co, or Zn ion with nanomolar affinity, which is consistent even at 55 °C, the optimal temperature for the methanogen's growth. At the center of the protein trimer, the metal ion is coordinated by six histidines, two from each protomer, leading to an octahedral geometry. Small-angle X-ray scattering analysis confirms that the trimer seen in the crystal lattice is a biological assembly; this assembly dissociates to monomers upon removal of the metal ion. The introduction of two histidines (S17H/S19H) into a homologous ßγ-crystallin, Clostrillin, allows it to bind nickel at the introduced site, though with micromolar affinity. However, because of the lack of a compatible interface, nickel binding could not induce trimerization, affirming that Methallin is a naturally occurring trimer for high-affinity transition metal binding. While ßγ-crystallins are known to bind Ca2+ and form homodimers and oligomers, the transition metal-binding, trimeric Methallin is a new paradigm for ßγ-crystallins. The distinct features of Methallin, such as nickel or iron binding, are also possible imprints of biogeochemical changes during the period of its origin.

Ca2+ and ßγ-crystallins: An affair that did not last?

BACKGROUND: During the last three decades, lens ß- and γ-crystallins have found a huge number of kin from numerous taxonomical sources. Most of these proteins from invertebrates and microbes have been demonstrated or predicted to bind Ca2+ involving a distinct double-clamp motif, which is largely degenerated in lens homologues. SCOPE OF REVIEW: The various aspects of transformation of ßγ-crystallins from a quintessential Ca2+-binding protein into a primarily structural molecule have been reviewed. MAJOR CONCLUSIONS: In lens members of ßγ-crystallins, the residues involved in Ca2+ binding have diverged considerably from the classical consensus with consequent reduction in their Ca2+-binding properties. This evolutionary change is congenial to their new role as robust constituents of lens. The exact functions of the residual affinity for Ca2+ are yet to be established. GENERAL SIGNIFICANCE: This review highlights the significance of reduction in Ca2+-binding ability of the ßγ-crystallins for lens physiology and why this residual affinity may be functionally important. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.

NMR solution structure of the terminal immunoglobulin-like domain from the leptospira host-interacting outer membrane protein, LigB.

A number of surface proteins specific to pathogenic strains of Leptospira have been identified. The Lig protein family has shown promise as a marker in typing leptospiral isolates for pathogenesis and as an antigen in vaccines. We used NMR spectroscopy to solve the solution structure of the twelfth immunoglobulin-like (Ig-like) repeat domain from LigB (LigB-12). The fold is similar to that of other bacterial Ig-like domains and comprised mainly of ß-strands that form a ß-sandwich based on a Greek-key folding arrangement. Based on sequence analysis and conservation of structurally important residues, homology models for the other LigB Ig-like domains were generated. The set of LigB models illustrates the electrostatic differences between the domains as well as the possible interactions between neighboring domains. Understanding the structure of the extracellular portion of LigB and related proteins is important for developing diagnostic methods and new therapeutics directed toward leptospirosis.

Serine protease activity of calnuc: regulation by Zn2+ and G proteins.

The functions of calnuc, a novel Ca(2+)-binding protein with multiple structural domains and diverse interacting partners, are yet unknown. We demonstrate unknown facets of calnuc, which is a serine protease in which Ser-378 of GXSXG motif, Asp-328 of DTG motif, and His-339 form the "catalytic triad," locating the enzyme active site in the C-terminal region. Analogous to the active site of Zn(2+) carboxypeptidases, calnuc has two high affinity (K(d) ∼ 20 nm), well conserved Zn(2+)-binding sites near its N terminus, although it is inactive as a peptidase. Zn(2+) binding allosterically and negatively regulates the serine protease activity of calnuc, inhibition being caused by an "open to close" change in its conformation not seen upon Ca(2+) binding. Most strikingly, interaction with G protein α subunit completely inhibits the enzymatic activity of calnuc. We thus illustrate that G proteins and Zn(2+) act as two "keys" that control enzymatic activity of calnuc, arresting it in "locked" state. Calnuc, therefore, exists dynamically in two different forms, (i) as a Ca(2+)-binding protein in Zn(2+)-bound form and (ii) as a protease in Zn(2+)-free form, commissioning it to perform multiple functions.

The perturbation of tryptophan fluorescence by phenylalanine to alanine mutations identifies the hydrophobic core in a subset of bacterial Ig-like domains.

Many host-parasite interactions are mediated via surface-exposed proteins containing bacterial immunoglobulin-like (Big) domains. Here, we utilize the spectral properties of a conserved Trp to provide evidence that, along with a Phe, these residues are positioned within the hydrophobic core of a subset of Big_2 domains. The mutation of the Phe to Ala decreases Big_2 domain stability and impairs the ability of LigBCen2 to bind to the host protein, fibronectin.

An Evolutionary Attention-Based Network for Medical Image Classification.

Deep learning has become a primary choice in medical image analysis due to its powerful representation capability. However, most existing deep learning models designed for medical image classification can only perform well on a specific disease. The performance drops dramatically when it comes to other diseases. Generalizability remains a challenging problem. In this paper, we propose an evolutionary attention-based network (EDCA-Net), which is an effective and robust network for medical image classification tasks. To extract task-related features from a given medical dataset, we first propose the densely connected attentional network (DCA-Net) where feature maps are automatically channel-wise weighted, and the dense connectivity pattern is introduced to improve the efficiency of information flow. To improve the model capability and generalizability, we introduce two types of evolution: intra- and inter-evolution. The intra-evolution optimizes the weights of DCA-Net, while the inter-evolution allows two instances of DCA-Net to exchange training experience during training. The evolutionary DCA-Net is referred to as EDCA-Net. The EDCA-Net is evaluated on four publicly accessible medical datasets of different diseases. Experiments showed that the EDCA-Net outperforms the state-of-the-art methods on three datasets and achieves comparable performance on the last dataset, demonstrating good generalizability for medical image classification.

Chaperone-mediated autophagy in neuronal dendrites utilizes activity-dependent lysosomal exocytosis for protein disposal.

The complex morphology of neurons poses a challenge for proteostasis because the majority of lysosomal degradation machinery is present in the cell soma. In recent years, however, mature lysosomes were identified in dendrites, and a fraction of those appear to fuse with the plasma membrane and release their content to the extracellular space. Here, we report that dendritic lysosomes are heterogeneous in their composition and that only those containing lysosome-associated membrane protein (LAMP) 2A and 2B fuse with the membrane and exhibit activity-dependent motility. Exocytotic lysosomes dock in close proximity to GluN2B-containing N-methyl-D-aspartate-receptors (NMDAR) via an association of LAMP2B to the membrane-associated guanylate kinase family member SAP102/Dlg3. NMDAR-activation decreases lysosome motility and promotes membrane fusion. We find that chaperone-mediated autophagy is a supplier of content that is released to the extracellular space via lysosome exocytosis. This mechanism enables local disposal of aggregation-prone proteins like TDP-43 and huntingtin.

A Minimally Invasive and Simple Technique of Superficial Quadriceps Tendon Graft Harvesting.

Quadriceps tendon (QT) graft is a versatile graft for anterior and posterior cruciate ligament reconstruction. Advantages of quadriceps tendon autograft are the superior quality of graft, customization of graft size, reduced anterior knee pain, reduced risk of neurovascular injury, reduced incidence of arthrofibrosis compared to BPTB graft, preservation of ACL agonists, i.e., hamstrings, implantation of thicker graft and better patella mobility. Considering its advantages over other available autografts, its popularity is now increasing among surgeons. Conventionally, quadriceps tendon graft is harvested by an open technique, which produces an ugly scar and delays rehabilitation. In this article, we describe a minimally invasive technique of quadriceps tendon graft harvesting with a 2-2.5-cm vertical skin incision. Our technique does not require any specialized instrumentation, unlike other reported minimally invasive QT graft harvesting techniques, as we harvest the graft with a close tendon stripper.

Neddylation-dependent protein degradation is a nexus between synaptic insulin resistance, neuroinflammation and Alzheimer's disease.

BACKGROUND: The metabolic syndrome is a consequence of modern lifestyle that causes synaptic insulin resistance and cognitive deficits and that in interaction with a high amyloid load is an important risk factor for Alzheimer's disease. It has been proposed that neuroinflammation might be an intervening variable, but the underlying mechanisms are currently unknown. METHODS: We utilized primary neurons to induce synaptic insulin resistance as well as a mouse model of high-risk aging that includes a high amyloid load, neuroinflammation, and diet-induced obesity to test hypotheses on underlying mechanisms. RESULTS: We found that neddylation and subsequent activation of cullin-RING ligase complexes induced synaptic insulin resistance through ubiquitylation and degradation of the insulin-receptor substrate IRS1 that organizes synaptic insulin signaling. Accordingly, inhibition of neddylation preserved synaptic insulin signaling and rescued memory deficits in mice with a high amyloid load, which were fed with a 'western diet'. CONCLUSIONS: Collectively, the data suggest that neddylation and degradation of the insulin-receptor substrate is a nodal point that links high amyloid load, neuroinflammation, and synaptic insulin resistance to cognitive decline and impaired synaptic plasticity in high-risk aging.

Monteggia fracture dislocation equivalents--analysis of eighteen cases treated by open reduction and internal fixation.

OBJECTIVE: Monteggia fracture dislocation equivalent, though already described by Bado, is still an unclassified entity. We aimed to retrospectively analyze 18 cases of Monteggia variants and discuss the injury mechanisms, management, and outcome along with a review of the literature. METHODS: A retrospective record of Monteggia fracture dislocation (2003-2008) was reviewed from medical record department of our institute. Classic Monteggia fracture dislocation, children below 12 years or adults over 50 years, as well as open grade II and III cases were excluded from this study. Monteggia variant inclusion criteria included fracture of the proximal ulna together with a fracture of the radial head or neck and skeletal maturity. Totally 26 patients were identified with Monteggia variants and 18 were available for follow-up, including 11 males and 7 females with the mean age of 35 years. The ulna fracture was treated by compression plating along with tension band wiring. Radial head/neck was reconstructed in 12 patients while excised in 6 patients. RESULTS: Follow-up ranged from 1-4 years, mean 2.6 years. Patients were assessed clinicoradiologically. Mayo Elbow Performance Score was employed to assess the outcomes. At final follow-up, the results were excellent in 10 patients, good in 4, fair in 2 and poor in 2. Mean range of motion of the elbow was 20 degree 116 degree, 50 degree and 55 degree for extension, flexion, pronation and supination, respectively. Two patients had complications in the form of heterotopic ossification and stiffness of the elbow. One nonunion ulna, primarily treated by tension band wiring, was managed by refixation with locking reconstruction plate and bone grafting. Bone grafting was only required in this patient for nonunion. Another patient had implants removed on his request. The results in our series closely correlated with extent of intraarticular damage, coronoid fracture and comminuted fractures. CONCLUSIONS: Monteggia fracture dislocation equivalents are rare injuries and pre-surgery recognition by radiographs and 3-D CT helps make optimal plan. The poor results usually relate to intraarticular damage, coronoid fractures and comminution of the ulna and radial head fractures.

The nuclear lamina is a hub for the nuclear function of Jacob.

Jacob is a synapto-nuclear messenger protein that couples NMDAR activity to CREB-dependent gene expression. In this study, we investigated the nuclear distribution of Jacob and report a prominent targeting to the nuclear envelope that requires NMDAR activity and nuclear import. Immunogold electron microscopy and proximity ligation assay combined with STED imaging revealed preferential association of Jacob with the inner nuclear membrane where it directly binds to LaminB1, an intermediate filament and core component of the inner nuclear membrane (INM). The association with the INM is transient; it involves a functional nuclear export signal in Jacob and a canonical CRM1-RanGTP-dependent export mechanism that defines the residing time of the protein at the INM. Taken together, the data suggest a stepwise redistribution of Jacob within the nucleus following nuclear import and prior to nuclear export.

Radiological evaluation of tunnel position in single bundle anterior cruciate ligament reconstruction in the Indian population and their clinical correlation.

BACKGROUND: Proper positioning of osseous tunnels during single bundle arthroscopic ACL reconstruction, which gives reproducibly good clinical outcome, is a matter of concern. Little evidence is there correlating tunnel position in arthroscopic ACL reconstruction with their clinical outcome in Indian population. Our aim in this study was to examine if the radiological tunnel-positions were significantly associated to the clinical outcomes. METHODS: ACL reconstruction was performed in 147 young patients with an isolated ACL tear. They were followed up prospectively for the next two years. Clinical assessment of each patient was done using the International Knee Documentation Committee (IKDC) evaluation form before surgery and at two years later the surgery. At the same time, the radiological assessment was done on standard digital radiographs. RESULTS: Considering the anterior and posterior-most points on the Blumensaat's line as 0% and 100% respectively the average position of the femoral tunnel was at 84.8%. Similarly, the tibial tunnel was at 46.8% along the tibial plateau. On the coronal plane the average position of the tibial tunnel was at 45.6% point along the tibial plateau (measured from the medial-most point towards laterally). The mean position of the femoral tunnel in the coronal plane was at 43.2% along the broadest part of the distal femur (measured from the lateral extent). The average inclination angle of the graft measured 19.6° (along the coronal plane). CONCLUSION: Ideal clinical outcome was significantly associated with the placement of the femoral tunnel along the sagittal plane. Placement of the femoral tunnel should not be beyond the 85% mark along the Blumensaat's line from the anterior-most point. No correlation was established between clinical results and any of the remaining radiological parameters described above.

SIPA1L2 controls trafficking and local signaling of TrkB-containing amphisomes at presynaptic terminals.

Amphisomes are organelles of the autophagy pathway that result from the fusion of autophagosomes with late endosomes. While biogenesis of autophagosomes and late endosomes occurs continuously at axon terminals, non-degradative roles of autophagy at boutons are barely described. Here, we show that in neurons BDNF/TrkB traffick in amphisomes that signal locally at presynaptic boutons during retrograde transport to the soma. This is orchestrated by the Rap GTPase-activating (RapGAP) protein SIPA1L2, which connects TrkB amphisomes to a dynein motor. The autophagosomal protein LC3 regulates RapGAP activity of SIPA1L2 and controls retrograde trafficking and local signaling of TrkB. Following induction of presynaptic plasticity, amphisomes dissociate from dynein at boutons enabling local signaling and promoting transmitter release. Accordingly, sipa1l2 knockout mice show impaired BDNF-dependent presynaptic plasticity. Taken together, the data suggest that in hippocampal neurons, TrkB-signaling endosomes are in fact amphisomes that during retrograde transport have local signaling capacity in the context of presynaptic plasticity.