Bridging Protein Research Articles

Structural Basis for LMO2-Driven Recruitment of the SCL:E47bHLH Heterodimer to Hematopoietic-Specific Transcriptional Targets

SummaryCell fate is governed by combinatorial actions of transcriptional regulators assembling into multiprotein complexes. However, the molecular details of how these complexes form are poorly understood. One such complex, which contains the basic-helix-loop-helix heterodimer SCL:E47 and bridging proteins LMO2:LDB1, critically regulates hematopoiesis and induces T cell leukemia. Here, we report the crystal structure of (SCL:E47)bHLH:LMO2:LDB1LID bound to DNA, providing a molecular account of the network of interactions assembling this complex. This reveals an unexpected role for LMO2. Upon binding to SCL, LMO2 induces new hydrogen bonds in SCL:E47, thereby strengthening heterodimer formation. This imposes a rotation movement onto E47 that weakens the heterodimer:DNA interaction, shifting the main DNA-binding activity onto additional protein partners. Along with biochemical analyses, this illustrates, at an atomic level, how hematopoietic-specific SCL sequesters ubiquitous E47 and associated cofactors and supports SCL’s reported DNA-binding-independent functions. Importantly, this work will drive the design of small molecules inhibiting leukemogenic processes.

Lactadherin/MFG‐E8 is essential for microglia‐mediated neuronal loss and phagoptosis induced by amyloid β

Nanomolar β-amyloid peptide (Aβ) can induce neuronal loss in culture by activating microglia to phagocytose neurons. We report here that this neuronal loss is mediated by the bridging protein lactadherin/milk-fat globule epidermal growth factor-like factor 8 (MFG-E8), which is released by Aβ-activated microglia, binds to co-cultured neurons and opsonizes neurons for phagocytosis by microglia. Aβ stimulated microglial phagocytosis, but did not opsonize neurons for phagocytosis. Aβ (250nM) induced delayed neuronal loss in mixed glial-neuronal mouse cultures that required microglia and occurred without increasing neuronal apoptosis or necrosis. This neuronal death/loss was prevented by antibodies to MFG-E8 and was absent in cultures from Mfge8 knockout mice (leaving viable neurons), but was reconstituted by addition of recombinant MFG-E8. Thus, nanomolar Aβ caused neuronal death by inducing microglia to phagocytose otherwise viable neurons via MFG-E8. The direct neurotoxicity of micromolar Aβ was not affected by MFG-E8. The essential role of MFG-E8 in Aβ-induced phagoptosis, suggests this bridging protein as a potential therapeutic target to prevent neuronal loss in Alzheimer's disease.

Identification of a Novel Complex AβPP:Fe65:PP1 that Regulates AβPP Thr668 Phosphorylation Levels

The amyloid-β protein precursor (AβPP) binds several proteins determining metabolism, processing, and the physiological fate of the former. Among these is Fe65, a protein with specific functional significance for AβPP, in particular conferring stability when the latter is dephosphorylated on Thr668. Thus, it follows that phosphatases like protein phosphatase 1 (PP1) are relevant to AβPP processing. Consequently, the identification of AβPP binding proteins, which can be modulated directly or indirectly by PP1, take on added relevance in terms of biological significance. Using the yeast tri-hybrid system and co-immunoprecipitation assays, we describe a novel tri-complex comprising AβPP, Fe65 and PP1. We show that the trimeric complex (AβPP:Fe65:PP1γ) occurs in COS-7 cells, rat hippocampal and cortical primary neurons, and in adult rat hippocampus and cortex. Using overlay assays, we demonstrate that Fe65 is in fact the bridging protein in the complex formed and thus we simultaneously describe another PP1 binding protein. This is singularly important given that PP1 binding proteins determine and confer subcellular localization, as well as substrate specificity, thus regulating the phosphatase activity and subsequent intracellular events. Additionally, we show that this interaction correlates with AβPP Thr668 phosphorylation state, consistent with the role of protein (de)phosphorylation as a key mechanism in regulating cellular events.

Probing the role of interfacial waters in protein–DNA recognition using a hybrid implicit/explicit solvation model

When proteins bind to their DNA target sites, ordered water molecules are often present at the protein-DNA interface bridging protein and DNA through hydrogen bonds. What is the role of these ordered interfacial waters? Are they important determinants of the specificity of DNA sequence recognition, or do they act in binding in a primarily nonspecific manner, by improving packing of the interface, shielding unfavorable electrostatic interactions, and solvating unsatisfied polar groups that are inaccessible to bulk solvent? When modeling details of structure and binding preferences, can fully implicit solvent models be fruitfully applied to protein-DNA interfaces, or must the individualistic properties of these interfacial waters be accounted for? To address these questions, we have developed a hybrid implicit/explicit solvation model that specifically accounts for the locations and orientations of small numbers of DNA-bound water molecules, while treating the majority of the solvent implicitly. Comparing the performance of this model with that of its fully implicit counterpart, we find that explicit treatment of interfacial waters results in a modest but significant improvement in protein side-chain placement and DNA sequence recovery. Base-by-base comparison of the performance of the two models highlights DNA sequence positions whose recognition may be dependent on interfacial water. Our study offers large-scale statistical evidence for the role of ordered water for protein-DNA recognition, together with detailed examination of several well-characterized systems. In addition, our approach provides a template for modeling explicit water molecules at interfaces that should be extensible to other systems.

The Role of Cav2.2 C-Terminus in Calcium Channel Anchoring at the Presynaptic Transmitter Release Sites

Voltage gated neuronal calcium channels (Cav2.2) gate neurotransmitter release at specialized regions within the presynaptic nerve terminals, known as transmitter release sites (TRS). Published studies have predicted that calcium channels are closely attached to the TRS in an organized structure, supporting a channel-channel anchoring mechanism. Although calcium channels have been linked to a broad range of presynaptic proteins, molecular components of the channel-channel anchor are poorly understood. Our objective is to identify the mechanism of Ca2+ channel anchoring at the TRS and will explore the role of channel cytoplasmic regions. GST or strep tagged Cav2.2 cytoplasmic region fusion proteins were generated and used for pull-down assays from which distal C-terminus region (C3strep) captured the channel from brain lysate. In order to determine the specific region responsible for Cav2.2 pull-down, GST fusion proteins comprising overlapping proximal (C3proximal) and distal (C3distal) regions of C3strep were generated. Our data show that C3distal region captures the channel from avian synaptosome membrane lysate. This finding was supported by a reduction of channel pull-down from brain lysate in the presence of a blocking peptide mimicking the last four amino acids of long splice variant Cav2.2 distal C-terminus which code a PDZ-ligand domain. C3strep protein was also able to capture transfected Cav2.2 from TSA201 cell lysate, narrowing down the possibilities of a channel-channel bridging protein(s). Overall, these data suggest a key role for Cav2.2 distal C-terminus in channel anchoring at TRS.

Structural and biochemical analysis of the assembly and function of the yeast pre-mRNA 3′ end processing complex CF I

The accuracy of the 3'-end processing by cleavage and polyadenylation is essential for mRNA biogenesis and transcription termination. In yeast, two poorly conserved neighboring elements upstream of cleavage sites are important for accuracy and efficiency of this process. These two RNA sequences are recognized by the RNA binding proteins Hrp1 and Rna15, but efficient processing in vivo requires a bridging protein (Rna14), which forms a stable dimer of hetero-dimers with Rna15 to stabilize the RNA-protein complex. We earlier reported the structure of the ternary complex of Rna15 and Hrp1 bound to the RNA processing element. We now report the use of solution NMR to study the interaction of Hrp1 with the Rna14-Rna15 heterodimer in the presence and absence of 3'-end processing signals. By using methyl selective labeling on Hrp1, in vivo activity and pull-down assays, we were able to study this complex of several hundred kDa, identify the interface within Hrp1 responsible for recruitment of Rna14 and validate the functional significance of this interaction through structure-driven mutational analysis.

Extractability and Molecular Modifications of Gliadin and Glutenin Proteins Withdrawn from Different Stages of a Commercial Ethanol Fuel/Distillers Dried Grains with Solubles Process Using a Wheat Feedstock

ABSTRACTExtractability and molecular modifications of gliadin and glutenin proteins withdrawn from different stages of a commercial ethanol fuel/distillers dried grains with solubles (DDGS) process using a wheat feedstock were investigated. Materials were taken postliquefaction (PL), postdistillation (whole stillage), and postdrying (DDGS) during the process and then fractionated to separate the gliadins and the soluble high‐ and low‐molecular‐weight glutenins following a modified Verbruggen extraction method. Each fraction was characterized based on the extraction efficiencies within various aqueous alcohols of propan‐1‐ol, electrophoretic patterns, intrinsic and extrinsic fluorescence, free and total sulfhydryl content, and total disulfide bond levels. Findings indicated significant changes to the composition of extracted proteins and modifications to the protein structure (i.e., surface properties and conformation) throughout the ethanol/DDGS process, beginning with the first step of production (PL, ≈83°C). Overall, processing resulted in a shift toward an unextractable gluten matrix, accompanied by increases in hydrophobicity, disulfide bridging, and excessive protein aggregation.

Hepatocyte odd protein shuttling (HOPS) is a bridging protein in the nucleophosmin-p19Arf network

Nucleophosmin (NPM), a ubiquitously and abundantly expressed protein, occurs in the nucleolus, shuttling between the nucleoplasm and cytoplasm. The NPM gene is mutated in almost 30% of human acute myeloid leukemia cells. NPM interacts with p53 and p19(Arf), directs localization of p19(Arf) in the nucleolus and protects the latter from degradation. Hepatocyte odd protein shuttling (HOPS) is also a ubiquitously expressed protein that moves between the nucleus and cytoplasm. Within the nucleus of resting cells, HOPS overexpression causes cell cycle arrest in G0/G1. HOPS knockdown causes centrosome hyperamplification leading to multinucleated cells and the formation of micronuclei. We demonstrate a direct interaction of HOPS with NPM and p19(Arf), resulting in a functionally active trimeric complex. NPM appeared to regulate HOPS half-life, which, in turn, stabilized p19(Arf) and controlled its localization in the nucleolus. These findings suggest that HOPS acts as a functional bridge in the interaction between NPM and p19(Arf), providing new mechanistic insight into how NPM and p19(Arf) will oppose tumor cell proliferation.

Innate and adaptive immune responses to dead and dying cells

Under steady state conditions, billions of dead and dying cells are removed by extrusion from epithelial surfaces as well as by phagocytosis. Cells such as macrophages and dendritic cells have specialized receptors that directly recognize altered protein or lipids on apoptotic cells or opsonins that bind to the dying cell. Once engulfed, phagosomes containing apoptotic cells are rapidly acidified and the contents degraded by proteases and nucleases in lysozymes. During necrosis, cellular material is released prior to engulfment and extracellular nucleases as well as intracellular sensors dictate the inflammatory potential of the cellular debris. The outcome may be release of TNF-a, IL-1-b or interferon (IFN)-a depending upon the type of phagocyte, molecular nature of the cellular particle and the intracellular sensor engaged. In addition to responses by cells of the innate immune system, we have recently defined a link between processing of apoptotic cells and their debris to T cell activation [1]. MFG-E8 is an opsonin (bridging protein) that binds to phosphatidylserine on apoptotic cells and facilitates their removal through interaction with integrins on phagocytes. Mice deficient in MFG-E8 develop lupus like autoimmunity associated with accumulation of apoptotic cells in vivo. We observed that older MFG-8-/- mice spontaneously developed a dermatitis associated with CD8 T cell infiltration and striking activation of effector memory CD8 T cells. T cell responses to both exogenous and endogenous apoptotic cell associated antigens were enhanced in MFG-E8 deficient mice and transfer of ovalbumin (OVA) reactive OT-I CD8 T cells caused accelerated diabetes in MFG-E8-/- RIP-mOVA mice and skin disease in kmOVA transgenic mice. The enhanced CD8 T cell response was attributed to increased cross-presentation by dendritic cells (DCs) associated with increased detection of antigen peptide MHCI complexes. Investigation of intracellular trafficking revealed that, whereas intact apoptotic cells ingested by wild type DC rapidly fused with lysosomes, in the absence of MFG-E8, smaller apoptotic cell fragments persisted in endosomal compartments and failed to fuse with lysosomes. These observations suggest that in addition to altering the rate of clearance of apoptotic cells, MFG-E8 deficiency promotes immune responses to self antigens by altered intracellular processing leading to enhanced antigen presentation. Thus, handling of dead and dying cells impacts both innate and adaptive immune responses to self antigens.

Excitation energy transfer in GFP‐X‐CFP model peptides (X = amino acids): Direct Versus through‐bridge energy transfers

AbstractThe excitation energy transfer (EET) between a fluorescent resonance energy transfer (FRET) pair, cyan fluorescent protein chromophore (CFPc)/ green fluorescent protein chromophore (GFPc), is used for measuring molecular proximity in experimental studies. In this article, the role of the bridging protein media has been investigated using the quantum chemical calculations for the EET electronic coupling. We adopted a model peptide connecting GFPc and CFPc, a GFPc‐X‐CFPc model, and examined the X (amino acids) dependence. The major part of the EET coupling element arises from the direct interaction between GFPc and CFPc via the Förster mechanism. The magnitude of the through‐X interactions vary for the type of residues. The contribution of the X was, however, at most 10% of the total value. Together with the contributions from through‐peptide and charge‐transfer types, X gives minor modifications to the total coupling via the super‐exchange mechanism. © 2012 Wiley Periodicals, Inc.

The cellular antiviral protein APOBEC3G interacts with HIV-1 reverse transcriptase and inhibits its function during viral replication.

The cytidine deaminase APOBEC3G (A3G) exerts a multifaceted antiviral effect against HIV-1 infection. First, A3G was shown to be able to terminate HIV infection by deaminating the cytosine residues to uracil in the minus strand of the viral DNA during reverse transcription. Also, a number of studies have indicated that A3G inhibits HIV-1 reverse transcription by a non-editing-mediated mechanism. However, the mechanism by which A3G directly disrupts HIV-1 reverse transcription is not fully understood. In the present study, by using a cell-based coimmunoprecipitation (Co-IP) assay, we detected the direct interaction between A3G and HIV-1 reverse transcriptase (RT) in produced viruses and in the cotransfected cells. The data also suggested that their interaction did not require viral genomic RNA bridging or other viral proteins. Additionally, a deletion analysis showed that the RT-binding region in A3G was located between amino acids 65 and 132. Overexpression of the RT-binding polypeptide A3G(65-132) was able to disrupt the interaction between wild-type A3G and RT, which consequently attenuated the anti-HIV effect of A3G on reverse transcription. Overall, this paper provides evidence for the physical and functional interaction between A3G and HIV-1 RT and demonstrates that this interaction plays an important role in the action of A3G against HIV-1 reverse transcription.

β1 Integrin Gene Excision in the Adult Murine Cardiac Myocyte Causes Defective Mechanical and Signaling Responses

How mechanical signals are transmitted in the cardiac myocyte is poorly understood. In this study, we produced a tamoxifen-inducible mouse model in which β1 integrin could be reduced specifically in the adult cardiomyocyte, so that the function of this integrin could be assessed in the postnatal and mechanically stressed heart. The expression of β1 integrin was reduced to 35% of control levels, but function remained normal at baseline. With aortic constriction, the knockout mice survived but had a blunted hypertrophic response. Integrin knockout myocytes, in contrast to controls, showed reduced integrin-linked kinase expression both at baseline and after hemodynamic stress; focal adhesion kinase expression was reduced after stress. Alterations in multiple signaling pathways were detected in the integrin knockout group after acute and chronic hemodynamic stress. Most remarkably, when we challenged the knockout mice with short-term loading, the robust responses of several kinases (extracellular signal-regulated kinase 1/2, p38, and Akt) evident in control mice were essentially abolished in the knockout mice. We also found that reduction of myocyte β1 integrin expression modified adrenergic-mediated signaling through extracellular signal-regulated kinase, p38, and Akt. Reduction of β1 integrin expression in the mature cardiac myocyte leads to a varied response compared with when this protein is reduced during either the embryonic or perinatal period. These results show that β1 integrin expression is required for proper mechanotransductive and adrenergic responses of the adult heart.

Neuronal Death Induced by Nanomolar Amyloid β Is Mediated by Primary Phagocytosis of Neurons by Microglia

Alzheimer disease is characterized by neuronal loss and brain plaques of extracellular amyloid β (Aβ), but the means by which Aβ may induce neuronal loss is not entirely clear. Although high concentrations of Aβ (μM) can induce direct toxicity to neurons, we find that low concentration (nM) induce neuronal loss through a microglia-mediated mechanism. In mixed neuronal-glial cultures from rat cerebellum, 250 nM Aβ1-42 (added as monomers, oligomers or fibers) induced about 30% loss of neurons between 2 and 3 days. This neuronal loss occurred without any increase in neuronal apoptosis or necrosis, and no neuronal loss occurred with Aβ42-1. Aβ greatly increased the phagocytic capacity of microglia and induced phosphatidylserine exposure (an "eat-me" signal) on neuronal processes. Blocking exposed phosphatidylserine by adding annexin V or an antibody to phosphatidylserine or inhibiting microglial phagocytosis by adding either cytochalasin D (to block actin polymerization) or cyclo(RGDfV) (to block vitronectin receptors) significantly prevented neuronal loss. Loss of neuronal synapses occurred in parallel with loss of cell bodies and was also prevented by blocking phagocytosis. Inhibition of phagocytosis prevented neuronal loss with no increase in neuronal death, even after 7 days, suggesting that microglial phagocytosis was the primary cause of neuronal death induced by nanomolar Aβ.

BRCA1 is required for hMLH1 stabilization following doxorubicin-induced DNA damage

Human DNA mismatch repair (MMR) is involved in the removal of DNA base mismatches that arise either during DNA replication or are caused by DNA damage. In this study, we show that the activation of the MMR component hMLH1 in response to doxorubicin (DOX) treatment requires the presence of BRCA1 and that this phenomenon is mediated by an ATM/ATR dependent phosphorylation of the hMLH1 Ser-406 residue. BRCA1 is an oncosuppressor protein with a central role in the DNA damage response and it is a critical component of the ATM/ATR mediated checkpoint signaling. Starting from a previous finding in which we demonstrated that hMLH1 is able to bind to BRCA1, in this study we asked whether BRCA1 might be the bridge for ATM/ATR dependent phosphorylation of the hMLH1 molecular partner. We found that: ( i) the negative modulation of BRCA1 expression is able to produce a remarkable reversal of hMLH1 stabilization, ( ii) BRCA1 is required for post-translational modification produced by DOX treatment on hMLH1 which is, in turn, attributed to the ATM/ATR activity, ( iii) the serine 406 phosphorylatable residue is critical for hMLH1 activation by ATM/ATR via BRCA1. Taken together, our data lend support to the hypothesis suggesting an important role of this oncosuppressor as a scaffold or bridging protein in DNA-damage response signaling via downstream phosphorylation of the ATM/ATR substrate hMLH1.

DAZAP1, an RNA-binding protein required for development and spermatogenesis, can regulate mRNA translation.

DAZ-associated protein 1 (DAZAP1) is an RNA-binding protein required for normal growth, development, and fertility in mice. However, its molecular functions have not been elucidated. Here we find that Xenopus laevis and human DAZAP1, which are each expressed as short and long forms, act as mRNA-specific activators of translation in a manner that is sensitive to the number of binding sites present within the 3' UTR. Domain mapping suggests that this conserved function is mainly associated with C-terminal regions of DAZAP1. Interestingly, we find that the expression of xDAZAP1 and its polysome association are developmentally controlled, the latter suggesting that the translational activator function of DAZAP1 is regulated. However, ERK phosphorylation of DAZAP1, which can alter protein interactions with its C terminus, does not play a role in regulating its ability to participate in translational complexes. Since relatively few mRNA-specific activators have been identified, we explored the mechanism by which DAZAP1 activates translation. By utilizing reporter mRNAs with internal ribosome entry sites, we establish that DAZAP1 stimulates translation initiation. Importantly, this activity is not dependent on the recognition of the 5' cap by initiation factors, showing that it functions downstream from this frequently regulated event, but is modulated by changes in the adenylation status of mRNAs. This suggests a function in the formation of "end-to-end" complexes, which are important for efficient initiation, which we show to be independent of a direct interaction with the bridging protein eIF4G.

Cyclin-dependent Kinase-mediated Phosphorylation of RBP1 and pRb Promotes Their Dissociation to Mediate Release of the SAP30·mSin3·HDAC Transcriptional Repressor Complex

Eukaryotic cell cycle progression is mediated by phosphorylation of protein substrates by cyclin-dependent kinases (CDKs). A critical substrate of CDKs is the product of the retinoblastoma tumor suppressor gene, pRb, which inhibits G(1)-S phase cell cycle progression by binding and repressing E2F transcription factors. CDK-mediated phosphorylation of pRb alleviates this inhibitory effect to promote G(1)-S phase cell cycle progression. pRb represses transcription by binding to the E2F transactivation domain and recruiting the mSin3·histone deacetylase (HDAC) transcriptional repressor complex via the retinoblastoma-binding protein 1 (RBP1). RBP1 binds to the pocket region of pRb via an LXCXE motif and to the SAP30 subunit of the mSin3·HDAC complex and, thus, acts as a bridging protein in this multisubunit complex. In the present study we identified RBP1 as a novel CDK substrate. RBP1 is phosphorylated by CDK2 on serines 864 and 1007, which are N- and C-terminal to the LXCXE motif, respectively. CDK2-mediated phosphorylation of RBP1 or pRb destabilizes their interaction in vitro, with concurrent phosphorylation of both proteins leading to their dissociation. Consistent with these findings, RBP1 phosphorylation is increased during progression from G(1) into S-phase, with a concurrent decrease in its association with pRb in MCF-7 breast cancer cells. These studies provide new mechanistic insights into CDK-mediated regulation of the pRb tumor suppressor during cell cycle progression, demonstrating that CDK-mediated phosphorylation of both RBP1 and pRb induces their dissociation to mediate release of the mSin3·HDAC transcriptional repressor complex from pRb to alleviate transcriptional repression of E2F.

When the human viral infectome and diseasome networks collide: towards a systems biology platform for the aetiology of human diseases

BackgroundComprehensive understanding of molecular mechanisms underlying viral infection is a major challenge towards the discovery of new antiviral drugs and susceptibility factors of human diseases. New advances in the field are expected from systems-level modelling and integration of the incessant torrent of high-throughput "-omics" data.ResultsHere, we describe the Human Infectome protein interaction Network, a novel systems virology model of a virtual virus-infected human cell concerning 110 viruses. This in silico model was applied to comprehensively explore the molecular relationships between viruses and their associated diseases. This was done by merging virus-host and host-host physical protein-protein interactomes with the set of genes essential for viral replication and involved in human genetic diseases. This systems-level approach provides strong evidence that viral proteomes target a wide range of functional and inter-connected modules of proteins as well as highly central and bridging proteins within the human interactome. The high centrality of targeted proteins was correlated to their essentiality for viruses' lifecycle, using functional genomic RNAi data. A stealth-attack of viruses on proteins bridging cellular functions was demonstrated by simulation of cellular network perturbations, a property that could be essential in the molecular aetiology of some human diseases. Networking the Human Infectome and Diseasome unravels the connectivity of viruses to a wide range of diseases and profiled molecular basis of Hepatitis C Virus-induced diseases as well as 38 new candidate genetic predisposition factors involved in type 1 diabetes mellitus.ConclusionsThe Human Infectome and Diseasome Networks described here provide a unique gateway towards the comprehensive modelling and analysis of the systems level properties associated to viral infection as well as candidate genes potentially involved in the molecular aetiology of human diseases.

Milk Fat Globule-EGF Factor VIII in Sepsis and Ischemia-Reperfusion Injury

Sepsis and ischemia-reperfusion (I/R) injury are among the leading causes of death in critically ill patients at the surgical intensive care unit setting. Both conditions are marked by the excessive inflammatory response which leads to a lethal disease complex such as acute lung injury, systemic inflammatory response syndrome and multiple organ dysfunction syndrome. Despite the advances in the understanding of the pathophysiology of those conditions, very little progress has been made toward therapeutic interventions. One of the key aspects of these conditions is the accumulation of apoptotic cells that have the potential to release toxic and proinflammatory contents due to secondary necrosis without appropriate clearance by phagocytes. Along with the prevention of apoptosis, that is reported to be beneficial in sepsis and I/R injury, thwarting the development of secondary necrosis through the active removal of apoptotic cells via phagocytosis may offer a novel therapy. Milk fat globule-EGF factor VIII (MFG-E8), which is mainly produced by macrophages and dendritic cells, is an opsonin for apoptotic cells and acts as a bridging protein between apoptotic cells and phagocytes. Recently, we have shown that MFG-E8 expression is decreased in experimental sepsis and I/R injury models. Exogenous administration of MFG-E8 attenuated the inflammatory response as well as tissue injury and mortality through the promotion of phagocytosis of apoptotic cells. In this review, we describe novel information available about the involvement of MFG-E8 in the pathophysiology of sepsis and I/R injury, and the therapeutic potential of exogenous MFG-E8 treatment for those conditions.

Mammalian Sperm Head Formation Involves Different Polarization of Two Novel LINC Complexes

BackgroundLINC complexes are nuclear envelope bridging protein structures formed by interaction of SUN and KASH proteins. They physically connect the nucleus with the peripheral cytoskeleton and are critically involved in a variety of dynamic processes, such as nuclear anchorage, movement and positioning and meiotic chromosome dynamics. Moreover, they are shown to be essential for maintaining nuclear shape.FindingsBased on detailed expression analysis and biochemical approaches, we show here that during mouse sperm development, a terminal cell differentiation process characterized by profound morphogenic restructuring, two novel distinctive LINC complexes are established. They consist either of spermiogenesis-specific Sun3 and Nesprin1 or Sun1η, a novel non-nuclear Sun1 isoform, and Nesprin3. We could find that these two LINC complexes specifically polarize to opposite spermatid poles likely linking to sperm-specific cytoskeletal structures. Although, as shown in co-transfection/immunoprecipitation experiments, SUN proteins appear to arbitrarily interact with various KASH partners, our study demonstrates that they actually are able to confine their binding to form distinct LINC complexes.ConclusionsFormation of the mammalian sperm head involves assembly and different polarization of two novel spermiogenesis-specific LINC complexes. Together, our findings suggest that theses LINC complexes connect the differentiating spermatid nucleus to surrounding cytoskeletal structures to enable its well-directed shaping and elongation, which in turn is a critical parameter for male fertility.

Intrinsic molecular properties of the protein–protein bridge facilitate ratchet-like motion of the ribosome

The ribosomal intersubunit bridges maintain the overall architecture of the ribosome and thereby play a pivotal role in the dynamics of translation. The only protein–protein bridge, b1b, is formed by the two proteins, S13 and L5 of the small and large ribosomal subunits, respectively. B1b absorbs the largest movement during ratchet-like motion, and its two proteins reorganize in different constellations during this motion of the ribosome.Our results in this study of b1b in the Escherichia coli 70S ribosome suggest that the intrinsic molecular features of the bridging proteins allow the bridge to modulate the ratchet-like motion in a controlled manner. Additionally, another large subunit protein, L31, seems to participate with S13 and L5 in the formation, dynamics, and stabilization of this bridge.