Search

Antibodies are fundamental effectors of humoral immunity, and have become a highly successful class of therapeutics. There is increasing evidence that antibodies utilize transient homotypic interactions to enhance function, and elucidation of such interactions can provide insights into their biology and new opportunities for their optimization as drugs. Yet the transitory nature of weak interactions makes them difficult to investigate. Capitalizing on their rich structural data and high conservation, we have characterized all the ways that antibody Fab regions interact crystallographically. This approach led to the discovery of previously unrealized interfaces between antibodies. While diverse interactions exist, β-sheet dimers and variable-constant elbow dimers are recurrent motifs. Disulfide engineering enabled interactions to be trapped and investigated structurally and functionally, providing experimental validation of the interfaces and illustrating their potential for optimization. This work provides first insight into previously undiscovered oligomeric interactions between antibodies, and enables new opportunities for their biotherapeutic optimization.

Hefty structures of IgA and IgM complexes Immunoglobulin M (IgM) and IgA are antibody isotypes that can form higher-order secretory complexes (sIgM and sIgA), which allows them to effectively bind and neutralize antigens with low-affinity repetitive epitopes, such as those found on the surface of many bacteria and viruses. The assembly and transport of these molecules is also dependent on the joining chain (J-chain) and the polymeric immunoglobulin receptor (pIgR) secretory component (SC). The architecture of these complex, multimeric structures has remained elusive. Li et al. resolved cryo–electron microscopy structures of the sIgM-Fc pentamer in complex with the J-chain and SC. Using similar techniques, Kumar et al. visualized dimeric, tetrameric, and pentameric structures of secretory sIgA-Fc interacting with the J-chain and SC. Both groups report highly similar mechanisms wherein the J-chain serves as a template for antibody oligomerization. An unanticipated, amyloid-like assembly of the oligomerized structure is present in both cases, with the J-chain conferring asymmetry for pIgR binding and transcytosis. These studies may inform structure-based engineering of these molecules for future therapeutic purposes. Science , this issue p. 1014 , p. 1008

During macroautophagic stress, autophagosomes can be produced continuously and in high numbers. Many different organelles have been reported as potential donor membranes for this sustained autophagosome growth, but specific machinery to support the delivery of lipid to the growing autophagosome membrane has remained unknown. Here we show that the autophagy protein, ATG2, without a clear function since its discovery over 20 yr ago, is in fact a lipid-transfer protein likely operating at the ER–autophagosome interface. ATG2A can bind tens of glycerophospholipids at once and transfers lipids robustly in vitro. An N-terminal fragment of ATG2A that supports lipid transfer in vitro is both necessary and fully sufficient to rescue blocked autophagosome biogenesis in ATG2A/ATG2B KO cells, implying that regulation of lipid homeostasis is the major autophagy-dependent activity of this protein and, by extension, that protein-mediated lipid transfer across contact sites is a principal contributor to autophagosome formation.

Immunoglobulins (Ig) A and M are the only human antibodies that form oligomers and undergo transcytosis to mucosal secretions via the polymeric Ig receptor (pIgR). When complexed with the J-chain (JC) and the secretory component (SC) of pIgR, secretory IgA and IgM (sIgA and sIgM) play critical roles in host-pathogen defense. Recently, we determined the structure of sIgA-Fc which elucidated the mechanism of polymeric IgA assembly and revealed an extensive binding interface between IgA-Fc, JC, and SC. Despite low sequence identity shared with IgA-Fc, IgM-Fc also undergoes JC-mediated assembly and binds pIgR. Here, we report the structure of sIgM-Fc and carryout a systematic comparison to sIgA-Fc. Our structural analysis reveals a remarkably conserved mechanism of JC-templated oligomerization and SC recognition of both IgM and IgA through highly a conserved network of interactions. These studies reveal the structurally conserved features of sIgM and sIgA required for function in mucosal immunity.

The phosphoinositide PI(3,5)P(2), generated exclusively by the PIKfyve lipid kinase complex, is key for lysosomal biology. Here we explore how PI(3,5)P(2) levels within cells are regulated. We find the PIKfyve complex comprises 5 copies of the scaffolding protein Vac14, and one copy each of the lipid kinase PIKfyve, generating PI(3,5)P(2) from PI3P, and the lipid phosphatase Fig4, reversing the reaction. Fig4 is active as a lipid phosphatase in the ternary complex, whereas PIKfyve within the complex cannot access membrane-incorporated phosphoinositides due to steric constraints. We find further that the phosphoinositide-directed activities of both PIKfyve and Fig4 are regulated by protein-directed activities within the complex. PIKfyve autophosphorylation represses its lipid kinase activity and stimulates Fig4 lipid phosphatase activity. Further, Fig4 is also a protein phosphatase acting on PIKfyve to stimulate its lipid kinase activity, explaining why catalytically active Fig4 is required for maximal PI(3,5)P(2) production by PIKfyve in vivo.

Structural and biochemical studies show that VPS13 family proteins are lipid transporters. VPS13A and VPS13C tether the ER to mitochondria and to endosomes/lysosomes, respectively, suggesting lipid dyshomeostasis as the cause of chorea acanthocytosis and Parkinson’s disease resulting from their mutations., Mutations in the human VPS13 genes are responsible for neurodevelopmental and neurodegenerative disorders including chorea acanthocytosis (VPS13A) and Parkinson’s disease (VPS13C). The mechanisms of these diseases are unknown. Genetic studies in yeast hinted that Vps13 may have a role in lipid exchange between organelles. In this study, we show that the N-terminal portion of VPS13 is tubular, with a hydrophobic cavity that can solubilize and transport glycerolipids between membranes. We also show that human VPS13A and VPS13C bind to the ER, tethering it to mitochondria (VPS13A), to late endosome/lysosomes (VPS13C), and to lipid droplets (both VPS13A and VPS13C). These findings identify VPS13 as a lipid transporter between the ER and other organelles, implicating defects in membrane lipid homeostasis in neurological disorders resulting from their mutations. Sequence and secondary structure similarity between the N-terminal portions of Vps13 and other proteins such as the autophagy protein ATG2 suggest lipid transport roles for these proteins as well.

Objective— To study atherosclerosis regression in mice after plasma lipid reduction to moderately elevated apolipoprotein B (apoB)–lipoprotein levels. Approach and Results— Chow-fed hypomorphic Apoe mice deficient in low-density lipoprotein receptor expression ( Apoe h/h Ldlr –/– Mx1-cre mice) develop hyperlipidemia and atherosclerosis. These mice were studied before and after inducible cre-mediated Apoe gene repair. By 1 week, induced mice displayed a 2-fold reduction in plasma cholesterol and triglyceride levels and a decrease in the non–high-density lipoprotein:high-density lipoprotein-cholesterol ratio from 87%:13% to 60%:40%. This halted atherosclerotic lesion growth and promoted macrophage loss and accumulation of thick collagen fibers for up to 8 weeks. Concomitantly, blood Ly-6C high monocytes were decreased by 2-fold but lesional macrophage apoptosis was unchanged. The expression of several genes involved in extracellular matrix remodeling and cell migration was changed in lesional macrophages 1 week after Apoe gene repair. However, mRNA levels of numerous genes involved in cholesterol efflux and inflammation were not significantly changed at this time point. Conclusions— Restoring apoE expression in Apoe h/h Ldlr –/– Mx1-cre mice resulted in lesion stabilization in the context of a human-like ratio of non–high-density lipoprotein:high-density lipoprotein-cholesterol. Our data suggest that macrophage loss derived in part from reduced blood Ly-6C high monocytes levels and genetic reprogramming of lesional macrophages.

Kindlins are essential FERM-domain-containing focal adhesion (FA) proteins required for proper integrin activation and signaling. Despite the widely accepted importance of each of the three mammalian kindlins in cell adhesion, the molecular basis for their function has yet to be fully elucidated, and the functional differences between isoforms have generally not been examined. Here, we report functional differences between kindlin-2 and -3 (also known as FERMT2 and FERMT3, respectively); GFP-tagged kindlin-2 localizes to FAs whereas kindlin-3 does not, and kindlin-2, but not kindlin-3, can rescue α5β1 integrin activation defects in kindlin-2-knockdown fibroblasts. Using chimeric kindlins, we show that the relatively uncharacterized kindlin-2 F2 subdomain drives FA targeting and integrin activation. We find that the integrin-linked kinase (ILK)–PINCH–parvin complex binds strongly to the kindlin-2 F2 subdomain but poorly to that of kindlin-3. Using a point-mutated kindlin-2, we establish that efficient kindlin-2-mediated integrin activation and FA targeting require binding to the ILK complex. Thus, ILK-complex binding is crucial for normal kindlin-2 function and differential ILK binding contributes to kindlin isoform specificity.

FTY720, an analogue of sphingosine-1-phosphate, is cardioprotective during acute injury. Whether long-term FTY720 affords cardioprotection is unknown. Here, we report the effects of oral FTY720 on ischemia/reperfusion injury and in hypomorphic apoE mice deficient in SR-BI receptor expression (ApoeR61(h/h)/SRB1(-/- mice), a model of diet-induced coronary atherosclerosis and heart failure. We added FTY720 (0.3 mg·kg(-1)·d(-1)) to the drinking water of C57BL/6J mice. After ex vivo cardiac ischemia/reperfusion injury, these mice had significantly improved left ventricular (LV) developed pressure and reduced infarct size compared with controls. Subsequently, ApoeR61(h/h)/SRB1(-/-) mice fed a high-fat diet for 4 weeks were treated or not with oral FTY720 (0.05 mg·kg(-1)·d(-1)). This sharply reduced mortality (P < 0.02) and resulted in better LV function and less LV remodeling compared with controls without reducing hypercholesterolemia and atherosclerosis. Oral FTY720 reduced the number of blood lymphocytes and increased the percentage of CD4+Foxp3+ regulatory T cells (Tregs) in the circulation, spleen, and lymph nodes. FTY720-treated mice exhibited increased TGF-β and reduced IFN-γ expression in the heart. Also, CD4 expression was increased and strongly correlated with molecules involved in natural Treg activity, such as TGF-β and GITR. Our data suggest that long-term FTY720 treatment enhances LV function and increases longevity in mice with heart failure. These benefits resulted not from atheroprotection but from systemic immunosuppression and a moderate reduction of inflammation in the heart.

FTY720 is an analogue of sphingosine-1-phosphate, which is cardioprotective during acute injury. Whether FTY720 affords cardioprotection and prevents chronic heart failure (CHF) after myocardial infarction (MI) is unknown. Initially we added FTY720 (0.3mg/kg/day) to the drinking water of C57BL/6J mice. After ex vivo cardiac I/R injury these mice had significantly improved left ventricular (LV) developed pressure and reduced infarct size compared with controls. Next, we fed FTY720 to a previously described mouse model of diet-induced coronary atherosclerosis, MI and premature death. This model was developed by breeding our hypomorphic apoE mice, also referred to as “HypoE” mice, which express sub-physiological apoE in all tissues due to Apoeh/h alleles, to mice deficient in the SR-BI receptor (Apoeh/h/SRB1-/- mice). These mice were given a high fat diet (HFD) for 4 weeks and treated or not with oral FTY720 (0.05mg/kg/day). This therapy sharply reduced mortality (p

Apolipoprotein (apo) E can suppress atherosclerosis by regulating myeloid cell proliferation and monocyte activation. However, little is known about its capacity to regulate adaptive immunity. To explore this question, we studied our recently described HypoE mice deficient in the LDL receptor (Apoeh/hLdlr−/− mice) that spontaneously develop a hyperlipidemia similar to that of Apoe-/-Ldlr-/- mice when fed a chow diet, but accumulate apoE in plasma and display reduced atherosclerosis. Twenty-week old Apoeh/hLdlr-/- mice (n=10) displayed smaller spleen and lymph nodes that contained fewer leukocytes (p

Objectives— Apolipoprotein (apo) E4 is an established risk factor for atherosclerosis, but the structural components underlying this association remain unclear. ApoE4 is characterized by 2 biophysical properties: domain interaction and molten globule state. Substituting Arg-61 for Thr-61 in mouse apoE introduces domain interaction without molten globule state, allowing us to delineate potential proatherogenic effects of domain interaction in vivo. Methods and Results— We studied atherosclerosis susceptibility of hypomorphic Apoe mice expressing either Thr-61 or Arg-61 apoE ( Apoe T h/h or Apoe R h/h mice). On a chow diet, both mouse models were normolipidemic with similar levels of plasma apoE and lipoproteins. However, on a high-cholesterol diet, Apoe R h/h mice displayed increased levels of total plasma cholesterol and very-low-density lipoprotein as well as larger atherosclerotic plaques in the aortic root, arch, and descending aorta compared with Apoe T h/h mice. In addition, evidence of cellular dysfunction was identified in peritoneal Apoe R h/h macrophages which released lower amounts of apoE in culture medium and displayed increased expression of major histocompatibility complex class II molecules. Conclusions— These data indicate that domain interaction mediates proatherogenic effects of apoE4 in part by modulating lipoprotein metabolism and macrophage biology. Pharmaceutical targeting of domain interaction could lead to new treatments for atherosclerosis in apoE4 individuals.

Background Apolipoprotein (apo) E is best known for its ability to lower plasma cholesterol and protect against atherosclerosis. Although the liver is the major source of plasma apoE, extra-hepatic sources of apoE, including from macrophages, account for up to 10% of plasma apoE levels. This study examined the contribution of macrophage-derived apoE expression levels in diet-induced hyperlipidemia and atherosclerosis. Methodology/Principal Findings Hypomorphic apoE (Apoe h/h) mice expressing wildtype mouse apoE at ∼2–5% of physiological levels in all tissues were derived by gene targeting in embryonic stem cells. Cre-mediated gene repair of the Apoe h/h allele in Apoe h/hLysM-Cre mice raised apoE expression levels by 26 fold in freshly isolated peritoneal macrophages, restoring it to 37% of levels seen in wildtype mice. Chow-fed Apoe h/hLysM-Cre and Apoe h/h mice displayed similar plasma apoE and cholesterol levels (55.53±2.90 mg/dl versus 62.70±2.77 mg/dl, n = 12). When fed a high-cholesterol diet (HCD) for 16 weeks, Apoe h/hLysM-Cre mice displayed a 3-fold increase in plasma apoE and a concomitant 32% decrease in plasma cholesterol when compared to Apoe h/h mice (602.20±22.30 mg/dl versus 888.80±24.99 mg/dl, n = 7). On HCD, Apoe h/hLysM-Cre mice showed increased apoE immunoreactivity in lesional macrophages and liver-associated Kupffer cells but not hepatocytes. In addition, Apoe h/hLysM-Cre mice developed 35% less atherosclerotic lesions in the aortic root than Apoe h/h mice (167×103±16×103 µm2 versus 259×103±56×103 µm2, n = 7). This difference in atherosclerosis lesions size was proportional to the observed reduction in plasma cholesterol. Conclusions/Significance Macrophage-derived apoE raises plasma apoE levels in response to diet-induced hyperlipidemia and by such reduces atherosclerosis proportionally to the extent to which it lowers plasma cholesterol levels.

Diabetic patients are known to be more susceptible to atherosclerosis and its associated cardiovascular complications. However, the effects of hyperglycemia on atherosclerosis regression remain unclear. We hypothesized that hyperglycemia impairs atherosclerosis regression by modulating the biological function of lesional macrophages. HypoE ( Apoe h/h Mx1-Cre) mice express low levels of apolipoprotein E (apoE) and develop atherosclerosis when fed a high-fat diet. Atherosclerosis regression occurs in these mice upon plasma lipid lowering induced by a change in diet and the restoration of apoE expression. We examined the morphological characteristics of regressed lesions and assessed the biological function of lesional macrophages isolated with laser-capture microdissection in euglycemic and hyperglycemic HypoE mice. Hyperglycemia induced by streptozotocin treatment impaired lesion size reduction (36% versus 14%) and lipid loss (38% versus 26%) after the reversal of hyperlipidemia. However, decreases in lesional macrophage content and remodeling in both groups of mice were similar. Gene expression analysis revealed that hyperglycemia impaired cholesterol transport by modulating ATP-binding cassette A1, ATP-binding cassette G1, scavenger receptor class B family member (CD36), scavenger receptor class B1, and wound healing pathways in lesional macrophages during atherosclerosis regression. Hyperglycemia impairs both reduction in size and loss of lipids from atherosclerotic lesions upon plasma lipid lowering without significantly affecting the remodeling of the vascular wall.