Astrid Weins

Abatacept in B7-1–Positive Proteinuric Kidney Disease

Abatacept (cytotoxic T-lymphocyte-associated antigen 4-immunoglobulin fusion protein [CTLA-4-Ig]) is a costimulatory inhibitor that targets B7-1 (CD80). The present report describes five patients who had focal segmental glomerulosclerosis (FSGS) (four with recurrent FSGS after transplantation and one with primary FSGS) and proteinuria with B7-1 immunostaining of podocytes in kidney-biopsy specimens. Abatacept induced partial or complete remissions of proteinuria in these patients, suggesting that B7-1 may be a useful biomarker for the treatment of some glomerulopathies. Our data indicate that abatacept may stabilize β1-integrin activation in podocytes and reduce proteinuria in patients with B7-1-positive glomerular disease.

Mutational and Biological Analysis of {alpha}-Actinin-4 in Focal Segmental Glomerulosclerosis

Mutations in the alpha-actinin-4 gene (ACTN4) cause an autosomal dominant form of focal segmental glomerulosclerosis (FSGS). A mutational analysis was performed of ACTN4 in DNA from probands with a family history of FSGS as well as in individuals with nonfamilial FSGS. The possible contribution of noncoding variation in ACTN4 to the development of FSGS also was assessed. Multiple nucleotide variants were identified in coding and noncoding sequence. The segregation of nonsynonymous coding sequence variants was examined in the relevant families. Only a small number of nucleotide changes that seemed likely to be causing (or contributing to) disease were identified. Sequence changes that predicted I149del, W59R, V801M, R348Q, R837Q, and R310Q changes were identified. For studying their biologic relevance and their potential roles in the pathogenesis of FSGS, these variants were expressed as GFP-fusion proteins in cultured podocytes. F-actin binding assays also were performed. Three of these variants (W59R, I149del, and V801M) showed clear cellular mislocalization in the form of aggregates adjacent to the nucleus. Two of these mislocalized variants (W59R and I149del) also showed an increased actin-binding activity. The I149del mutation segregated with disease; W59R was found to be a de novo mutation in the proband. A total of five ACTN4 mutations that are believed to be disease causing (three reported previously and two novel) as well as a number of variants with unclear contribution to disease now have been identified. The possibility that some of these other variants increase the susceptibility to FSGS cannot be excluded. ACTN4 mutations seem to account for approximately 4% of familial FSGS.

Direct dynamin–actin interactions regulate the actin cytoskeleton

The large GTPase dynamin assembles into higher order structures that are thought to promote endocytosis. Dynamin also regulates the actin cytoskeleton through an unknown, GTPase‐dependent mechanism. Here, we identify a highly conserved site in dynamin that binds directly to actin filaments and aligns them into bundles. Point mutations in the actin‐binding domain cause aberrant membrane ruffling and defective actin stress fibre formation in cells. Short actin filaments promote dynamin assembly into higher order structures, which in turn efficiently release the actin‐capping protein (CP) gelsolin from barbed actin ends in vitro, allowing for elongation of actin filaments. Together, our results support a model in which assembled dynamin, generated through interactions with short actin filaments, promotes actin polymerization via displacement of actin‐CPs.

Disease-associated mutant α-actinin-4 reveals a mechanism for regulating its F-actin-binding affinity

α-Actinin-4 is a widely expressed protein that employs an actin-binding site with two calponin homology domains to crosslink actin filaments (F-actin) in a Ca2+-sensitive manner in vitro. An inherited, late-onset form of kidney failure is caused by point mutations in the α-actinin-4 actin-binding domain. Here we show that α-actinin-4/F-actin aggregates, observed in vivo in podocytes of humans and mice with disease, likely form as a direct result of the increased actin-binding affinity of the protein. We document that exposure of a buried actin-binding site 1 in mutant α-actinin-4 causes an increase in its actin-binding affinity, abolishes its Ca2+ regulation in vitro, and diverts its normal localization from actin stress fibers and focal adhesions in vivo. Inactivation of this buried actin-binding site returns the affinity of the mutant to that of the WT protein and abolishes aggregate formation in cells. In vitro, actin filaments crosslinked by the mutant α-actinin-4 exhibit profound changes of structural and biomechanical properties compared with WT α-actinin-4. On a molecular level, our findings elucidate the physiological importance of a dynamic interaction of α-actinin with F-actin in podocytes in vivo. We propose that a conformational change with full exposure of actin-binding site 1 could function as a switch mechanism to regulate the actin-binding affinity of α-actinin and possibly other calponin homology domain proteins under physiological conditions.

Differentiation- and stress-dependent nuclear cytoplasmic redistribution of myopodin, a novel actin-bundling protein

We report the cloning and functional characterization of myopodin, the second member of the synaptopodin gene family. Myopodin shows no significant homology to any known protein except synaptopodin. Northern blot analysis resulted in a 3.6-kb transcript for mouse skeletal and heart muscle. Western blots showed an 80-kD signal for skeletal and a 95-kD signal for heart muscle. Myopodin contains one PPXY motif and multiple PXXP motifs. Myopodin colocalizes with α-actinin and is found at the Z-disc as shown by immunogold electron microscopy. In myoblasts, myopodin shows preferential nuclear localization. During myotube differentiation, myopodin binds to stress fibers in a punctuated pattern before incorporation into the Z-disc. Myopodin can directly bind to actin and contains a novel actin binding site in the center of the protein. Myopodin has actin-bundling activity as shown by formation of latrunculin-A–sensitive cytosolic actin bundles and nuclear actin loops in transfected cells expressing green fluorescent protein–myopodin. Under stress conditions, myopodin accumulates in the nucleus and is depleted from the cytoplasm. Nuclear export of myopodin is sensitive to leptomycin B, despite the absence of a classical nuclear export sequence. We propose a dual role for myopodin as a structural protein also participating in signaling pathways between the Z-disc and the nucleus.

Inhibition of the TRPC5 ion channel protects the kidney filter

An intact kidney filter is vital to retention of essential proteins in the blood and removal of waste from the body. Damage to the filtration barrier results in albumin loss in the urine, a hallmark of cardiovascular disease and kidney failure. Here we found that the ion channel TRPC5 mediates filtration barrier injury. Using Trpc5-KO mice, a small-molecule inhibitor of TRPC5, Ca2+ imaging in isolated kidney glomeruli, and live imagining of podocyte actin dynamics, we determined that loss of TRPC5 or its inhibition abrogates podocyte cytoskeletal remodeling. Inhibition or loss of TRPC5 prevented activation of the small GTP-binding protein Rac1 and stabilized synaptopodin. Importantly, genetic deletion or pharmacologic inhibition of TRPC5 protected mice from albuminuria. These data reveal that the Ca2+-permeable channel TRPC5 is an important determinant of albuminuria and identify TRPC5 inhibition as a therapeutic strategy for the prevention or treatment of proteinuric kidney disease.

Control of signaling-mediated clearance of apoptotic cells by the tumor suppressor p53

Tumor suppressor p53 linked to immune function We thought we knew all we needed to about the tumor suppressor p53. However, Yoon et al. now describe a previously unrecognized function of p53 (see the Perspective by Zitvogel and Kroemer). p53 induces expression of the gene encoding DD1α, a receptor-like transmembrane protein of the immunoglobulin superfamily. In conditions of stress, p53 activation can lead to cell death. p53-induced expression of DD1α also promotes the clearance of dead cells by promoting engulfment by macrophages. Furthermore, expression of DD1α on T cells inhibits T cell function. Thus, p53 offers protection from inflammatory disease caused by the accumulation of apoptotic cells, and its suppression of T cells might help cancer cells to escape immune detection. Science, this issue 10.1126/science.1261669; see also p. 476 p53 promotes clearance of dead cells and proper immune function. [Also see Perspective by Zitvogel and Kroemer] INTRODUCTION Programmed cell death occurs throughout life in all tissues of the body, and more than a billion cells die every day as part of normal processes. Thus, rapid and efficient clearance of cell corpses is a vital prerequisite for homeostatic maintenance of tissue health. Failure to clear dying cells can lead to the accumulation of autoantigens in tissues that foster diseases, such as chronic inflammation, autoimmunity, and developmental abnormalities. In the normal immune system, phagocytic engulfment of apoptotic cells is accompanied by induction of a certain degree of immune tolerance in order to prevent self-antigen recognition. Over the past few decades, enormous efforts have been made toward understanding various mechanisms of tumor suppressor p53–mediated apoptosis. However, the involvement of p53 in postapoptosis has yet to be addressed. RATIONALE One of the most intriguing, yet enigmatic, questions in studying homeostatic control of efficient dead cell clearance and proper immune tolerance is how these two essential activities are interrelated: The complexity of these processes is demonstrated by the many receptors and signaling pathways involved in the engulfment of apoptotic cells and stringent discrimination of self antigens from nonself antigens. Thus, there must be key connection(s) linking the balance between immune homeostasis and inflammation. In addition to the antitumor functions of p53, p53 has been implicated in immune responses and inflammatory diseases, with various roles in the immune system becoming apparent. We identified a postapoptotic target gene of p53, Death Domain1α (DD1α), that is responsive to genotoxic stresses and expressed in immune cells. DD1α appears to function as an immunoregulator of T cell tolerance. We hypothesized that p53 controls signaling-mediated phagocytosis of apoptotic cells through its target, DD1α. We determined that DD1α functions as an engulfment ligand or receptor that is involved in homophilic intermolecular interaction at intercellular junctions of apoptotic cells and macrophages. We also addressed whether DD1α deficiency caused any defects in dead cell clearance in vivo. RESULTS DD1α has similarity with several members of the immunoglobulin superfamily with the extracellular immunoglobulin V (IgV) domain, such as TIM family proteins and an immune checkpoint regulator, PD-L1. We found that the p53 induction and maintenance of DD1α expression in apoptotic cells and its subsequent functional intercellular homophilic interaction between apoptotic cells and macrophages are required for engulfment of apoptotic cells. DD1α-deficient mice showed less reduction in organ size and cell number after ionizing radiation (IR), owing to defective dead cell clearance. DD1α-null mice are viable and indistinguishable in appearance from wild-type littermates at an early age. However, at a later age, DD1α deficiency resulted in the development of autoimmune phenotypes and prominent formation of immune infiltrates in the skin, lung, and kidney, which indicated an immune dysregulation and breakdown of self-tolerance in DD1α-null mice. We demonstrated that DD1α also plays an important role as an intercellular homophilic receptor on T cells, which suggests that DD1α is a key-connecting molecule linking postapoptotic processes to immune surveillance. We found that DD1α deficiency in T cells impaired DD1α-mediated inhibitory activity of T cell proliferation. These data indicate that potential homophilic DD1α interactions are important for the DD1α-mediated T cell inhibitory role. Therefore, the results indicate a role for p53 in regulating expression of immune checkpoint regulators, including PD-1, PD-L1, and DD1α. CONCLUSION We found that the tumor suppressor p53 controls signaling-mediated phagocytosis of apoptotic cells through its target DD1α, which suggests that p53 promotes both the proapoptotic pathway and postapoptotic events. DD1α functions as an engulfment ligand that engages in homophilic intermolecular interaction at intercellular junctions of apoptotic cells and macrophages. DD1α-deficient mice showed in vivo defects in clearing dying cells that led to damage to multiple organs indicative of immune dysfunction. p53-induced expression of DD1α is a vital phase for the phagocytic engulfment process of dead cells and then facilitates the stepwise priming of immune surveillance. As a downstream target of the tumor suppressor p53, DD1α activation may extend the repertoire of p53 activities to “guardian of the immune integrity.” p53-dependent accumulation of DD1α and its involvement in dead cell clearance and immune tolerance. DD1α functions as an engulfment ligand that participates in homophilic intermolecular interaction at intercellular junctions of apoptotic cells and phagocytes. p53 induction of DD1α is a critical step in ensuring proper clearance of cell corpses to warrant the efficient generation of precise immune responses, leading to immune tolerance. The inefficient clearance of dying cells can lead to abnormal immune responses, such as unresolved inflammation and autoimmune conditions. We show that tumor suppressor p53 controls signaling-mediated phagocytosis of apoptotic cells through its target, Death Domain1α (DD1α), which suggests that p53 promotes both the proapoptotic pathway and postapoptotic events. DD1α appears to function as an engulfment ligand or receptor that engages in homophilic intermolecular interaction at intercellular junctions of apoptotic cells and macrophages, unlike other typical scavenger receptors that recognize phosphatidylserine on the surface of dead cells. DD1α-deficient mice showed in vivo defects in clearing dying cells, which led to multiple organ damage indicative of immune dysfunction. p53-induced expression of DD1α thus prevents persistence of cell corpses and ensures efficient generation of precise immune responses.

Dynamic Viscoelasticity of Actin Cross-Linked with Wild-Type and Disease-Causing Mutant α-Actinin-4

The actin cross-linker alpha-actinin-4 has been found to be indispensable for the structural and functional integrity of podocytes; deficiency or alteration of this protein due to mutations results in kidney disease. To gain insight into the effect of the cross-linker on cytoskeletal mechanics, we studied the macroscopic rheological properties of actin networks cross-linked with wild-type and mutant alpha-actinin-4. The frequency-dependent viscoelasticity of the networks is characterized by an elastic plateau at intermediate frequencies, and relaxation toward fluid properties at low frequencies. The relaxation frequencies of networks with mutant alpha-actinin-4 are an order of magnitude lower than that with the wild-type, suggesting a slower reaction rate for the dissociation of actin and alpha-actinin for the mutant, consistent with a smaller observed equilibrium dissociation constant. This difference can be attributed to an additional binding site that is exposed as a result of the mutation, and can be interpreted as a difference in binding energy barriers. This is further supported by the Arrhenius-like temperature dependence of the relaxation frequencies.

Mutations in PAX2 Associate with Adult-Onset FSGS

FSGS is characterized by the presence of partial sclerosis of some but not all glomeruli. Studies of familial FSGS have been instrumental in identifying podocytes as critical elements in maintaining glomerular function, but underlying mutations have not been identified for all forms of this genetically heterogeneous condition. Here, exome sequencing in members of an index family with dominant FSGS revealed a nonconservative, disease-segregating variant in the PAX2 transcription factor gene. Sequencing in probands of a familial FSGS cohort revealed seven rare and private heterozygous single nucleotide substitutions (4% of individuals). Further sequencing revealed seven private missense variants (8%) in a cohort of individuals with congenital abnormalities of the kidney and urinary tract. As predicted by in silico structural modeling analyses, in vitro functional studies documented that several of the FSGS-associated PAX2 mutations perturb protein function by affecting proper binding to DNA and transactivation activity or by altering the interaction of PAX2 with repressor proteins, resulting in enhanced repressor activity. Thus, mutations in PAX2 may contribute to adult-onset FSGS in the absence of overt extrarenal manifestations. These results expand the phenotypic spectrum associated with PAX2 mutations, which have been shown to lead to congenital abnormalities of the kidney and urinary tract as part of papillorenal syndrome. Moreover, these results indicate PAX2 mutations can cause disease through haploinsufficiency and dominant negative effects, which could have implications for tailoring individualized drug therapy in the future.

Crystal structure of the actin-binding domain of α-actinin-4 Lys255Glu mutant implicated in focal segmental glomerulosclerosis

Mutations in alpha-actinin-4 have been linked to familial focal segmental glomerulosclerosis (FSGS), a common renal disorder in humans, and produce an apparent increase in the actin-binding affinity of alpha-actinin-4 in vitro. One of the mutations, in particular, Lys255Glu, falls in the middle of the actin-binding interface of the actin-binding domain (ABD). The ABD consists of tandem calponin homology (CH) domains (CH1 and CH2). The crystal structures of most ABDs display a compact conformation, characterized by extensive inter-CH interactions. However, the conformation of F-actin-bound ABDs is unsettled. Some electron microscopy studies find that the compact conformation is preserved upon binding to F-actin, whereas other studies suggest that the CHs separate and the ABD becomes extended. The Lys255Glu mutation in CH2 is significant in this regard since it removes a crucial inter-CH interaction with Trp147 of CH1, thought to stabilize the compact conformation. Together, the increased actin-binding affinity and the removal of this important inter-CH contact suggested that the Lys255Glu mutation might facilitate the transition toward the open ABD conformation proposed by some of the electron microscopy studies. However, the crystal structure of the ABD of alpha-actinin-4 Lys255Glu mutant described here displays the canonical compact conformation. Furthermore, the sedimentation coefficients by analytical ultracentrifugation of wild-type and FSGS mutant ABDs (Lys255Glu, Ser262Pro, and Thr259Ile) are nearly identical (2.50+/-0.03 S) and are in good agreement with the theoretical value calculated from the crystal structure (2.382 S), implying that the compact conformation is retained in solution. The absence of a structural change suggests that the compact ABD conformation observed in the majority of the structures is highly stable and is preserved in solution, even in FSGS mutant ABDs.