A highly conserved pocket on PP2A-B56 is required for hSgo1 binding and cohesion protection during mitosis

The shugoshin proteins are universal protectors of centromeric cohesin during mitosis and meiosis. The binding of human hSgo1 to the PP2A‐B56 phosphatase through a coiled‐coil (CC) region mediates cohesion protection during mitosis. Here we undertook a structure function analysis of the PP2A‐B56‐hSgo1 complex, revealing unanticipated aspects of complex formation and function. We establish that a highly conserved pocket on the B56 regulatory subunit is required for hSgo1 binding and cohesion protection during mitosis in human somatic cells. Consistent with this, we show that hSgo1 blocks the binding of PP2A‐B56 substrates containing a canonical B56 binding motif. We find that PP2A‐B56 bound to hSgo1 dephosphorylates Cdk1 sites on hSgo1 itself to modulate cohesin interactions. Collectively our work provides important insight into cohesion protection during mitosis

Structural studies on the membrane attack complex and staphylococcal immune evasion : C8α-MACPF, C5b6 and FLIPr-like-FcγRIIa

The human body is continuously at battle with bacterial pathogens from its surrounding environment. This leads to a strong selective pressure on both the pathogen and the host. Accordingly, humans and bacteria have evolved elaborate measures to fight one another. In humans the complement system forms the first line of defense against invading bacteria. These trigger the activation of the complement system. This leads to various effector functions one of which entails the formation of large lytic pores on the bacterial membrane, thereby directly killing Gram-negative bacteria. These pores are called the membrane attack complex MAC) and their formation is mediated by a group of proteins collectively referred to as the MACPF family which act in concert to drill a hole through the membrane of target cells. We have determined crystal structures of two components of the MAC, the complex between C5b and C6 and the MACPF domain of C8a?. These structures yielded the unexpected result that the MACPF domain resembles cholesterol dependent cytolysins (CDCs), a group of well studied bacterial pore-forming toxins. These structure show for the first time how formation of the MAC is initiated and allow us to construct a model for how the proteins of the MAC go from a water soluble state in the blood to a membrane inserted pore on the surface of bacterial targets. Like CDCs, MACPF domains contain two amphipathic segments folded against the protein. Upon activation these segments can unfold to form extended ?-hairpins that ultimately form the lining of a large ?-barrel pore. The structures have greatly expanded our understanding of how the proteins of the MAC function together to form a bactericidal pore but also form a starting point of biochemical studies that will increase our understanding even further. A second key component humans use to fight of infections is phagocytosis. In this process, white blood cells like neutrophils, recognize antibodies bound to e.g. bacteria through so called Fc? receptors. Staphylococcus aureas, a Gram-positive bacteria that is well known for its numerous immune evasion strategies, has evolved a protein, FLIPr and its homologue, FLIPr-like, that are able to bind to the Fc? receptor most important for phagocytosis by neutrophils, Fc?RIIa This binding inhibits phagocytosis thereby conferring a strong advantage for invading bacteria. We have solved the crystal structure of the complex between FLIPr-like and Fc?RIIa and characterized the binding of the proteins with isothermal calorimetry. The structure give a clear picture of how FLIPr-like inhibits antibody binding to the receptor as their binding sites almost fully overlap. Furthermore, the structure also gives insight into the specificity FLIPr-like has for the different isoforms of Fc? receptors. Collectively the data could aid in the design of novel receptor isoform specific inhibitors to aid in the treatment of auto-immune diseases and inflammatory disorders

Structural studies on the membrane attack complex and staphylococcal immune evasion : C8α-MACPF, C5b6 and FLIPr-like-FcγRIIa

The human body is continuously at battle with bacterial pathogens from its surrounding environment. This leads to a strong selective pressure on both the pathogen and the host. Accordingly, humans and bacteria have evolved elaborate measures to fight one another. In humans the complement system forms the first line of defense against invading bacteria. These trigger the activation of the complement system. This leads to various effector functions one of which entails the formation of large lytic pores on the bacterial membrane, thereby directly killing Gram-negative bacteria. These pores are called the membrane attack complex MAC) and their formation is mediated by a group of proteins collectively referred to as the MACPF family which act in concert to drill a hole through the membrane of target cells. We have determined crystal structures of two components of the MAC, the complex between C5b and C6 and the MACPF domain of C8a?. These structures yielded the unexpected result that the MACPF domain resembles cholesterol dependent cytolysins (CDCs), a group of well studied bacterial pore-forming toxins. These structure show for the first time how formation of the MAC is initiated and allow us to construct a model for how the proteins of the MAC go from a water soluble state in the blood to a membrane inserted pore on the surface of bacterial targets. Like CDCs, MACPF domains contain two amphipathic segments folded against the protein. Upon activation these segments can unfold to form extended ?-hairpins that ultimately form the lining of a large ?-barrel pore. The structures have greatly expanded our understanding of how the proteins of the MAC function together to form a bactericidal pore but also form a starting point of biochemical studies that will increase our understanding even further. A second key component humans use to fight of infections is phagocytosis. In this process, white blood cells like neutrophils, recognize antibodies bound to e.g. bacteria through so called Fc? receptors. Staphylococcus aureas, a Gram-positive bacteria that is well known for its numerous immune evasion strategies, has evolved a protein, FLIPr and its homologue, FLIPr-like, that are able to bind to the Fc? receptor most important for phagocytosis by neutrophils, Fc?RIIa This binding inhibits phagocytosis thereby conferring a strong advantage for invading bacteria. We have solved the crystal structure of the complex between FLIPr-like and Fc?RIIa and characterized the binding of the proteins with isothermal calorimetry. The structure give a clear picture of how FLIPr-like inhibits antibody binding to the receptor as their binding sites almost fully overlap. Furthermore, the structure also gives insight into the specificity FLIPr-like has for the different isoforms of Fc? receptors. Collectively the data could aid in the design of novel receptor isoform specific inhibitors to aid in the treatment of auto-immune diseases and inflammatory disorders

Structure of C8α-MACPF Reveals Mechanism of Membrane Attack in Complement Immune Defense

Membrane attack is important for mammalian immune defense against invading microorganisms and infected host cells. Proteins of the complement membrane attack complex (MAC) and the protein perforin share a common MACPF domain that is responsible for membrane insertion and pore formation. We determined the crystal structure of the MACPF domain of complement component C8α at 2.5 angstrom resolution and show that it is structurally homologous to the bacterial, pore-forming, cholesterol-dependent cytolysins. The structure displays two regions that (in the bacterial cytolysins) refold into transmembrane b hairpins, forming the lining of a barrel pore. Local hydrophobicity explains why C8Membrane attack is important for mammalian immune defense against invading microorganisms and infected host cells. Proteins of the complement membrane attack complex (MAC) and the protein perforin share a common MACPF domain that is responsible for membrane insertion and pore formation. We determined the crystal structure of the MACPF domain of complement component C8α at 2.5 angstrom resolution and show that it is structurally homologous to the bacterial, pore-forming, cholesterol-dependent cytolysins. The structure displays two regions that (in the bacterial cytolysins) refold into transmembrane b hairpins, forming the lining of a barrel pore. Local hydrophobicity explains why C8α is the first complement protein to insert into the membrane. The size of the MACPF domain is consistent with known C9 pore sizes. These data imply that these mammalian and bacterial cytolytic proteins share a common mechanism of membrane insertion. is the first complement protein to insert into the membrane. The size of the MACPF domain is consistent with known C9 pore sizes. These data imply that these mammalian and bacterial cytolytic proteins share a common mechanism of membrane insertion

The ins and outs of Aurora B inner centromere localization

Error-free chromosome segregation is essential for the maintenance of genomic integrity during cell division. Aurora B, the enzymatic subunit of the Chromosomal Passenger Complex (CPC), plays a crucial role in this process. In early mitosis Aurora B localizes predominantly to the inner centromere, a specialized region of chromatin that lies at the crossroads between the inter-kinetochore and inter-sister chromatid axes. Two evolutionarily conserved histone kinases, Haspin and Bub1, control the positioning of the CPC at the inner centromere and this location is thought to be crucial for the CPC to function. However, recent studies sketch a subtler picture, in which not all functions of the CPC require strict confinement to the inner centromere. In this review we discuss the molecular pathways that direct Aurora B to the inner centromere and deliberate if and why this specific localization is important for Aurora B function

Structure of C8α-MACPF Reveals Mechanism of Membrane Attack in Complement Immune Defense

Membrane attack is important for mammalian immune defense against invading microorganisms and infected host cells. Proteins of the complement membrane attack complex (MAC) and the protein perforin share a common MACPF domain that is responsible for membrane insertion and pore formation. We determined the crystal structure of the MACPF domain of complement component C8α at 2.5 angstrom resolution and show that it is structurally homologous to the bacterial, pore-forming, cholesterol-dependent cytolysins. The structure displays two regions that (in the bacterial cytolysins) refold into transmembrane b hairpins, forming the lining of a barrel pore. Local hydrophobicity explains why C8Membrane attack is important for mammalian immune defense against invading microorganisms and infected host cells. Proteins of the complement membrane attack complex (MAC) and the protein perforin share a common MACPF domain that is responsible for membrane insertion and pore formation. We determined the crystal structure of the MACPF domain of complement component C8α at 2.5 angstrom resolution and show that it is structurally homologous to the bacterial, pore-forming, cholesterol-dependent cytolysins. The structure displays two regions that (in the bacterial cytolysins) refold into transmembrane b hairpins, forming the lining of a barrel pore. Local hydrophobicity explains why C8α is the first complement protein to insert into the membrane. The size of the MACPF domain is consistent with known C9 pore sizes. These data imply that these mammalian and bacterial cytolytic proteins share a common mechanism of membrane insertion. is the first complement protein to insert into the membrane. The size of the MACPF domain is consistent with known C9 pore sizes. These data imply that these mammalian and bacterial cytolytic proteins share a common mechanism of membrane insertion

Assembly and Regulation of the Membrane Attack Complex Based on Structures of C5b6 and sC5b9

Activation of the complement system results in formation of membrane attack complexes (MACs), pores that disrupt lipid bilayers and lyse bacteria and other pathogens. Here, we present the crystal structure of the first assembly intermediate, C5b6, together with a cryo-electron microscopy reconstruction of a soluble, regulated form of the pore, sC5b9. Cleavage of C5 to C5b results in marked conformational changes, distinct from those observed in the homologous C3-to-C3b transition. C6 captures this conformation, which is preserved in the larger sC5b9 assembly. Together with antibody labeling, these structures reveal that complement components associate through sideways alignment of the central MAC-perforin (MACPF) domains, resulting in a C5b6-C7-C8β-C8α-C9 arc. Soluble regulatory proteins below the arc indicate a potential dual mechanism in protection from pore formation. These results provide a structural framework for understanding MAC pore formation and regulation, processes important for fighting infections and preventing complement-mediated tissue damage

Delineation of a KDM2B-related neurodevelopmental disorder and its associated DNA methylation signature.

PURPOSE: Pathogenic variants in genes involved in the epigenetic machinery are an emerging cause of neurodevelopment disorders (NDDs). Lysine-demethylase 2B (KDM2B) encodes an epigenetic regulator and mouse models suggest an important role during development. We set out to determine whether KDM2B variants are associated with NDD. METHODS: Through international collaborations, we collected data on individuals with heterozygous KDM2B variants. We applied methylation arrays on peripheral blood DNA samples to determine a KDM2B associated epigenetic signature. RESULTS: We recruited a total of 27 individuals with heterozygous variants in KDM2B. We present evidence, including a shared epigenetic signature, to support a pathogenic classification of 15 KDM2B variants and identify the CxxC domain as a mutational hotspot. Both loss-of-function and CxxC-domain missense variants present with a specific subepisignature. Moreover, the KDM2B episignature was identified in the context of a dual molecular diagnosis in multiple individuals. Our efforts resulted in a cohort of 21 individuals with heterozygous (likely) pathogenic variants. Individuals in this cohort present with developmental delay and/or intellectual disability; autism; attention deficit disorder/attention deficit hyperactivity disorder; congenital organ anomalies mainly of the heart, eyes, and urogenital system; and subtle facial dysmorphism. CONCLUSION: Pathogenic heterozygous variants in KDM2B are associated with NDD and a specific epigenetic signature detectable in peripheral blood