Modulation of histone deposition by the karyopherin kap114

The nuclear import of histones is a prerequisite for the downstream deposition of histones to form chromatin. However, the coordinate regulation of these processes remains poorly understood. Here we demonstrate that Kap114p, the primary karyopherin/importin responsible for the nuclear import of histones H2A and H2B, modulates the deposition of histones H2A and H2B by the histone chaperone Nap1p. We show that a complex comprising Kap114p, histones H2A and H2B, and Nap1p is present in the nucleus and that the presence of this complex is specifically promoted by Nap1p. This places Kap114p in a position to modulate Nap1p function, and we demonstrate by the use of two different assay systems that Kap114p inhibits Nap1p-mediated chromatin assembly. The inhibition of H2A and H2B deposition by Kap114p results in the concomitant inhibition of RCC1 loading onto chromatin. Biochemical evidence suggests that the mechanism by which Kap114p modulates histone deposition primarily involves direct histone binding, while the interaction between Kap114p and Nap1p plays a secondary role. Furthermore, we found that the inhibition of histone deposition by Kap114p is partially reversed by RanGTP. Our results indicate a novel mechanism by which cells can regulate histone deposition and establish a coordinate link between histone nuclear import and chromatin assembly.     

Defining amino acid residues involved in DNA-protein interactions and revelation of 3'-exonuclease activity in endonuclease V

Endonuclease V is an enzyme that initiates a conserved DNA repair pathway by making an endonucleolytic incision at the 3' side one nucleotide from a deaminated base lesion. Site-directed mutagenesis analysis was conducted at seven conserved motifs of the thermostable Thermotoga maritima endonuclease V to probe for residues that affect DNA-protein interactions. Y80, G83, and L85 in motif III, H116 and G121 in motif IV, A138 in motif V, and S182 in motif VI affect binding of both the double-stranded inosine-containing DNA substrate and the nicked double-stranded inosine-containing DNA product, resulting in multiple enzymatic turnovers. The substantially reduced DNA cleavage activity observed in G113 in motif IV and G136 in motif V can be partly attributed to their defect in metal cofactor coordination. Alanine substitution at amino acid 118 primarily reduces the level of binding to the nicked product, suggesting that R118 plays a significant role in postcleavage DNA-protein interaction. Binding and cleavage analyses of multiple mutants at positions Y80 and H116 underscore the role these residues play in protein-DNA interaction and implicate their potential involvement as a hydrogen bond donor in recognition of deaminated DNA bases. DNA cleavage analysis using mutants defective in DNA binding reveals a novel 3'-exonuclease activity in endonuclease V. An alternative model is proposed that entails lesion specific cleavage and endonuclease to 3'-exonuclease mode switch by endonuclease V for removal of deaminated base lesions during endonuclease V-mediated repair.     

The Interaction Between Intrathecal Administration of Low Doses of Palmitoylethanolamide and AM251 in Formalin-Induced Pain Related Behavior and Spinal Cord IL1-β Expression in Rats

Most of the modulating effects of cannabinoids on pain are through putative cannabinoid CB1 and CB2 receptors. However, the involvement of other receptors is also suggested. Cannabinoid compounds with analgesic activity such as palmitoylethanolamide (PEA) show low affinity to CB1 and CB2 receptors, yet selectively activate GPR55 receptors. The objective of the present study was to evaluate the possible role of spinal CB1 and GPR55 receptors on antinociceptive activity of PEA in formalin test as well as in the spinal expression of IL1-β in rat. Intrathecal (i.t.) administration of PEA (1, 10 μg) significantly decreased both pain-related scores in formalin test and IL1-β expression in rat spinal cord. Pretreatment of rats with low doses of CB1 receptor antagonist/GPR55 receptor agonist AM251 (10, 100 ng; i.t.), did not attenuated the effect of PEA, yet even significantly increased the effect of PEA on IL1-β expression in rat spinal cord. Interestingly, i.t. administration of low doses of AM251 per se significantly decreased both pain related behavior and spinal IL1-β expression in formalin test. These findings suggest the possible involvement of receptors other than CB1 receptors in spinal pain pathways, such as GPR55, in pain modulating activity of cannabinoids.     

Deleterious mutations in LRBA are associated with a syndrome of immune deficiency and autoimmunity

Most autosomal genetic causes of childhood-onset hypogammaglobulinemia are currently not well understood. Most affected individuals are simplex cases, but both autosomal-dominant and autosomal-recessive inheritance have been described. We performed genetic linkage analysis in consanguineous families affected by hypogammaglobulinemia. Four consanguineous families with childhood-onset humoral immune deficiency and features of autoimmunity shared genotype evidence for a linkage interval on chromosome 4q. Sequencing of positional candidate genes revealed that in each family, affected individuals had a distinct homozygous mutation in LRBA (lipopolysaccharide responsive beige-like anchor protein). All LRBA mutations segregated with the disease because homozygous individuals showed hypogammaglobulinemia and autoimmunity, whereas heterozygous individuals were healthy. These mutations were absent in healthy controls. Individuals with homozygous LRBA mutations had no LRBA, had disturbed B cell development, defective in vitro B cell activation, plasmablast formation, and immunoglobulin secretion, and had low proliferative responses. We conclude that mutations in LRBA cause an immune deficiency characterized by defects in B cell activation and autophagy and by susceptibility to apoptosis, all of which are associated with a clinical phenotype of hypogammaglobulinemia and autoimmunity.     

Hematopoietic stem cell subtypes expand differentially during development and display distinct lymphopoietic programs

Adult hematopoietic stem cells (HSCs) with serially transplantable activity comprise two subtypes. One shows a balanced output of mature lymphoid and myeloid cells; the other appears selectively lymphoid deficient. We now show that both of these HSC subtypes are present in the fetal liver (at a 1:10 ratio) with the rarer, lymphoid-deficient HSCs immediately gaining an increased representation in the fetal bone marrow, suggesting that the marrow niche plays a key role in regulating their ensuing preferential amplification. Clonal analysis of HSC expansion posttransplant showed that both subtypes display an extensive but variable self-renewal activity with occasional interconversion. Clonal analysis of their differentiation programs demonstrated functional and molecular as well as quantitative HSC subtype-specific differences in the lymphoid progenitors they generate but an indistinguishable production of multipotent and myeloid-restricted progenitors. These findings establish a level of heterogeneity in HSC differentiation and expansion control that may have relevance to stem cell populations in other hierarchically organized tissues.