Ayman Antoun

How initiation factors tune the rate of initiation of protein synthesis in bacteria

The kinetics of initiator transfer RNA (tRNA) interaction with the messenger RNA (mRNA)‐programmed 30S subunit and the rate of 50S subunit docking to the 30S preinitiation complex were measured for different combinations of initiation factors in a cell‐free Escherichia coli system for protein synthesis with components of high purity. The major results are summarized by a Michaelis–Menten scheme for initiation. All three initiation factors are required for maximal efficiency (kcat/KM) of initiation and for maximal in vivo rate of initiation at normal concentration of initiator tRNA. Spontaneous release of IF3 from the 30S preinitiation complex is required for subunit docking. The presence of initiator tRNA on the 30S subunit greatly increases the rate of 70S ribosome formation by increasing the rate of IF3 dissociation from the 30S subunit and the rate of 50S subunit docking to the IF3‐free 30S preinitiation complex. The reasons why IF1 and IF3 are essential in E. coli are discussed in the light of the present observations.

Ribosome formation from subunits studied by stopped-flow and Rayleigh light scattering

Light scattering and standard stopped-flow techniques were used to monitor rapid association of ribosomal subunits during initiation of eubacterial protein synthesis. The effects of the initiation factors IF1, IF2, IF3 and buffer conditions on subunit association were studied along with the role of GTP in this process. The part of light scattering theory that is essential for kinetic measurements is highlighted in the main text and a more general treatment of Rayleigh scattering from macromolecules is given in an appendix.

The roles of initiation factor 2 and guanosine triphosphate in initiation of protein synthesis

The role of IF2 from Escherichia coli was studied in vitro using a system for protein synthesis with purified components. Stopped flow experiments with light scattering show that IF2 in complex with guanosine triphosphate (GTP) or a non‐cleavable GTP analogue (GDPNP), but not with guanosine diphosphate (GDP), promotes fast association of ribosomal subunits during initiation. Biochemical experiments show that IF2 promotes fast formation of the first peptide bond in the presence of GTP, but not GDPNP or GDP, and that IF2–GDPNP binds strongly to post‐initiation ribosomes. We conclude that the GTP form of IF2 accelerates formation of the 70S ribosome from subunits and that GTP hydrolysis accelerates release of IF2 from the 70S ribosome. The results of a recent report, suggesting that GTP and GDP promote initiation equally fast, have been addressed. Our data, indicating that eIF5B and IF2 have similar functions, are used to rationalize the phenotypes of GTPase‐deficient mutants of eIF5B and IF2.

Ethnic variability in human leukocyte antigen‐E haplotypes

Human leukocyte antigen-E (HLA-E) is an important nonclassical major histocompatibility complex (MHC) class I (Ib) molecule that acts as the ligand for NKG2A/B/C receptors expressed on natural killer (NK) cells and T cells. Unlike the classical class I molecules, HLA-E is highly conserved in evolution and the biological significance of polymorphism is therefore unclear. Our aim was to investigate the polymorphism in HLA-E gene in three ethnic groups in the UK and to obtain population data relating to any variations observed at this locus. We developed a polymerase chain reaction-sequence-specific primer (PCR-SSP) method for identifying HLA-E single nucleotide polymorphisms (SNPs) in genomic DNA. This was used to investigate the genotype distribution and allele frequency of nine published SNPs in the coding region of HLA-E in 223 Euro-Caucasoid, 60 Afro-Caribbean and 52 Asian healthy individuals. Genotype frequencies were in Hardy-Weinberg equilibrium. No polymorphism was observed for seven previously reported SNPs and these should not be considered polymorphic. However, positions 1114 and 1446 were confirmed as polymorphic and different genotype frequencies were identified at nucleotide position 1114 between the three studied ethnic groups. We present these data together with the intragene haplotype frequencies in these populations. To our knowledge, this is the first description of population frequencies of nine different SNPs in HLA-E in three main large ethnic groups. The data generated from this study will be of importance in the context of describing the effect of HLA-E polymorphism in clinical settings such as transplantation and autoimmune diseases.

Single nucleotide polymorphism analysis of the NKG2D ligand cluster on the long arm of chromosome 6: Extensive polymorphisms and evidence of diversity between human populations.

NKG2D is an important activating receptor on NK cells and T-cells and has a diverse panel of ligands (NKG2DL) which include the ULBP and RAET1 proteins. Several NKG2DL exhibit a considerable degree of genetic polymorphism, and although the functional significance of such allelic variation remains unclear, genetic variants have been implicated in susceptibility to infection and auto-immune disease. We used sequence-specific primer polymerase chain reaction to determine the frequency of 25 single nucleotide polymorphisms (SNPs) in the promoter and coding regions of genes of the RAET1/ULBP cluster in 223 Euro-Caucasoid, 60 Afro-Caribbean, and 52 Indo-Asian individuals to determine NKG2DL allele and haplotype frequencies within these populations. We show marked differences in the frequency of NKG2DL SNPs and haplotypes among the three ethnic groups, and certain haplotypes were observed almost exclusively in Afro-Caribbean compared with the Euro-Caucasoid and Indo-Asian populations. Interestingly, variation was focused within the RAET1E (ULBP4), RAET1L, and ULBP3 genes, whereas the ULBP1, ULBP2 and RAET1G (ULBP5) genes were highly conserved. These findings suggest that individual NKG2DL alleles have been subject to divergent selective pressures during the migration of Homo sapiens. This information will be of importance in understanding the biology and clinical significance of NKG2DL polymorphism.

The genotype of RAET1L (ULBP6), a ligand for human NKG2D (KLRK1), markedly influences the clinical outcome of allogeneic stem cell transplantation

NKG2D (KLRK1) is an activating receptor on natural killer (NK) and T‐cells and binds a diverse panel of polymorphic ligands encoded by the MIC and RAET1 gene families. We studied the clinical importance of retinoic acid early transcript‐1 (RAET1) polymorphism in allogeneic stem cell transplantation (SCT) by determining the frequency of 18 single nucleotide polymorphisms (SNPs) and individual RAET1 alleles in 371 patient‐donor pairs and relating this to clinical outcome. A strong association was observed between the presence of five SNPs within the patient RAET1L (ULBP6) gene and relapse‐free survival and overall survival. Two common alleles of RAET1L were determined and the presence of the protective RAET1L*02 allele in the patient was associated with a relapse‐free survival of 44% at 8 years compared with just 25% in patients who lacked a RAET1L*02 allele (P < 0·001). Overall survival at this time was 55% in those with RAET1L*02 allele compared to 39% in patients who lacked a RAET1L*02 allele (P = 0·003). These novel findings indicate a critical role for NKG2D–RAET1L interactions in determining SCT clinical outcome and show RAET1L may have an important influence on regulating the strength of the alloreactive immune response. The data will be of value in guiding the development of future transplant therapy protocols.

A disease-linked ULBP6 polymorphism inhibits NKG2D-mediated target cell killing by enhancing the stability of NKG2D ligand binding

A disease-associated variant of an activating ligand engages receptors so strongly that it impairs NK cell–mediated killing. Distracting natural killer cells Natural killer (NK) cells target virally infected and transformed cells for cytolysis. When sufficient activating receptors on the NK cell surface, such as NKG2D, are engaged by ligands on the target cell, such as ULBP proteins, the NK cell kills the target. Polymorphisms within ULBP-encoding genes are associated with immune dysfunction. Zuo et al. found that the affinity of a commonly occurring ULBP6 variant for NKG2D was greater than that of the wild-type protein, which impaired NK cell activation. A soluble form of this protein variant bound so tightly to NKG2D that it suppressed receptor activation and target cell killing in response to other NKG2D ligands. Together, these data suggest that targeting NK cell–ligand interactions may provide therapies to modulate the strength of immune responses. NKG2D (natural killer group 2, member D) is an activating receptor found on the surface of immune cells, including natural killer (NK) cells, which regulates innate and adaptive immunity through recognition of the stress-induced ligands ULBP1 (UL16 binding protein 1) to ULBP6 and MICA/B. Similar to class I human leukocyte antigen (HLA), these NKG2D ligands have a major histocompatibility complex–like fold and exhibit pronounced polymorphism, which influences human disease susceptibility. However, whereas class I HLA polymorphisms occur predominantly in the α1α2 groove and affect antigen binding, the effects of most NKG2D ligand polymorphisms are unclear. We studied the molecular and functional consequences of the two major alleles of ULBP6, the most polymorphic ULBP gene, which are associated with autoimmunity and relapse after stem cell transplantation. Surface plasmon resonance and crystallography studies revealed that the arginine-to-leucine polymorphism within ULBP0602 affected the NKG2D-ULBP6 interaction by generating an energetic hotspot. This resulted in an NKG2D-ULBP0602 affinity of 15.5 nM, which is 10- to 1000-fold greater than the affinities of other ULBP-NKG2D interactions and limited NKG2D-mediated activation. In addition, soluble ULBP0602 exhibited high-affinity competitive binding for NKG2D and partially suppressed NKG2D-mediated activation of NK cells by other NKG2D ligands. These effects resulted in a decrease in a range of NKG2D-mediated effector functions. Our results reveal that ULBP polymorphisms affect the strength of human lymphocyte responses to cellular stress signals and may offer opportunities for therapeutic intervention.