Brian F.C. Clark

Crystal Structure of the Ternary Complex of Phe-tRNAPhe, EF-Tu, and a GTP Analog

The structure of the ternary complex consisting of yeast phenylalanyl-transfer RNA (Phe-tRNAPhe), Thermus aquaticus elongation factor Tu (EF-Tu), and the guanosine triphosphate (GTP) analog GDPNP was determined by x-ray crystallography at 2.7 angstrom resolution. The ternary complex participates in placing the amino acids in their correct order when messenger RNA is translated into a protein sequence on the ribosome. The EF-Tu-GDPNP component binds to one side of the acceptor helix of Phe-tRNAPhe involving all three domains of EF-Tu. Binding sites for the phenylalanylated CCA end and the phosphorylated 5′ end are located at domain interfaces, whereas the T stem interacts with the surface of the β-barrel domain 3. The binding involves many conserved residues in EF-Tu. The overall shape of the ternary complex is similar to that of the translocation factor, EF-G-GDP, and this suggests a novel mechanism involving “molecular mimicry” in the translational apparatus.

Structural details of the binding of guanosine diphosphate to elongation factor Tu from E. coli as studied by X-ray crystallography.

Structural details of the guanosine diphosphate binding to a modified form of elongation factor Tu from Escherichia coli, resulting from X‐ray crystallographic studies, are reported. The protein elements that take part in the nucleotide binding are located in four loops connecting beta‐strands with alpha‐helices. These loops correspond to regions in primary sequences which show a high degree of homology when compared with other prokaryotic and eukaryotic elongation factors and initiation factor 2.

Eukaryotic protein elongation factors.

In eukaryotes, peptide chain elongation is mediated by elongation factors EF-1 and EF-2. EF-1 is composed of a nucleotide-binding protein EF-1 alpha, and a nucleotide exchange protein complex, EF-1 beta gamma, while EF-2 catalyses the translocation of peptidyl-tRNA on the ribosome. Elongation factors are highly conserved among different species and may be involved in functions other than protein synthesis, such as organization of the mitotic apparatus, signal transduction, developmental regulation, ageing and transformation. Yeast contains a third factor, EF-3, whose structure and function is not yet well understood.

Millennial-scale trends in west Pacific warm pool hydrology since the Last Glacial Maximum

Models and palaeoclimate data suggest that the tropical Pacific climate system plays a key part in the mechanisms underlying orbital-scale and abrupt climate change. Atmospheric convection over the western tropical Pacific is a major source of heat and moisture to extratropical regions, and may therefore influence the global climate response to a variety of forcing factors. The response of tropical Pacific convection to changes in global climate boundary conditions, abrupt climate changes and radiative forcing remains uncertain, however. Here we present three absolutely dated oxygen isotope records from stalagmites in northern Borneo that reflect changes in west Pacific warm pool hydrology over the past 27,000 years. Our results suggest that convection over the western tropical Pacific weakened 18,000–20,000 years ago, as tropical Pacific and Antarctic temperatures began to rise during the early stages of deglaciation. Convective activity, as inferred from oxygen isotopes, reached a minimum during Heinrich event 1 (ref. 10), when the Atlantic meridional overturning circulation was weak, pointing to feedbacks between the strength of the overturning circulation and tropical Pacific hydrology. There is no evidence of the Younger Dryas event in the stalagmite records, however, suggesting that different mechanisms operated during these two abrupt deglacial climate events. During the Holocene epoch, convective activity appears to track changes in spring and autumn insolation, highlighting the sensitivity of tropical Pacific convection to external radiative forcing. Together, these findings demonstrate that the tropical Pacific hydrological cycle is sensitive to high-latitude climate processes in both hemispheres, as well as to external radiative forcing, and that it may have a central role in abrupt climate change events.

The GTP binding motif: variations on a theme.

GTP binding proteins (G‐proteins) have wide‐ranging functions in biology, being involved in cell proliferation, signal transduction, protein synthesis, and protein targeting. Common to their functioning is that they are active in the GTP‐bound form and inactive in the GDP‐bound form. The protein synthesis elongation factor EF‐Tu was the first G‐protein whose nucleotide binding domain was solved structurally by X‐ray crystallography to yield a structural definition of the GDP‐bound form, but a still increasing number of new structures of G‐proteins are appearing in the literature, in both GDP and GTP bound forms. A common structural core for nucleotide binding is present in all these structures, and this core has long been known to include common consensus sequence elements involved in binding of the nucleotide. Nevertheless, subtle changes in the common sequences reflect functional differences. Therefore, it becomes increasingly important to focus on how these differences are reflected in the structures, and how these structural differences are related to function. The aim of this review is to describe to what extent this structural motif for GDP/GTP binding is common to other known structures of this class of proteins. We first describe the common structural core of the G‐proteins. Next, examples are based on information available on the Ras protein superfamily, the targeting protein ARF, elongation factors EF‐Tu and EF‐G, and the heterotrimeric G‐proteins. Finally, we discuss the important structures of complexes between GTP binding proteins and their substrates that have appeared in the literature recently.—Kjeldgaard, M., Nyborg, J., Clark, B. F. C. The GTP binding motif: variations on a theme. FASEB J. 10, 1347‐1368 (1996)

HEAT SHOCK RESPONSE AND AGEING: MECHANISMS AND APPLICATIONS

Ageing is associated with a decrease in the ability of cells to cope with environmental challenges. This is due partly to the attenuation of a primordial stress response, the so‐called heat shock (HS) response, which induces the expression of heat shock proteins (HSPs), composed of chaperones and proteases. The attenuation of the HS response during ageing may be responsible for the accumulation of damaged proteins as well as abnormal regulation of cell death. Maintenance of the HS response by repeated mild heat stress causes anti‐ageing hormetic effects on cells and organisms. Here, we describe the molecular mechanism and the state of the HS response as well as the role of specific HSPs during ageing, and discuss the possibility of hormetic modulation of ageing and longevity by repeated mild stress.

Protein Synthesis, Posttranslational Modifications, and Aging

Posttranslational modifications of proteins are involved in determining their activities, stability, and specificity of interaction. More than 140 major and minor modifications of proteins have been reported. Of these, only a few have been studied in relation to the aging of cells, tissues, and organisms. These include phosphorylation, methylation, ADP-ribosylation, oxidation, glycation, and deamidation. Several of these modifications occur on proteins involved in crucial cellular processes, such as DNA synthesis, protein synthesis, protein degradation, signal transduction, cytoskeletal organization, and the components of extracellular matrix. Some of the modifications are the markers of abnormal and altered proteins for rapid degradation. Others make them less susceptible to degradation by normal proteolytic enzymes, and hence these accumulate during aging.

Mild stress-induced stimulation of heat-shock protein synthesis and improved functional ability of human fibroblasts undergoing aging in vitro

Repeated mild heat-shock (RMHS) treatment has anti-aging hormetic effects on human fibroblasts undergoing aging in vitro. Since heat and various other stresses induce the transcription and translation of heat-shock proteins (Hsp), it was investigated if RMHS treatment affected the basal levels of four major stress proteins Hsp27, 70, 90 and Hsc70. The basal levels of Hsp27, Hsc70, and Hsp70 increased significantly in late passage senescent cells, which is indicative of an adaptive response to cumulative intracellular stress during aging. RMHS increased the levels of these Hsp even in early passage young cells and were maintained high throughout their replicative lifespan. In comparison, the amount of Hsp90 decreased both with aging and RMHS treatment in vitro. However, whereas the difference in the levels of Hsp70 and Hsp90 was statistically significant, the levels of Hsp27 and Hsc70 were statistically similar in normal and RMHS-treated serially passaged cells. These alterations were accompanied by an improved functional and survival ability of the cells in terms of increased proteasomal activities, increased ability to decompose H(2)O(2), reduced accumulation of lipofuscin and enhanced resistance to ethanol, H(2)O(2) and UV-A radiation.

Demonstration of cellular aging and senescence in serially passaged long-term cultures of human trabecular osteoblasts

The proliferative capacity and cellular and biochemical characteristics of human trabecular bone osteoblasts were analysed throughout their replicative lifespan in vitro. Like several other cell types, human osteoblasts demonstrated a typical Hayflick phenomenon of cellular aging comprising a period of rapid proliferation until cumulative population doubling level (CPDL) 22 to 24, followed by a phase of slow growth and the final cessation of cell division at CPDL 32 to 34. Comparing young cells (less than 20% lifespan completed) and old cells (more than 90% lifespan completed) revealed a progressive increase in population doubling (PD) time, a decrease in attachment frequency, a decrease in the number of S-phase positive cells, a decrease in the rates of DNA, RNA and protein synthesis, an increase in the protein content per cell and an increased proportion of senescence-specificβ-galactosidase positive cells. While osteoblastic production of collagen type I decreased progressively during aging, alkaline phosphatase activity dropped rapidly after the first few passages and then remained constant during the rest of the proliferative lifespan. Significant morphological changes from thin and spindle-shaped early passage young cells to large, flattened and irregularly shaped late passage old cells full of intracellular debris were observed. In comparison, osteoblasts established from an osteoporotic bone sample showed a maximum CPDL of less than 5, had a longer PD time and exhibited abnormal senescent morphology. Thus, we have demonstrated for the first time that human osteoblasts, like several other diploid cell types, have a limited proliferative capacity in vitro and undergo aging and senescence as measured by various cellular and biochemical markers. In addition, preliminary studies show that cells from osteoporotic bone have a severely reduced proliferative capacity. This model of bone cell aging facilitates study of the molecular mechanisms of osteoblast senescence as well as factors related to osteoblast dysfunction in patients with osteoporosis.

Reduced levels of oxidized and glycoxidized proteins in human fibroblasts exposed to repeated mild heat shock during serial passaging in vitro.

Repeated mild heat shock (RMHS) has beneficial hormesis-like effects on various characteristics of human skin fibroblasts undergoing replicative senescence in vitro. We have tested whether RMHS could reduce the accumulation of oxidized and glycoxidized proteins, which is a major age-related change. Levels of carbonylated proteins, furosine, N(epsilon)-carboxymethyl-lysine-rich proteins and advanced glycation end products increased during serial passaging of fibroblasts in culture. However, the extent of accumulation of oxidized and glycoxidized proteins was significantly reduced in RMHS cells. The basal concentration of reduced glutathione was higher and that of oxidized glutathione was lower in RMHS cells. Whereas the basal level of heat shock protein HSP27 decreased in both RMHS and control cells during serial passaging, the increase of the basal level of HSP70 with increasing passage level was significantly higher in RMHS cells. These results show that the slower accumulation of damaged proteins in fibroblasts exposed to RMHS results partly from the increased ability of these cells to cope with oxidative stress, and to synthesize HSP responsible for protein capping and refolding.