Nobel Lecture. Protein synthesis,proteolysis, and cell cycle transitions

The discovery of the role(s) of protein synthesis and degradation in the operation of the cell cycle is described.     

Protein synthesis, nitrogen excretion and long-term growth of juvenile Pleuronectes flesus

This study aimed to measure protein synthesis using a stable isotope method, investigate protein-nitrogen flux in a flatfish Pleuronectes flesus , and use the data to test the hypothesis that individual differences in growth efficiency were related to individual differences in protein-nitrogen flux mediated through differences in protein synthesis and degradation. Three measurements of protein-nitrogen flux via consumption, protein synthesis and nitrogenous excretion were made for individual flounder during a 212-day period and fractional rates of protein-nitrogen flux were scaled for a 50–g flounder to provide mean values for protein consumption (2·11 ± 0·21% day⁻¹), protein synthesis (2·08±0·23% day⁻¹), protein growth (0·71±0·06% day⁻¹) and protein degradation (1·37±0·24% day⁻¹). Mean rates of nitrogenous excretion were 0·142 mg N g⁻¹ day⁻¹ and 0·047 mg N g⁻¹ day⁻¹ for ammonia and urea, respectively. Individual flounder had different protein growth efficiencies and this was correlated negatively and significantly with mean rates of protein synthesis ( r - 0·70; P <0·05) and degradation ( r - 0·67; P < 0·05) and correlated positively and significantly with the efficiency of retaining synthesized protein ( r +0·63, P <0·05). This supported the proposed hypothesis that flounder which grow more efficiently achieve this through adopting a low protein turnover strategy.     

Protein synthesis in isolated cell nuclei

1. Nuclei prepared from calf thymus tissue in a sucrose medium actively incorporate labelled amino acids into their proteins. This is an aerobic process which is dependent on nuclear oxidative phosphorylation. 2. Evidence is presented to show that the uptake of amino acids represents nuclear protein synthesis. 3. The deoxyribonucleic acid of the nucleus plays a role in amino acid incorporation. Protein synthesis virtually ceases when the DNA is removed from the nucleus, and uptake resumes when the DNA is restored. 4. In the essential mechanism of amino acid incorporation, the role of the DNA can be filled by denatured or partially degraded DNA, by DNAs from other tissues, and even by RNA. Purine and pyrimidine bases, monoribonucleotides, and certain dinucleotides are unable to substitute for DNA in this system. 5. When the proteins of the nucleus are fractionated and classified according to their specific activities, one finds the histones to be relatively inert. The protein fraction most closely associated with the DNA has a very high activity. A readily extractable ribonucleoprotein complex is also extremely active, and it is tempting to speculate that this may be an intermediary in nucleocytoplasmic interaction. 6. The isolated nucleus can incorporate glycine into nucleic acid purines, and orotic acid into the pyrimidines of its RNA. Orotic acid uptake into nuclear RNA requires the presence of the DNA. 7. The synthesis of ribonucleic acid can be inhibited at any time by a benzimidazole riboside (DRB) (which also retards influenza virus multiplication (11)). 8. The incorporation of amino acids into nuclear proteins seems to require a preliminary activation of the nucleus. This can be inhibited by the same benzimidazole derivative (DRB) which interferes with RNA synthesis, provided that the inhibitor is present at the outset of the incubation. DRB added 30 minutes later has no effect on nuclear protein synthesis. These results suggest that the activation of the nucleus so that it actively incorporates amino acids into its proteins requires a preliminary synthesis of ribonucleic acid. 9. Together with earlier observations (27, 28) on the incorporation of amino acids by cytoplasmic particulates, these results show that protein synthesis can occur in both nucleus and cytoplasm.     

Protein synthesis, degradation, and retention: mechanisms of indeterminate growth in cephalopods

This study is the first to examine the underlying process of growth in a cephalopod, the southern dumpling squid (Euprymna tasmanica), to ascertain the mechanism by which indeterminate growth is achieved in this live-fast, die-young group of animals. This is the first study to estimate rates of protein synthesis and growth of squid from 7 to 140 d of age, providing an understanding of both the pattern and the process of growth throughout the lifetime of a squid species. Younger and smaller individuals had greater rates of protein synthesis and protein synthesis retention efficiency, as well as more RNA, than did older and larger individuals. Variation in growth rates among older, larger individuals was a function of individuals with faster growth rates having greater protein synthesis retention efficiency and also greater concentrations of protein. Critically, growth did not cease in the adults and, with an average of 10% of protein synthesized being retained, the mechanism to support the nonasymptotic growth model of cephalopods is provided.     

Protein synthesis inhibitors, like growth factors, may render resting 3T3 cells competent for DNA synthesis: a radioautographic and cell fusion study

Serum-deprived (0.1-0.2%) resting NIH 3T3 mouse fibroblasts pre-incubated with cycloheximide (7.5 micrograms/ml), or puromycin (10 micrograms/ml), were fused with stimulated cells taken 10 h after changing the medium to one containing 10% serum, and DNA synthesis was investigated in the nuclei of monokaryons, homodikaryons and heterodikaryons using radioautography with the double-labelling technique. Pre-incubation of resting cells with inhibitors of protein synthesis for 1-4 h abolished their ability to suppress DNA synthesis in stimulated nuclei in heterokaryons. Three hours after the removal of cycloheximide from the medium, the resting cells acquired once again the inhibitory capacity for entry of stimulated nuclei into the S period. This inhibitory influence disappeared also in the case of post-fusion cycloheximide application as well as following an 8-12 h pre-treatment of resting cells with actinomycin D (1 microgram/ml) prior to fusion. Pre-incubation of resting cells for 12 h with PDGF (1 u/ml-1) followed by an 8-48 h incubation in serum-free medium stimulated the onset of DNA synthesis. A brief exposure (45 min) of resting cells to cycloheximide (7.5 micrograms/ml), or puromycin (7.5 micrograms/ml), exerted a similar effect, inducing by itself the entry of cells into the S period. The results support the assumption that acquirement, by resting cells, of competence for DNA replication includes as a necessary step the down-regulation of intracellular growth inhibitors whose formation depends on protein synthesis.     

Relationships among dolichyl phosphate, glycoprotein synthesis, and cell culture growth

Following treatment of Chinese hamster ovary cells with inhibitors of mevalonate biosynthesis in the presence of exogenous cholesterol, the cellular concentration of phosphorylated dolichol and the incorporation of [3H]mannose into dolichol-linked saccharides and N-linked glycoproteins declined coincident with a decline in DNA synthesis. Addition of mevalonate to the culture medium increased rates of mannose incorporation into lipid-linked saccharides and restored mannose incorporation into N-linked glycoproteins to control levels within 4 h. After an additional 4 h, synchronized DNA synthesis began. Inhibition of the synthesis of lipid-linked oligosaccharides and N-linked glycoproteins by tunicamycin prevented the induction of DNA synthesis by mevalonate, indicating that glycoprotein synthesis was required for cell division. The results suggest that the rate of cell culture growth may be influenced by the level of dolichyl phosphate acting to limit the synthesis of N-linked glycoproteins.     

Protein synthesis persists during necrotic cell death

Cell death is an intrinsic part of metazoan development and mammalian immune regulation. Whereas the molecular events orchestrating apoptosis have been characterized extensively, little is known about the biochemistry of necrotic cell death. Here, we show that, in contrast to apoptosis, the induction of necrosis does not lead to the shut down of protein synthesis. The rapid drop in protein synthesis observed in apoptosis correlates with caspase-dependent breakdown of eukaryotic translation initiation factor (eIF) 4G, activation of the double-stranded RNA-activated protein kinase PKR, and phosphorylation of its substrate eIF2-alpha. In necrosis induced by tumor necrosis factor, double-stranded RNA, or viral infection, de novo protein synthesis persists and 28S ribosomal RNA fragmentation, eIF2-alpha phosphorylation, and proteolytic activation of PKR are absent. Collectively, these results show that, in contrast to apoptotic cells, necrotic dying cells retain the opportunity to synthesize proteins.     

Regulation of cell wall synthesis and growth

The replication and growth of Mycobacterium tuberculosis are fundamentally linked to the synthesis and extension of its complex cell wall. Incorporation of new wall material must be tightly regulated so that its deposition does not compromise the extant structure. M. tuberculosis also produces an impressive array of complex bioactive lipids that are intimately involved in pathogenesis and protective immunity. The profiles of these lipids are regulated appropriately to allow the bacterium to respond to the prevailing conditions it faces in vivo. A number of regulatory strategies employed by M. tuberculosis to control cell wall biosynthesis and cell division have now been elucidated. The review highlights the role of alternative sigma factors with extracytoplasmic function in the activation of genes for biosynthesis of complex lipids involved in pathogenicity. Rel(Mtb) and CRP(Mt) play roles in cell wall responses to general nutrient deprivation by synthesis and sensing of starvation second messengers, respectively. Recently, the importance of protein phosphorylation networks in cell wall biosynthesis has attracted considerable interest. A plethora of two-component and eukaryotic-like serine/threonine protein kinases systems have been discovered and several are implicated in cell-division, morphogenesis and regulation of the profile of complex bioactive lipids elaborated by the pathogen.     

Induction, differentiation and oncogenesis

Eukaroytic cytodifferentiation usually leads to stable end-states. However, evidence on embryonic induction in amphibia supports the view that the initial stages of cellular maturation following induction of differentiation are comparatively labile, stability being a property acquired during maturation.Early developing cells may therefore retain the capacity for reversion to their previous, less developed, condition and a proportion of any group of induced cells may do so. Possible implications of such a phenomenon are explored, using two kinetic models of reversion. It is concluded that reversion is a possible mechanism in leukaemogenesis and other forms of oncogenesis.     

Protein synthesis and secretion in a myogenic cell line

Myogenesis in a clonal myoblast cell line is accompanied by an increase in the specific activities of creatine phosphokinase and myokinase and in the rates of synthesis and accumulation of myosin heavy chain. Exponentially dividing myoblasts synthesize myosin heavy chain at a rate of about 1% of their rate of total protein synthesis; this rate increases 7-fold during the differentiation process. Both myoblasts and myotubes secrete a minimum of 12-soluble proteins. Although there is a quantitative change in the rates of appearance of five of these proteins during myogenesis, no qualitative changes in the profile of the secreted proteins are detected. Three of the secreted proteins share several properties of soluble collagen molecules. Basal laminae and polymerized collagen fibrils are associated with myotubes, but not with exponentially dividing myoblasts.     

Somatic cell hybridization and problems of viral oncogenesis

In Vitro Cellular &; Developmental Biology - Plant -     

Derangement of growth and differentiation control in oncogenesis.

Human neoplasms develop following the progressive accumulation of genetic and epigenetic alterations to oncogenes and tumor suppressor genes. These alterations confer a growth advantage to the cancer cell, leading to its clonal proliferation, invasion into surrounding tissues, and spread to distant organs. Genes that are altered in neoplasia affect three major biologic pathways that normally regulate cell growth and tissue homeostasis: the cell cycle, apoptosis, and differentiation. While each of these pathways can be defined by a unique set of molecular events, they are not biologically separate. Rather, they function more as an integrated molecular network, and perturbations in one pathway can have profound consequences on another. Insights into what distinguishes the regulation of growth and differentiation in a normal cell versus a cancer cell have led to the development of novel anticancer therapies.     

Protein synthesis during salivary gland development,ageing, and cell death in Chironomus tentans larvae

Salivary gland protein synthesis in Chironomus tentans larvae was analyzed from the mid-third instar to larval pupation. Correcting for stage specific variations in the specific activity of the amino acid pool revealed a 30–40% reduction in the rate of protein synthesis during the larval moult. Except for a transient increase early in the fourth instar, this low rate of protein synthesis was maintained until the pharate pupal period when protein synthesis dramatically increased: maximum synthesis occurred in mid-pharate pupae with a subsequent decline correlating with gland autolysis and cell death at pupation. Each developmental period was characterized by a particular pattern of secretory protein synthesis: high and 35,000 daltons peptides were maximally synthesized only at particular larval stages, being reduced or absent in post-ecdysis, diapause and autolysing salivary glands.Except for the ecdysone puffs, and as otherwise previously noted (Clever, 1961, 1962), puffing activity during the peri-moult period remained relatively constant and did not decrease by the 30–40% predicted from the decreased rate of protein synthesis. The nearly complete loss in synthesis of the 35,000 daltons peptide was not accompanied by regression in any puff.     

Protein synthesis requirements for nuclear division, cytokinesis, and cell separation in Saccharomyces cerevisiae.

Protein synthesis inhibitors have often been used to identify regulatory steps in cell division. We used cell division cycle mutants of the yeast Saccharomyces cerevisiae and two chemical inhibitors of translation to investigate the requirements for protein synthesis for completing landmark events after the G1 phase of the cell cycle. We show, using cdc2, cdc6, cdc7, cdc8, cdc17 (38 degrees C), and cdc21 (also named tmp1) mutants, that cells arrested in S phase complete DNA synthesis but cannot complete nuclear division if protein synthesis is inhibited. In contrast, we show, using cdc16, cdc17 (36 degrees C), cdc20, cdc23, and nocodazole treatment, that cells that arrest in the G2 stage complete nuclear division in the absence of protein synthesis. Protein synthesis is required late in the cell cycle to complete cytokinesis and cell separation. These studies show that there are requirements for protein synthesis in the cell cycle, after G1, that are restricted to two discrete intervals.