Reverse engineering of the giant muscle protein titin

Through the study of single molecules it has become possible to explain the function of many of the complex molecular assemblies found in cells. The protein titin provides muscle with its passive elasticity. Each titin molecule extends over half a sarcomere, and its extensibility has been studied both in situ and at the level of single molecules. These studies suggested that titin is not a simple entropic spring but has a complex structure-dependent elasticity. Here we use protein engineering and single-molecule atomic force microscopy to examine the mechanical components that form the elastic region of human cardiac titin. We show that when these mechanical elements are combined, they explain the macroscopic behaviour of titin in intact muscle. Our studies show the functional reconstitution of a protein from the sum of its parts.     

Insulin stimulates sugar transport in giant muscle fibres of the barnacle

Insulin stimulates sugar transport in vertebrate skeletal muscle but the mechanism of insulin action is unknown. It has been reported that Na transport in giant muscle fibers of the barnacle (Balanus nubilis) is sensitive to insulin but no one has examined the sensitivity of sugar tansport to insulin in this preparation. We show here that insulin does, indeed, stimulate sugar transport in barnacle muscle. The great advantage of barnacle muscle over all other muscles used so far for investigating the mechanism of insulin action is its large size, which facilitates measurements on single cells and permits the experimenter to control the intracellular environment of the muscle fibre by the technique of internal dialysis. Using single muscle fibres it is possible to show that acceleration of sugar transport by insulin is associated with a fall in ionized Ca, a fall in cyclic AMP and a rise in cyclic GMP. Working with internally dialysed muscle fibres we find that insulin only increases sugar transport when the dialysis solution contains ATP. In the absence of insulin, sugar transport is dialysed muscle is increased by a rise in ionized Ca, a fall in cyclic AMP and, when the internal Ca is elevated, by a rise in cyclic GMP.     

Hidden complexity in the mechanical properties of titin

Individual molecules of the giant protein titin span the A-bands and I-bands that make up striated muscle. The I-band region of titin is responsible for passive elasticity in such muscle, and contains tandem arrays of immunoglobulin domains. One such domain (I27) has been investigated extensively, using dynamic force spectroscopy and simulation. However, the relevance of these studies to the behaviour of the protein under physiological conditions was not established. Force studies reveal a lengthening of I27 without complete unfolding, forming a stable intermediate that has been suggested to be an important component of titin elasticity. To develop a more complete picture of the forced unfolding pathway, we use mutant titins--certain mutations allow the role of the partly unfolded intermediate to be investigated in more depth. Here we show that, under physiological forces, the partly unfolded intermediate does not contribute to mechanical strength. We also propose a unified forced unfolding model of all I27 analogues studied, and conclude that I27 can withstand higher forces in muscle than was predicted previously.     

Expression of a mammalian Na-H exchanger in muscle fibres of the giant barnacle

It is now well established that the internal pH (pHi) of mammalian cells is regulated by means of a plasma membrane transport system that exchanges extracellular Na+ for intracellular H+ (ref. 1). Furthermore, modulation of the activity of the Na-H exchanger seems to have a crucial role in the action of various mitogens and growth factors. The possibility that such a mammalian Na-H exchanger might be efficiently expressed in a giant invertebrate cell was suggested to us by recent results of Barnard and Miledi and colleagues, who demonstrated in frog oocytes the expression of various plasma membrane channels that presumably were encoded by the mammalian messenger RNA wih which the oocytes had been injected. We used muscle fibres of the giant barnacle, which normally have no demonstrable Na-H exchanger activity, and report here that, when injected with poly(A)+ RNA isolated from rabbit liver, the muscle fibres express a Na-H exchanger. No such expression is observed, however, when the injected material is pretreated with ribonuclease A. As hepatocytes are known to possess a Na-H exchanger, the most straightforward interpretation of our data is that a mammalian Na-H exchanger has been expressed in the muscle fibre of an invertebrate.     

A specific amyloid-beta protein assembly in the brain impairs memory

Memory function often declines with age, and is believed to deteriorate initially because of changes in synaptic function rather than loss of neurons. Some individuals then go on to develop Alzheimer's disease with neurodegeneration. Here we use Tg2576 mice, which express a human amyloid-beta precursor protein (APP) variant linked to Alzheimer's disease, to investigate the cause of memory decline in the absence of neurodegeneration or amyloid-beta protein amyloidosis. Young Tg2576 mice (< 6 months old) have normal memory and lack neuropathology, middle-aged mice (6-14 months old) develop memory deficits without neuronal loss, and old mice (> 14 months old) form abundant neuritic plaques containing amyloid-beta (refs 3-6). We found that memory deficits in middle-aged Tg2576 mice are caused by the extracellular accumulation of a 56-kDa soluble amyloid-beta assembly, which we term Abeta*56 (Abeta star 56). Abeta*56 purified from the brains of impaired Tg2576 mice disrupts memory when administered to young rats. We propose that Abeta*56 impairs memory independently of plaques or neuronal loss, and may contribute to cognitive deficits associated with Alzheimer's disease.     

Machinery for protein sorting and assembly in the mitochondrial outer membrane

Mitochondria contain translocases for the transport of precursor proteins across their outer and inner membranes. It has been assumed that the translocases also mediate the sorting of proteins to their submitochondrial destination. Here we show that the mitochondrial outer membrane contains a separate sorting and assembly machinery (SAM) that operates after the translocase of the outer membrane (TOM). Mas37 forms a constituent of the SAM complex. The central role of the SAM complex in the sorting and assembly pathway of outer membrane proteins explains the various pleiotropic functions that have been ascribed to Mas37 (refs 4, 11-15). These results suggest that the TOM complex, which can transport all kinds of mitochondrial precursor proteins, is not sufficient for the correct integration of outer membrane proteins with a complicated topology, and instead transfers precursor proteins to the SAM complex.     

蛋白质结构中氨基酸残基聚集体的识别与分析

在蛋白质结构中，氨基酸残基并不是单独行使其功能．几个残基通常聚集在一起，共同承担生物学角色．本文通过对蛋白质结构内残基的空间分布进行分析，提取出从两个残基到五个残基的组合，并统计出它们出现的频率和频率分布．二元组在维系蛋白质三级结构中起重要作用，而三元组、四元组和五元组与蛋白质的功能有着密切的关系．这些多元组可为蛋白质结构及功能的研究提供必要的信息．     

Ras regulates assembly of mitogenic signalling complexes through the effector protein IMP

The signal transduction cascade comprising Raf, mitogen-activated protein (MAP) kinase kinase (MEK) and MAP kinase is a Ras effector pathway that mediates diverse cellular responses to environmental cues and contributes to Ras-dependent oncogenic transformation. Here we report that the Ras effector protein Impedes Mitogenic signal Propagation (IMP) modulates sensitivity of the MAP kinase cascade to stimulus-dependent activation by limiting functional assembly of the core enzymatic components through the inactivation of KSR, a scaffold/adaptor protein that couples activated Raf to its substrate MEK. IMP is a Ras-responsive E3 ubiquitin ligase that, on activation of Ras, is modified by auto-polyubiquitination, which releases the inhibition of Raf-MEK complex formation. Thus, Ras activates the MAP kinase cascade through simultaneous dual effector interactions: induction of Raf kinase activity and derepression of Raf-MEK complex formation. IMP depletion results in increased stimulus-dependent MEK activation without alterations in the timing or duration of the response. These observations suggest that IMP functions as a threshold modulator, controlling sensitivity of the cascade to stimulus and providing a mechanism to allow adaptive behaviour of the cascade in chronic or complex signalling environments.     

A regular pattern of two types of 100-residue motif in the sequence of titin

Titin is the largest polypeptide yet described (relative molecular mass approximately 3 x 10(6); refs 1, 2) and an abundant protein of striated muscle. Its molecules are string-like and in vivo span from the M to Z-lines. I-band regions of titin are thought to make elastic connections between the thick filament and the Z-line, thereby forming a third type of sarcomere filament. These would centre the A-band in the sarcomere and provide structural continuity in relaxed myofibrils. The A-band region of titin seems to be bound to the thick filament, where it has been proposed to act as a 'molecular ruler' regulating filament length and assembly. Here, we show that partial titin complementary DNAs encode a regular pattern of two types of 100-residue motif, each of which probably folds into a separate domain type. Such motifs are present in several evolutionarily divergent muscle proteins, all of which are likely to interact with myosin. One or both of the domain types is therefore likely to bind to myosin.     

莱茵衣藻鞭毛内运输蛋白IFT81调控鞭毛组装

利用插入突变的方式获得了一株衣藻不运动突变体ift81,该突变体表现出鞭毛缺失或者短鞭毛的性状。基因序列分析表明,外源基因aphⅧ插入了突变体中IFT81( intraflagellar transport, IFT)基因的第五个外显子内,并导致该外显子原有的52个碱基对被替换。把含有完整IFT81基因的重组质粒导入突变体ift81后,其鞭毛恢复为野生型且可以检测到IFT81-HA融合蛋白的表达,这证明突变体的鞭毛缺陷确实是由于IFT81基因突变所导致。电镜观察显示突变体中鞭毛的显微结构发生改变,免疫荧光实验证实IFT81蛋白主要定位于基体和鞭毛部位。上述结果表明：IFT81蛋白缺失会导致衣藻鞭毛组装缺陷,在鞭毛组装所需蛋白的运输过程中,IFT81蛋白是必不可少的。