Trigger factor and DnaK cooperate in folding of newly synthesized proteins.

The role of molecular chaperones in assisting the folding of newly synthesized proteins in the cytosol is poorly understood. In Escherichia coli, GroEL assists folding of only a minority of proteins and the Hsp70 homologue DnaK is not essential for protein folding or cell viability at intermediate growth temperatures. The major protein associated with nascent polypeptides is ribosome-bound trigger factor, which displays chaperone and prolyl isomerase activities in vitro. Here we show that delta tig::kan mutants lacking trigger factor have no defects in growth or protein folding. However, combined delta tig::kan and delta dnaK mutations cause synthetic lethality. Depletion of DnaK in the delta tig::kan mutant results in massive aggregation of cytosolic proteins. In delta tig::kan cells, an increased amount of newly synthesized proteins associated transiently with DnaK. These findings show in vivo activity for a ribosome-associated chaperone, trigger factor, in general protein folding, and functional cooperation of this protein with a cytosolic Hsp70. Trigger factor and DnaK cooperate to promote proper folding of a variety of E. coli proteins, but neither is essential for folding and viability at intermediate growth temperatures.     

Rapid degradation of a large fraction of newly synthesized proteins by proteasomes

MHC class I molecules function to present peptides eight to ten residues long to the immune system. These peptides originate primarily from a cytosolic pool of proteins through the actions of proteasomes, and are transported into the endoplasmic reticulum, where they assemble with nascent class I molecules. Most peptides are generated from proteins that are apparently metabolically stable. To explain this, we previously proposed that peptides arise from proteasomal degradation of defective ribosomal products (DRiPs). DRiPs are polypeptides that never attain native structure owing to errors in translation or post-translational processes necessary for proper protein folding. Here we show, first, that DRiPs constitute upwards of 30% of newly synthesized proteins as determined in a variety of cell types; second, that at least some DRiPs represent ubiquitinated proteins; and last, that ubiquitinated DRiPs are formed from human immunodeficiency virus Gag polyprotein, a long-lived viral protein that serves as a source of antigenic peptides.     

Transient association of newly synthesized unfolded proteins with the heat-shock GroEL protein

It has been suggested that newly synthesized proteins are maintained in their unfolded state by cellular ATP-driven factors which may prevent or reverse the formation of misfolded structures or promote the correct assembly of oligomeric proteins or post-translational secretion. Using a photocross-linking approach, we have identified the 20S heat-shock GroEL protein as the major cytosolic component which forms a complex with the unfolded newly synthesized pre-beta-lactamase or chloramphenicol acetyltransferase in Escherichia coli. Dissociation of these complexes is ATP-dependent. The unfolded state of pre-beta-lactamase, maintained by the transient interaction with GroEL, may be essential for the secretion of this protein.     

Anti-pp60src antibodies are substrates for EGF-stimulated protein kinase

Epidermal growth factor (EGF) stimulates phosphorylation of its own receptor at a tyrosine residue. Similarly, the viral gene product pp60src, which is responsible for cellular transformation by avian sarcoma virus (ASV), phosphorylates itself and immunoglobulin directed against pp60src at tyrosine residues. This unusual site of phosphorylation catalysed by two membrane-associated protein kinases involved in growth control prompted us to study the immunological relatedness of the EGF-stimulated protein kinase and the pp60src. Using anti-pp60src antisera, we attempted to immunoprecipitate the EGF-stimulated protein kinase solubilized from plasma membranes. We report here that neither the EGF-stimulated kinase nor the EGF receptor were immunoprecipitable by anti-pp60src sera. However, anti-pp60src IgG served as a specific substrate for the EGF-stimulated kinase, suggesting a close similarity between the EGF-stimulated kinase and pp60src.     

In vivo somatic mutations in human lymphocytes frequently result from major gene alterations

Somatic mutations, either spontaneous or produced by identifiable mutagens, are thought to be important in the aetiology of cancer and in the ageing process. The study of somatic mutations in human cells in vivo has recently been made possible by the development of techniques for enumeration and clonal expansion of lymphocytes mutated at the chromosome X-linked hypoxanthine phosphoribosyl transferase (HPRT) locus. We have studied the molecular basis of in vivo hprt mutations in human lymphocytes and report here that a surprisingly high proportion (57%) involve substantial gene alterations which are not evident cytogenetically. These major gene alterations include deletions, exon amplifications and novel, sometimes amplified, bands on Southern analysis. Such changes emphasize the fluid nature of information in DNA and may be indicative of general mechanisms by which functional gene loss is involved in the aetiology of cancer and the homeostatic failure of ageing.     

新发现和正在研究中的细胞凋亡蛋白抑制剂(综述)

自从杆状病毒中分离出凋亡蛋白抑制剂 (inhibitorofapoptosisproteins ,IAPs)后 ,发现的凋亡蛋白抑制剂的种类和数量逐渐增多。迄今为止 ,在人体新发现的IAPs有HIAP - 1(humanIAP -1)、HIAP - 2 (humanIAP - 2 )、XIAP(Xchromosome -likedIAP)、ML -IAP(melanocytesIAP)、Survivin和Livin等。对IAPs的发现、定位、结构、分布和作用等方面的研究进展进行介绍     

Predominant naturally processed peptides bound to HLA-DR1 are derived from MHC-related molecules and are heterogeneous in size.

Peptides bound to class I molecules are 8-10 amino acids long, and possess a binding motif representative of peptides that bind to a given class I allele. In the only published study of naturally processed peptides bound to class II molecules (mouse I-Ab and I-Eb), these peptides were longer (13-17 amino acids) and had heterogenous carboxy terminals but precise amino-terminal truncations. Here we report the characterization of acid-eluted peptides bound to HLA-DR1 by high-performance liquid chromatography, mass spectrometry and microsequencing analyses. The relative molecular masses of the peptides varied between 1,602 and 2,996 (13-25 residues), the most abundant individual M(r) values being between 1,700 and 1,800, corresponding to an average peptide length of 15 residues. Complete sequence data were obtained for twenty peptides derived from five epitopes, of which all but one were from self proteins. These peptides represented sets nested at both the N- and C-terminal ends. Binding experiments confirmed that all of the isolated peptides had high affinity for the groove of DR1. Alignment of the peptides bound to HLA-DR1 and the sequences of 35 known HLA-DR1-binding peptides revealed a putative motif. Although peptides bound to class II molecules may have some related features (due to the nonpolymorphic HLA-DR alpha-chain), accounting for degenerate binding to different alleles, particular amino acids in the HLA-DR beta-chains presumably define allelic specificity of peptide binding.     

A distal enhancer and an ultraconserved exon are derived from a novel retroposon

Hundreds of highly conserved distal cis-regulatory elements have been characterized so far in vertebrate genomes. Many thousands more are predicted on the basis of comparative genomics. However, in stark contrast to the genes that they regulate, in invertebrates virtually none of these regions can be traced by using sequence similarity, leaving their evolutionary origins obscure. Here we show that a class of conserved, primarily non-coding regions in tetrapods originated from a previously unknown short interspersed repetitive element (SINE) retroposon family that was active in the Sarcopterygii (lobe-finned fishes and terrestrial vertebrates) in the Silurian period at least 410 million years ago (ref. 4), and seems to be recently active in the 'living fossil' Indonesian coelacanth, Latimeria menadoensis. Using a mouse enhancer assay we show that one copy, 0.5 million bases from the neuro-developmental gene ISL1, is an enhancer that recapitulates multiple aspects of Isl1 expression patterns. Several other copies represent new, possibly regulatory, alternatively spliced exons in the middle of pre-existing Sarcopterygian genes. One of these, a more than 200-base-pair ultraconserved region, 100% identical in mammals, and 80% identical to the coelacanth SINE, contains a 31-amino-acid-residue alternatively spliced exon of the messenger RNA processing gene PCBP2 (ref. 6). These add to a growing list of examples in which relics of transposable elements have acquired a function that serves their host, a process termed 'exaptation', and provide an origin for at least some of the many highly conserved vertebrate-specific genomic sequences.     

The helical s constant for alanine in water derived from template-nucleated helices.

Formation of alpha helices from disordered polypeptides depends on the degree to which amino acids favour the helical state. The folding of helical oligopeptides can be modelled by two parameters: sigma which reflects helix initiation and s which reflects propagation of a pre-existing helix and measures helical bias. Scheraga has reported s values for oligopeptides of about 1.1, implying a weak helical bias for amino-acid residues. By contrast, certain helical peptides studied by Baldwin seem to require much larger s values for alanine. Resolution of this inconsistency requires experiments that disentangle the ease of propagation from that of initiation. In this study varying lengths of polyalanine are linked to a 'template' that initiates helical structure and permits study solely of propagation. We report here that the s value for alanine in water is close to 1, supporting the earlier results of Scheraga but not the more recent results of Baldwin.     

Ribonucleotides in DNA newly synthesised in 3T6 cells in vivo.

Within the field of DNA replication, considerable interest has focused in recent years on the mechanism of initiation of synthesis of DNA molecules. In vitro replication systems from Escherichia coli have been instrumental in uncovering a priming function fo9r ribonucleotides on the earliest intermediates of DNA polymerisation in vitro and in identifying the proteins involved. In vitro replication systems from mammalian cells that permit the use of the phosphate-transfer method for detection of RNA-DNA junctions as well as direct labelling of the RNA moiety of the molecules have suggested a similar role for ribonucleotides in DNA synthesis in eukaryotes. However, the existence of this mechanism in mammalian cells in vivo has not been established. Here we report the first evidence that a significant proportion of the earliest intermediates in mammalian DNA polymerisation in vivo do, in fact, possess ribonucleotides, presumably because their synthesis was initiated with one or more ribonucleotides.     

溶剂法生产莠去津工艺的改进

以三氯乙烯做溶剂,以三聚氯氰、异丙胺与乙胺为原料,经过2次取代合成莠去津.通过探索和单因素实验得到最佳合成条件:三聚氯氰和三氯乙烯物料比为1∶6.25(g/mL);分2次加入三聚氯氰,一取代反应过程控温为(25±1)℃,保温反应为30 min;二取代反应过程控温为(45±1)℃,保温反应为90 min.产品纯度达94％以上,收率达92％以上.     

小鼠胎肝间充质干细胞体内向骨骼肌样细胞分化的研究

目的:了解小鼠胎肝间充质干细胞在体内向骨骼肌细胞分化的潜能。方法:无菌条件下将C57BL/6 J胎鼠肝脏制成单细胞悬液,雄性胎肝悬液体外贴壁培养纯化,传3代后将贴壁细胞移植于心肌毒素(card iotoxin)造成的雌鼠骨骼肌损伤部位,2月后处死受鼠,取相应骨骼肌组织固定、制片;用免疫组织化学染色和荧光原位杂交检测雌性受体小鼠骨骼肌组织内供体小鼠胎肝间充质干细胞向骨骼肌样细胞分化情况。结果:在骨骼肌组织内发现存在Y染色体阳性的供体来源的细胞,同时呈现骨骼肌组织的部分特征,表型为desm in /F lt-1-/CD4-5/F4-/80。结论:胎肝中分离出的间充质干细胞在体内可以分化为骨骼肌样细胞。     

Individual recognition in mice mediated by major urinary proteins.

The ability to recognize individuals is essential to many aspects of social behaviour, such as the maintenance of stable social groups, parent-offspring or mate recognition, inbreeding avoidance and the modulation of competitive relationships. Odours are a primary mediator of individuality signals among many mammals. One source of odour complexity in rodents, and possibly in humans, resides in the highly polymorphic major histocompatibility complex (MHC). The olfactory acuity of mice and rats allows them to distinguish between the urinary odours of congenic strains differing only in single genes within the MHC, although the chemical mediators or odorants are unknown. However, rodent urine also contains a class of proteins, termed major urinary proteins (MUPs), that bind and release small volatile pheromones. We have shown that the combinatorial diversity of expression of MUPs among wild mice might be as great as for MHC, and at protein concentrations a million times higher. Here we show in wild house mice (Mus domesticus) that urinary MUPs play an important role in the individual recognition mechanism.     

Protein targeting and degradation are coupled for elimination of mislocalized proteins

A substantial proportion of the genome encodes membrane proteins that are delivered to the endoplasmic reticulum by dedicated targeting pathways. Membrane proteins that fail targeting must be rapidly degraded to avoid aggregation and disruption of cytosolic protein homeostasis. The mechanisms of mislocalized protein (MLP) degradation are unknown. Here we reconstitute MLP degradation in vitro to identify factors involved in this pathway. We find that nascent membrane proteins tethered to ribosomes are not substrates for ubiquitination unless they are released into the cytosol. Their inappropriate release results in capture by the Bag6 complex, a recently identified ribosome-associating chaperone. Bag6-complex-mediated capture depends on the presence of unprocessed or non-inserted hydrophobic domains that distinguish MLPs from potential cytosolic proteins. A subset of these Bag6 complex 'clients' are transferred to TRC40 for insertion into the membrane, whereas the remainder are rapidly ubiquitinated. Depletion of the Bag6 complex selectively impairs the efficient ubiquitination of MLPs. Thus, by its presence on ribosomes that are synthesizing nascent membrane proteins, the Bag6 complex links targeting and ubiquitination pathways. We propose that such coupling allows the fast tracking of MLPs for degradation without futile engagement of the cytosolic folding machinery.     

Mimicking the alloantigenicity of proteins with chemically synthesized peptides differing in single amino acids

Recent studies have shown that short chemically synthesized peptides very often induce antibodies which react with the cognate sequence in the intact folded protein. Since such antibodies react with known regions of proteins, they are of predetermined specificity and offer a precision not previously possible with immunological probes. A basic concept emerging from the use of such antibodies in viral systems is that the differential immunogenicity of closely related proteins can be mimicked by short peptides which span the regions of sequence variation. To generalize this concept, we have studied the two Thy-1 proteins which vary by only a single amino acid. Chemically synthesized peptides differing in only one out of 19 amino acids were able to induce allospecific antisera. Thus, single amino acid changes have similar effects on the immunogenicity of proteins and small peptides, even though the latter are free from constraints provided by neighbouring structures in the tertiary configuration of the intact folded proteins.