Search

Thioredoxin (Trx) is a cytosolic, redox-active protein that is secreted from many cells and has several extracellular functions. In activated lymphocytes, the pathway of secretion does not involve the Golgi apparatus. Levels of extracellular Trx are decreased by the antioxidant N-acetylcysteine. Hence, the secretion of Trx could be altered by the redox status of the cell or the protein. To study Trx mutants, we characterized the secretion of human Trx from Chinese hamster ovary cells. Secretion of human Trx is unaffected by brefeldin A, slow but efficient, and sensitive to low temperature and factors in serum. We demonstrate that N-acetylcysteine reduces the cellular level of Trx but not the proportion secreted; thus this chemical does not block the nonclassic pathway for Trx secretion. Furthermore, we find that mutations in either the active site or the dimerization site of Trx do not alter its secretion. Thus the nonclassic secretion of Trx is not dependent on the redox status of either the cell or the protein.

Determinants for the recognition of a mitochondrial presequence by the mitochondrial processing peptidase (MPP) have been investigated using mutagenesis and bioinformatics approaches. All plant mit ...

The critical residues involved in targeting and processing of the soybean alternative oxidase to plant and animal mitochondria was investigated. Import of various site-directed mutants into soybean mitochondria indicated that positive residues throughout the length of the presequence were important for import, not just those in the predicted region of amphiphilicity. The position of the positive residues in the C-terminal end of the presequence was also important for import. Processing assays of the various constructs with purified spinach mitochondrial processing peptidase showed that all the -2-position mutants had a drastic effect on processing. In contrast to the import assay, the position of the positive residue could be changed for processing. Deletion mutants confirmed the site-directed mutagenesis data in that an amphiphilic alpha-helix was not the only determinant of mitochondrial import in this homologous plant system. Import of these constructs into rat liver mitochondria indicated that the degree of inhibition differed and that the predicted region of amphiphilic alpha-helix was more important with rat liver mitochondria. Processing with a rat liver matrix fraction showed little inhibition. These results are discussed with respect to targeting specificity in plant cells and highlight the need to carry out homologous studies and define the targeting requirements to plant mitochondria.

Mitochondrial biogenesis requires a coordinated expression of both the nuclear and the organellar genomes and specific intracellular protein trafficking, processing and assembly machinery. Most mitochondrial proteins are synthesised as precursor proteins containing an N-terminal extension which functions as a targeting signal, which is proteolytically cleaved off after import into mitochondria. We review our present knowledge on components and mechanisms involved in the mitochondrial protein import process in plants. This encompasses properties of targeting peptides, sorting of precursor proteins between mitochondria and chloroplasts, signal recognition, mechanism of translocation across the mitochondrial membranes and the role of cytosolic and organellar molecular chaperones in this process. The mitochondrial protein processing in plants is catalysed by the mitochondrial processing peptidase (MPP), which in contrast to other sources, is integrated into the bc1 complex of the respiratory chain. This is the most studied component of the plant import machinery characterised to date. What are the biochemical consequences of the integration of the MPP into an oligomeric protein complex and how are several hundred presequences of precursor proteins with no sequence similarities and no consensus for cleavage, specifically cleaved off by MPP? Finally we will address the emerging area of the control of protein import into mitochondria.

The effect of metal chelators on protein import was investigated using isolated soybean mitochondria and soybean precursor proteins. Adding 1,10-phenanthroline, a metal chelator that can cross both mitochondrial membranes abolished import of both the alternative oxidase, and the FAd subunit of the ATP synthase, a matrix located protein. Other metal chelators such as EDTA, 1,7-phenanthroline and 4,7-phenanthroline, which cannot cross the mitochondrial membranes, had no effect on import. When processing, a known metal-dependent step inside mitochondria, was inhibited using a mutagenesis approach (changing a - 2 arginine to a - 2 glycine in the pre-piece of the precursor), so was import. Thus it would appear that in soybean, at least, translocation of proteins across the mitochondrial membrane, as well as processing, relies on a metal dependent step. Taken together, the data suggest that the two processes may be directly connected in these mitochondria.

Cotransfection of a mixture of siRNAs species is typically used when simultaneous targeting of more than one mRNA is required. However, competition between siRNAs could occur and reduce the activity of some siRNAs within the mixture. To further study the factors affecting the degree of competition between siRNAs, we cotransfected luciferase targeting siRNAs with various irrelevant (ie, nonluciferase targeting) siRNAs into cells and examined differences in their competition profiles by assessing the effect on luciferase expression. We show that the degree of competition varies between irrelevant siRNAs and occurs at the point of RISC loading. Although the competition profile appears to be related to the calculated RNA-induced silencing complex (RISC) loading potential, empirical testing is required to confirm the competitive effects. We also observed reduced competition with siRNAs in the Dicer-substrate format, presumably due to more efficient RISC loading as a consequence of the physical transfer of the processed siRNA from Dicer.

A series of studies published in 2003 has challenged the essentiality of Cdk2. A recently published work indicates that cyclin E-Cdk1 compensates for Cdk2's function at G1/S transition in Cdk2(-/-) Mefs. In this study, we uncovered a redundant mechanism between Cdk1 and Cdk2 at G2 in multiple cancer cell lines. When either Cdk2 or Cdk1 is ablated using RNAi, there were complex shifts of cyclin A towards its reciprocal partner, i.e., when Cdk2 is ablated, cyclin A redistributes to Cdk1; when Cdk1 is ablated, cyclin A forms more abundant complexes with Cdk2. Further, cyclin B redistributes to Cdk2 upon Cdk1 knockdown. These redistributions bring about increased kinase activities of corresponding complexes. Elimination of the compensatory mechanism by knockdown of both Cdk1 and Cdk2 using RNAi reveals phenotypes at G2 phase. The results suggest that the redistributed complexes contribute to the cyclin B-Cdk1 activation when either Cdk1 or Cdk2 alone is ablated and this redundancy masks Cdk2's role when Cdk2 is singly ablated. It is also worth noting that the predominant G2 arrest described here, unlike those Cdk1-Cdk2 double ablated Mefs, raises a question of whether different Cdk activities are required for G1/S or G2/M progression in normal vs. cancer cells.

Centromeric protein-E (CENP-E) is a kinesin-like motor protein required for chromosome congression at prometaphase. Functional perturbation of CENP-E by various methods results in a consistent phenotype, i.e., unaligned chromosomes during mitosis. One unresolved question from previous studies is whether cells complete mitosis or sustain mitotic arrest in the presence of unaligned chromosomes. Using RNA interference and video-microscopy, we analyzed the dynamic process of mitotic progression of HeLa(H2B)-GFP cells lacking CENP-E. Our results demonstrate that these cells initiated anaphase after a delayed mitotic progression due to the presence of unaligned chromosomes. In some dividing cells, unaligned chromosomes are present during anaphase, causing nondisjunction of some sister chromatids producing aneuploid daughter cells. Unlike in Xenopus extract, the loss of CENP-E in HeLa cells does not impair gross checkpoint activation because cells were arrested in mitosis in response to microtubule-interfering agents. However, the lack of CENP-E at kinetochores reduced the hyperphosphorylation of BubR1 checkpoint protein during mitosis, which may explain the loss of sensitivity of a cell to a few unaligned chromosomes in the absence of CENP-E. We also found that presynchronization with nocodazole sensitizes cells to the depletion of CENP-E, leading to more unaligned chromosomes, longer arrest, and cell death.

The green fluorescent protein is a cytosolic protein frequently used as a molecular tag to study protein localization in intact cells. We discovered that this protein is secreted into the medium by several but not all cell lines through a non-classical secretory pathway that is insensitive to brefeldin A. Green fluorescent protein is secreted efficiently by Chinese hamster ovary cells, with 60% of synthesized proteins secreted over 8 hours. This pathway is sensitive to changes in temperature but not to factors in serum or chemicals known to affect other non-classical protein secretion pathways. Fluorescence is observed in cells expressing green fluorescent protein, indicating that some of the protein must be fully folded in the cytosol. However, secreted green fluorescent protein is not fluorescent and therefore not folded properly. Furthermore, cellular fluorescence does not change over 6 hours whereas a significant proportion of green fluorescent protein is secreted. Thus, nascent green fluorescent protein either is folded correctly or incorrectly, and the improperly folded molecules can be exported. Non-classical secretion might be a route by which cells remove an excess of improperly folded, cytosolic proteins.

Mitochondrial biogenesis requires a coordinated expression of both the nuclear and the organellar genomes and specific intracellular protein trafficking, processing and assembly machinery. Most mitochondrial proteins are synthesised as precursor proteins containing an N-terminal extension which functions as a targeting signal, which is proteolytically cleaved off after import into mitochondria. We review our present knowledge on components and mechanisms involved in the mitochondrial protein import process in plants. This encompasses properties of targeting peptides, sorting of precursor proteins between mitochondria and chloroplasts, signal recognition, mechanism of translocation across the mitochondrial membranes and the role of cytosolic and organellar molecular chaperones in this process. The mitochondrial protein processing in plants is catalysed by the mitochondrial processing peptidase (MPP), which in contrast to other sources, is integrated into the bc1 complex of the respiratory chain. This is the most studied component of the plant import machinery characterised to date. What are the biochemical consequences of the integration of the MPP into an oligomeric protein complex and how are several hundred presequences of precursor proteins with no sequence similarities and no consensus for cleavage, specifically cleaved off by MPP? Finally we will address the emerging area of the control of protein import into mitochondria.

Background Short interfering RNAs (siRNAs) have been shown to induce immune stimulation through a number of different receptors in a range of cell types. In primary cells, both TLR7 and TLR8 have been shown to recognise siRNAs however, despite the identification of a number of TLR7/8 stimulatory RNA motifs, the complete and definitive sequence determinants of TLR7 and TLR8 are yet to be elucidated. Results A total of 207 siRNA sequences were screened for TLR7/8 stimulation in human PBMCs. There was a significant correlation between the U count of the U-rich strand and the immunostimulatory activity of the duplex. Using siRNAs specifically designed to analyse the effect of base substitutions and hybridisation of the two strands, we found that sequence motifs and the thermodynamic properties of the duplexes appeared to be the major determinants of siRNA immunogenicity and that the strength of the hybridisation interaction between the two strands correlated negatively with immunostimulatory activity. Conclusion The data presented favour a model of TLR7/8 activation by siRNAs, in which the two strands are denatured in the endosome, and single-stranded, U-rich RNA species activate TLR7/8. These findings have relevance to the design of siRNAs, particularly for in vivo or clinical applications.

The identity of the multiple alternative oxidase bands detected in various soybean tissues was investigated to determine if any modification that can alter the mobility on SDS-PAGE of the alternative oxidase occurs after mitochondrial import other than removal of the presequence. Comparison of the mature, in vitro imported products of AOX1, AOX2 and AOX3 in soybean cotyledons and rat liver mitochondria indicated that they had an identical apparent molecular mass to their in vitro expressed mature forms. This suggests that no modification specific to plant alternative oxidase altering the mobility on SDS-PAGE, took place. Changing the –2 and/or –3 Arg residue resulted in the inhibition of the generation of this mature form, suggesting that processing was most likely by the general mitochondrial processing peptidase. Comparison of the in vitro expressed mature forms to that detected by immunoblots of soybean tissues, required the induction of AOX1. Treatment of soybean cultured cells with antimycin A resulted in the induction of an additional band cross-reacting to monoclonal antibodies against the alternative oxidase. Comparison of the in vitro expressed mature forms to the alternative oxidase detected by western blotting indicated that they were identical in apparent molecular mass. These results indicated that no modification other than presequence removal, which alters mobility on SDS-PAGE, was required to generate the mature functional alternative oxidase proteins.