Ubiquitination and SUMOylation of annexin A1 and helicase activity

Background

While annexin A1 in nuclei is proposed to be involved in cell transformation, its functions remain poorly understood. Since annexin A1 has the consensus motif, ¹⁶⁰LKRD, for SUMOylation as well as Ks, acceptors for ubiquitination that regulates localization and functions of proteins, we investigated SUMOylation and ubiquitination of annexin A1.

Methods

SUMOylation and ubiquitination of bovine annexin A1 were biochemically tested in vitro by purified proteins, and were confirmed by cell experiments with L5178 lymphoma cells. Effects of the modifications on DNA helicase activity were measured by ssDNA binding activity and by dsDNA unwinding activity.

Results

SUMOylation of annexin A1 was catalyzed by Ubc9, while its ubiquitination was by Rad6-Rad 18. Ubiquitinated annexin A1 had higher affinity for damaged DNA, and promoted in vitro translesion DNA synthesis by Pol β. In mouse lymphoma L5178Y tk(+/−) cells, levels of SUMOylated annexin A1 decreased by DNA damaging agents, MMS or As³⁺, whereas those of ubiquitinated annexin A1 increased under the same conditions.

Conclusion

These observations suggest but do not necessarily prove that ubiquitinated annexin A1 in nuclei may be involved in DNA damage response, while SUMOylated annexin A1 functions in proliferation–differentiation.

Significance

Ubiquitination of annexin A1 may play an important role in mutagenesis, an initial step of cell transformation.     

Mitochondrial proteolysis: Its emerging roles in stress responses

Background

Mitochondria are multifunctional organelles that not only serve as cellular energy stores but are also actively involved in several cellular stress responses, including apoptosis. In addition, mitochondria themselves are also continuously challenged by stresses such as reactive oxygen species (ROS), an inevitable by-product of oxidative phosphorylation. To exert various functions against these stresses, mitochondria must be equipped with appropriate stress responses that monitor and maintain their quality.

Scope of review

Interestingly, increasing evidence indicates that mitochondrial proteolysis has important roles in mitochondrial and cellular stress responses. In this review, we summarize current advances in mitochondrial proteolysis-mediated stress responses.

Major conclusions

Mitochondrial proteases do not only function as surveillance systems of protein quality control by degrading unfolded proteins but also regulate mitochondrial stress responses by processing specific mitochondrial proteins.

General significance

Studies on the regulation of mitochondrial proteolysis-mediated stress responses will provide the novel mechanistic insights into the stress response research fields.     

DNA-maleimide: An improved maleimide compound for electrophoresis-based titration of reactive thiols in a specific protein

Background

Thiol-mediated redox regulation of proteins plays a key role in many cellular processes.

Methods

To understand the redox status of cysteinyl thiol groups of the desired proteins, we developed a new maleimide reagent: a maleimide-conjugated single strand DNA, DNA-maleimide (DNA-Mal).

Results

DNA-Mal labelled proteins run as a distinct band on SDS-PAGE, with a discrete 9.32 kDa mobility shift per label regardless of the protein species or electrophoretic conditions.

Conclusions

DNA-Mal labels free thiols like standard maleimide reagents, but possesses practical advantages in titration of the number and relative content of free thiols in a protein.

General significance

The versatility of DNA molecule enhances the application of DNA-Mal in a broader range of cysteine containing proteins.     

Iron Translocation into and out of Listeria innocua Dps and Size Distribution of the Protein-enclosed Nanomineral Are Modulated by the Electrostatic Gradient at the 3-fold ��Ferritin-like�� Pores

Elucidating pore function at the 3-fold channels of 12-subunit, microbial Dps proteins is important in understanding their role in the management of iron/hydrogen peroxide. The Dps pores are called “ferritin-like” because of the structural resemblance to the 3-fold channels of 24-subunit ferritins used for iron entry and exit to and from the protein cage. In ferritins, negatively charged residues lining the pores generate a negative electrostatic gradient that guides iron ions toward the ferroxidase centers for catalysis with oxidant and destined for the mineralization cavity. To establish whether the set of three aspartate residues that line the pores in Listeria innocua Dps act in a similar fashion, D121N, D126N, D130N, and D121N/D126N/D130N proteins were produced; kinetics of iron uptake/release and the size distribution of the iron mineral in the protein cavity were compared. The results, discussed in the framework of crystal growth in a confined space, indicate that iron uses the hydrophilic 3-fold pores to traverse the protein shell. For the first time, the strength of the electrostatic potential is observed to modulate kinetic cooperativity in the iron uptake/release processes and accordingly the size distribution of the microcrystalline iron minerals in the Dps protein population.The widely distributed bacterial Dps proteins (1, 2) belong to the ferritin superfamily and are characterized by strong similarities (3) but also distinctive differences with respect to “canonical” ferritins, the ubiquitous iron storage, and detoxification proteins found in biological systems. The structural resemblance is apparent in the overall molecular assemblage because both Dps proteins and ferritins are shell-like oligomers constructed from four-helix bundle monomers. However, Dps proteins are 12-mers of identical subunits that assemble with 23 symmetry, whereas ferritins are built by 24 highly similar or identical subunits related by 432 symmetry. The functional similarities consist in the common capacity to remove Fe(II) from cytoplasm, catalyze its oxidation, and store Fe(III) thus produced in the protein cavity, wherefrom the metal can be mobilized when required by the organism. However, ferritins use molecular oxygen as iron oxidant with the production of hydrogen peroxide, whereas Dps proteins prefer hydrogen peroxide, which is typically about 100-fold more efficient than molecular oxygen (1). This difference is of major importance because it renders Dps proteins capable of removing concomitantly Fe(II) and H₂O₂ whose combination leads to the production of reactive oxygen species via Fenton chemistry (4). This capacity confers H₂O₂ resistance and hence may be a virulence factor in certain pathogens (e.g. Campylobacter jejuni, Streptococcus mutans, and Porphyromonas gingivalis) because the H₂O₂ burst represents one of the first defense lines of the host during infection (5–7).Key to a full understanding of the iron uptake and release processes at a molecular level is the route by which iron enters and exits the protein shell. In both 24-subunit ferritins and 12-subunit Dps proteins, the subunit assemblage creates pores across the protein shell that put the internal cavity in communication with the external medium. In ferritins there are two types of pores: largely hydrophobic ones along the axes with 4-fold symmetry and hydrophilic ones along the axes with 3-fold symmetry. The latter channels are funnel-shaped, with the smaller opening toward the protein cavity, and are lined with conserved glutamic and aspartic residues located in the narrow region of the funnel (8). These 3-fold pores were recognized to provide the route for iron entry into the protein cavity soon after resolution of the horse ferritin x-ray crystal structure (9). Later site-directed mutagenesis studies defined the role of specific residues (Asp¹³¹ and Glu¹³⁴) that line the pore (10, 11), whereas electrostatic calculations related the passage of iron to the existence of a gradient that drives metal ions toward the protein interior cavity (12, 13). More recently, the 3-fold symmetry pores were shown to be involved also in the exit process of iron from the protein cavity. Thus, in H-frog ferritin used as model system, iron exit is affected by local protein unfolding promoted by site-specific mutagenesis of individual amino acid residues (14, 15), by the use of chaotropes (16), and by means of selected peptides designed to bind at these channels (17).In Dps proteins, the protein shell is breached by two types of pores along the 3-fold axes, one type is formed by the N-terminal portion of the monomers and bears a strong similarity to the typical 3-fold channels of 24-subunit ferritins in that it is funnel-shaped, hydrophilic, and lined by conserved, negatively charged residues. It was therefore named “ferritin-like” and assumed to be involved in iron entry into the protein cavity upon resolution of the Listeria innocua x-ray crystal structure (18). The other type of pore is formed by the C-terminal ends of the monomers and was called “Dps type” because it is created at a subunit interface that is unique to Dps proteins. Although somewhat variable in length and in the size of the openings, the Dps type pore is mainly hydrophobic in nature (19).The present paper investigates the role of the ferritin-like pores in the iron uptake and release processes in Dps proteins using the well characterized L. innocua Dps (LiDps) as a model system (18, 20–22). In LiDps, the ferritin-like pores contain a set of three aspartate residues, Asp¹²¹, Asp¹²⁶, and Asp¹³⁰ that would be encountered in succession by a metal ion that is attracted by the electrostatic gradient they create and moves down the funnel-shaped pore toward the protein cavity (Fig. 1). Asp¹³⁰, which is located in the narrowest part of the funnel, is conserved significantly among Dps proteins (∼ 80%), whereas Asp¹²¹ and Asp¹²⁶ are less conserved (Fig. 1). Such considerations were used in the design of site-specific variants D121N, D126N, D130N, and D121N/D126N/D130N to elucidate the function of the ferritin-like pores in Dps proteins.Open in a separate windowFIGURE 1.Dps proteins sequences and conservation of the aspartate residues that line the 3-fold ferritin-like pores in L. innocua Dps. A, alignment of multiple Dps sequences from different bacteria: LiDps, non-heme iron-binding ferritin (L. innocua Clip11262]; EcDps, DNA-binding protein Dps (E. coli); HpDps, neutrophil-activating protein (Helicobacter pylori); YpDps, ferritin family protein (Yersinia pestis Angola); GtDps, DNA-protecting protein (Geobacillus thermodenitrificans NG80–2); RmDps, ferritin, and Dps (Ralstonia metallidurans CH34); AtDps, DNA protection during starvation conditions (Agrobacterium tumefaciens str. C58); TeDps Dps family DNA-binding stress response protein (Thermosynechococcus elongatus BP-1); PaDps, putative DNA-binding protein, Dps (Psychrobacter arcticus 273-4); TfDps, hypothetical protein Tfu_0799 (Thermobifida fusca YX); PhDps, DNA-binding DPS protein (Pseudoalteromonas haloplanktis TAC125); SoDps, Dps family protein (Shewanella oneidensis MR-1); BaDps1, general stress protein 20U (Bacillus anthracis str. Ames); BaDps2 general stress protein (B. anthracis str. Ames); LlDps, non-heme iron-binding ferritin (Lactococcus lactis subsp. lactis Il1403); VcDps, DPS family protein (Vibrio cholerae MZO-3); and StDps, DNA-binding ferritin-like protein (oxidative damage protectant) (Streptococcus thermophilus LMD-9). Residues forming the Dps catalytic center are highlighted in pale blue (His³¹, His⁴³, Asp⁵⁸, and Glu⁶² in LiDps); 3-fold pores aspartate residues are highlighted in yellow and marked in bold type. Alignment has been created with ClustalW2 (34). B, view of the junction of three monomers forming the 3-fold ferritin-like pore. C, Asp¹²¹, Asp¹²⁶, and Asp¹³⁰ aspartate residues comprised the pore area. D, three-dimensional view of the pore colored by charge. Red, negatively charged residues; blue, positively charged residues; white, uncharged residues. E, schematic representation of the vertical section of the pore. The images were created with PyMol (35).The results demonstrate that iron uses the LiDps ferritin-like pores to enter and leave the protein shell and hence that these pores have the same role as the structurally similar 3-fold channels in 24-subunit ferritins. LiDps residue Asp¹³⁰ is the most important determinant of the negative electrostatic gradient because of its location in the narrow part of the pores. Importantly, the data show for the first time that the electrostatic gradient at the pores modulates cooperativity in the iron uptake process and influences the size distribution of the iron core (23). The effect of the electrostatic gradient can be explained in terms of the electrostatic interaction effects between the fixed negative charges of the aspartate residues at the pores and the mobile positive charges of iron ions.     

Detection and interpretation of 8-oxodG and 8-oxoGua in urine,plasma and cerebrospinal fluid

Background

DNA and RNA oxidations have been linked to diseases such as cancer, arteriosclerosis, neurodegeneration and diabetes. The prototype base modification studied is the 8-hydroxylation of guanine. DNA integrity is maintained by elaborate repair systems and RNA integrity is less studied but relies mainly on degradation.

Scope of review

DNA and RNA oxidations are measured by very similar techniques. The scope of this review is to highlight the preferred methods of measurement of oxidized nucleic acid metabolites, to highlight novel findings particularly in RNA oxidation, and to present the interpretation of the measurements.

Major conclusions

Tissue levels are snap-shots of the level in a specific organ or cell system and reflect the balance between formation rate and elimination rate (repair), and must be interpreted as such. Urinary excretion is a global measure of oxidative stress in an organism and is therefore best suited for situations or diseases where large parts or the entire organism is stressed by oxidation. It represents the body average rate by which either RNA or DNA is oxidized and is interpreted as oxidative stress. Oxidations of RNA and DNA precursors have been demonstrated and the quantitative importance is debated.

General significance

Careful experimental designs and appropriate choice of methodology are paramount for correct testing of hypotheses related to oxidative stress, and pitfalls are plentiful. There is accumulating evidence that DNA oxidation is associated with disease, particularly cancer, and recent evidence points at an association between RNA oxidation and neurodegenerative diseases and diabetes. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

The Ferritin-like superfamily: Evolution of the biological iron storeman from a rubrerythrin-like ancestor

Background

The Ferritins are part of the extensive ‘Ferritin-like superfamily’ which have diverse functions but are linked by the presence of a common four-helical bundle domain. The role performed by Ferritins as the cellular repository of excess iron is unique. In many ways Ferritins act as tiny organelles in their ability to secrete iron away from the delicate machinery of the cell, and then to release it again in a controlled fashion avoiding toxicity. The Ferritins are ancient proteins, being common in all three domains of life. This ubiquity reflects the key contribution that Ferritins provide in achieving iron homeostasis.

Scope of the review

This review compares the features of the different Ferritins and considers how they, and other members of the Ferritin-like superfamily, have evolved. It also considers relevant features of the eleven other known families within the Ferritin-like superfamily, particularly the highly diverse rubrerythrins.

Major conclusions

The Ferritins have travelled a considerable evolutionary journey, being derived from far more simplistic rubrerythrin-like molecules which play roles in defence against toxic oxygen species. The forces of evolution have moulded such molecules into three distinct types of iron storing (or detoxifying) protein: the classical and universal 24-meric ferritins; the haem-containing 24-meric bacterioferritins of prokaryotes; and the prokaryotic 12-meric Dps proteins. These three Ferritin types are similar, but also possess unique properties that distinguish them and enable then to achieve their specific physiological purposes.

General significance

A wide range of biological functions have evolved from a relatively simple structural unit.     

The effects of selenium and the GPx-1 selenoprotein on the phosphorylation of H2AX

Background

Significant data supports the health benefits of selenium although supplementation trials have yielded mixed results. GPx-1, whose levels are responsive to selenium availability, is implicated in cancer etiology by human genetic data. Selenium's ability to alter the phosphorylation of the H2AX, a histone protein that functions in the reduction of DNA damage by recruiting repair proteins to the damage site, following exposure to ionizing radiation and bleomycin was investigated.

Methods

Human cell lines that were either exposed to selenium or were transfected with a GPx-1 expression construct were exposed to ionizing radiation or bleomycin. Phosphorylation of histone H2AX was quantified by flow cytometry and survival by the MTT assay. Phosphorylation of the Chk1 and Chk2 checkpoint proteins was quantified by western blotting.

Results

In colon-derived cells, selenium increases GPx-1 and attenuated H2AX phosphorylation following genotoxic exposures while the viability of these cells was unaffected. MCF-7 cells and transfectants that express high GPx-1 levels were exposed to ionizing radiation and bleomycin, and H2AX phosphorylation and cell viability were assessed. GPx-1 increased H2AX phosphorylation and viability following the induction of DNA damage while enhancing the levels of activated Chk1 and Chk2.

Conclusions

Exposure of mammalian cells to selenium can alter the DNA damage response and do so by mechanisms that are dependent and independent of its effect on GPx-1.

General significance

Selenium and GPx-1 may stimulate the repair of genotoxic DNA damage and this may account for some of the benefits attributed to selenium intake and elevated GPx-1 activity.     

Loss of HuR leads to senescence-like cytokine induction in rodent fibroblasts by activating NF-κB

Background

HuR (human antigen R) is a ubiquitously expressed member of the Hu/ELAV family of proteins that is involved in diverse biological processes. HuR has also been shown to play an important role in cell cycle arrest during replicative senescence in both human and mouse cells. Senescent cells not only halt their proliferation, but also activate the secretion of proinflammatory cytokines. A persistent DNA damage response is essential for the senescence-associated secretory phenotype (SASP), and increasing evidence has suggested that the SASP is associated with malignancy.

Methods

Senescence-associated phenotypes were analyzed in MEFs and other cell line in which HuR expression is inhibited by sh-RNA-mediated knockdown.

Results

RNAi-mediated HuR inhibition resulted in an increase in SASP-related cytokines. The induction of SASP factors did not depend on ARF–p53 pathway-mediated cell cycle arrest, but required NF-κB activity. In the absence of HuR, cells were defective in the DNA-damage response, and single strand DNA breaks accumulated, which may have caused the activation of NF-κB and subsequent cytokine induction.

Conclusions

In the absence of HuR, cells exhibit multiple senescence-associated phenotypes. Our findings suggest that HuR regulates not only the replicative lifespan, but also the expression of SASP-related cytokines in mouse fibroblasts.

General significance

RNA-binding protein HuR protects cells from undergoing senescence. Senescence-associated phenotypes are accelerated in HuR-deficient cells.     

Prediction of DNA-binding protein based on statistical and geometric features and support vector machines

Background

Previous studies on protein-DNA interaction mostly focused on the bound structure of DNA-binding proteins but few paid enough attention to the unbound structures. As more new proteins are discovered, it is useful and imperative to develop algorithms for the functional prediction of unbound proteins. In our work, we apply an alpha shape model to represent the surface structure of the protein-DNA complex and extract useful statistical and geometric features, and use structural alignment and support vector machines for the prediction of unbound DNA-binding proteins.

Results

The performance of our method is evaluated by discriminating a set of 104 DNA-binding proteins from 401 non-DNA-binding proteins. In the same test, the proposed method outperforms the other method using conditional probability. The results achieved by our proposed method for; precision, 83.33%; accuracy, 86.53%; and MCC, 0.5368 demonstrate its good performance.

Conclusions

In this study we develop an effective method for the prediction of protein-DNA interactions based on statistical and geometric features and support vector machines. Our results show that interface surface features play an important role in protein-DNA interaction. Our technique is able to predict unbound DNA-binding protein and discriminatory DNA-binding proteins from proteins that bind with other molecules.

     

Comparison between the effects of diallyl tetrasulfide on human retina pigment epithelial cells (ARPE-19) and HCT116 cells

Background

Diallyl mono- and polysulfanes from garlic are known to induce an adaptive cell response and the formation of antioxidants in cancer cells. In the case of a severe ER stress and a failure in the response, cancer cells eventually go into apoptosis. Only little is known about the response of normal cells upon treatment.

Methods

Normal ARPE-19 cells were treated with diallyl tetrasulfide to study their cellular response and the results were compared with those of HCT116 cancer cells. Cell viability was checked by an MTT assay and cytofluorimetry. The formation of superoxide radicals, H₂O₂ and thiols were determined and proteins involved in the ER stress response were also detected by Western blot analysis.

Results

We found that diallyl tetrasulfide induced reactive oxygen species (ROS) in normal cells similar to cancer cells in a time (0 to 60 min) and dose dependent manner (0 to 50 μM). The level of heme oxigenase-1 (HO-1) was up-regulated in both cell types. Initially, we found a decrease in the total thiol level in both cell types but in contrast to cancer cells, normal cells recovered from the decrease in the total thiol concentration within 60 min of treatment.

Conclusions

The recovery of the thiol concentration in normal cells treated with diallyl tetrasulfide seems to be responsible for the failure to induce the ER stress signalling pathway and finally apoptosis in normal cells.

General Significance

The difference in the recovery of the thiol status might be an explanation for the anti-carcinogenic effects of garlic compounds.     

DNA damage dependent activation of checkpoint kinase-1 and mitogen-activated protein kinase-p38 are required in malabaricone C-induced mitochondrial cell death

Background

Given that lung cancer is the second leading cause of cancer-related deaths with low survival rates, the project was aimed to formulate an efficient drug with minimum side effects, and rationalize its action mechanistically.

Methods

Mitochondria deficient cells, shRNA-mediated BCL2 and ATM depleted cells and pharmacological inhibition of DNA-damage response proteins were employed to explore the signaling mechanism governed between nucleus and mitochondria in response to mal C.

Results

Mal C decreased cell viability in three lung carcinoma cells, associated with DNA damage, p38-MAPK activation, imbalance in BAX/BCL2 expression, mitochondrial dysfunction and cytochrome-c release. Mitochondria depletion and p38-MAPK inhibition made A549 cells extremely resistant, but BCL2 knock-down partially sensitized the cells to mal C treatment. The mal C-induced apoptosis in A549 cells was initiated by DNA single strand breaks that led to double strand breaks (DSBs). DSB generation paralleled the induction of ATM- and ATR-mediated CHK1 phosphorylation. ATM silencing and ATR inhibition partially attenuated the mal C-induced p38-MAPK activation, CHK1 phosphorylation and apoptosis, which were completely suppressed by CHK1 inhibition.

Conclusions

Mal C activates the ATM-CHK1-p38 MAPK cascade to cause mitochondrial cell death in lung carcinoma cells.

General significance

Given that mal C has appreciable natural abundance and is non-toxic to mice, further in vivo evaluation would help in establishing its anti-cancer property.     

DNA double‐strand break response in stem cells: Mechanisms to maintain genomic integrity

Background

Embryonic stem cells (ESCs) represent the point of origin of all cells in a given organism and must protect their genomes from both endogenous and exogenous genotoxic stress. DNA double-strand breaks (DSBs) are one of the most lethal forms of damage, and failure to adequately repair DSBs would not only compromise the ability of SCs to self-renew and differentiate, but will also lead to genomic instability and disease.

Scope of Review

Herein, we describe the mechanisms by which ESCs respond to DSB-inducing agents such as reactive oxygen species (ROS) and ionizing radiation, compared to somatic cells. We will also discuss whether the DSB response is fully reprogrammed in induced pluripotent stem cells (iPSCs) and the role of the DNA damage response (DDR) in the reprogramming of these cells.

Major Conclusions

ESCs have distinct mechanisms to protect themselves against DSBs and oxidative stress compared to somatic cells. The response to damage and stress is crucial for the maintenance of self-renewal and differentiation capacity in SCs. iPSCs appear to reprogram some of the responses to genotoxic stress. However, it remains to be determined if iPSCs also retain some DDR characteristics of the somatic cells of origin.

General Significance

The mechanisms regulating the genomic integrity in ESCs and iPSCs are critical for its safe use in regenerative medicine and may shed light on the pathways and factors that maintain genomic stability, preventing diseases such as cancer. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Crystal structures of CbpF complexed with atropine and ipratropium reveal clues for the design of novel antimicrobials against Streptococcus pneumoniae

Background

Streptococcus pneumoniae is a major pathogen responsible of important diseases worldwide such as pneumonia and meningitis. An increasing resistance level hampers the use of currently available antibiotics to treat pneumococcal diseases. Consequently, it is desirable to find new targets for the development of novel antimicrobial drugs to treat pneumococcal infections. Surface choline-binding proteins (CBPs) are essential in bacterial physiology and infectivity. In this sense, esters of bicyclic amines (EBAs) such as atropine and ipratropium have been previously described to act as choline analogs and effectively compete with teichoic acids on binding to CBPs, consequently preventing in vitro pneumococcal growth, altering cell morphology and reducing cell viability.

Methods

With the aim of gaining a deeper insight into the structural determinants of the strong interaction between CBPs and EBAs, the three-dimensional structures of choline-binding protein F (CbpF), one of the most abundant proteins in the pneumococcal cell wall, complexed with atropine and ipratropium, have been obtained.

Results

The choline analogs bound both to the carboxy-terminal module, involved in cell wall binding, and, unexpectedly, also to the amino-terminal module, that possesses a regulatory role in pneumococcal autolysis.

Conclusions

Analysis of the complexes confirmed the importance of the tropic acid moiety of the EBAs on the strength of the binding, through π–π interactions with aromatic residues in the binding site.

General significance

These results represent the first example describing the molecular basis of the inhibition of CBPs by EBA molecules and pave the way for the development of new generations of antipneumococcal drugs.     

Supernumerary proteins of mitochondrial ribosomes

Background

Messenger RNAs encoded by mitochondrial genomes are translated on mitochondrial ribosomes that have unique structure and protein composition compared to prokaryotic and cytoplasmic ribosomes. Mitochondrial ribosomes are a patchwork of core proteins that share homology with prokaryotic ribosomal proteins and new, supernumerary proteins that can be unique to different organisms. In mammals, there are specific supernumerary ribosomal proteins that are not present in other eukaryotes.

Scope of review

Here we discuss the roles of supernumerary proteins in the regulation of mitochondrial gene expression and compare them among different eukaryotic systems. Furthermore, we consider if differences in the structure and organization of mitochondrial genomes may have contributed to the acquisition of mitochondrial ribosomal proteins with new functions.

Major conclusions

The distinct and diverse compositions of mitochondrial ribosomes illustrate the high evolutionary divergence found between mitochondrial genetic systems.

General significance

Elucidating the role of the organism-specific supernumerary proteins may provide a window into the regulation of mitochondrial gene expression through evolution in response to distinct evolutionary paths taken by mitochondria in different organisms. This article is part of a Special Issue entitled Frontiers of Mitochondrial Research.     

Lessons from computer simulations of Ras proteins in solution and in membrane

Background

A great deal has been learned over the last several decades about the function of Ras proteins in solution and membrane environments. While much of this knowledge has been derived from a plethora of experimental techniques, computer simulations have also played a substantial role.

Scope of review

Our goal here is to summarize the contribution of molecular simulations to our current understanding of normal and aberrant Ras function. We focus on lessons from molecular dynamics simulations in aqueous and membrane environments.

Major conclusions

The central message is that a close interaction between theory and simulation on the one hand and cell-biological, spectroscopic and other experimental approaches on the other has played, and will likely continue to play, a vital role in Ras research.

General significance

Atomistic insights emerging from detailed simulations of Ras in solution and in bilayers may be the key to unlock the secret that to date prevented development of selective anti-Ras inhibitors for cancer therapy.     

In vivo formation of Plasmodium falciparum ribosomal stalk — A unique mode of assembly without stable heterodimeric intermediates

Background

The ribosomal stalk composed of P-proteins constitutes a structure on the large ribosomal particle responsible for recruitment of translation factors and stimulation of factor-dependent GTP hydrolysis during translation. The main components of the stalk are P-proteins, which form a pentamer. Despite the conserved basic function of the stalk, the P-proteins do not form a uniform entity, displaying heterogeneity in the primary structure across the eukaryotic lineage. The P-proteins from protozoan parasites are among the most evolutionarily divergent stalk proteins.

Methods

We have assembled P-stalk complex of Plasmodium falciparum in vivo in bacterial system using tricistronic expression cassette and provided its characteristics by biochemical and biophysical methods.

Results

All three individual P-proteins, namely uL10/P0, P1 and P2, are indispensable for acquisition of a stable structure of the P stalk complex and the pentameric uL10/P0-(P1-P2)₂ form represents the most favorable architecture for parasite P-proteins.

Conclusion

The formation of P. falciparum P-stalk is driven by trilateral interaction between individual elements which represents unique mode of assembling, without stable P1–P2 heterodimeric intermediate.

General significance

On the basis of our mass-spectrometry analysis supported by the bacterial two-hybrid assay and biophysical analyses, a unique pathway of the parasite stalk assembling has been proposed. We suggest that the absence of P1/P2 heterodimer, and the formation of a stable pentamer in the presence of all three proteins, indicate a one-step formation to be the main pathway for the vital ribosomal stalk assembly, whereas the P2 homo-oligomer may represent an off-pathway product with physiologically important nonribosomal role.