Monday, 16 April 2012 11:51
In all organisms, tRNAs undergo numerous post-transcriptional modifications. In general, modifications that occur away from the anticodon loop play more of a structural role, and their absence could lead to destabilization of the tRNA. Modifications that affect the anticodon nucleotides usually have a direct bearing on decoding.
A special type of modification known as RNA editing can replace one nucleotide for a nother, directly changing the amino acid decoding capacity of the tRNA and effectively reassigning codons without the need for changes at the DNA level. In P. falciparum, the complete set of tRNAs used in both cytoplasmic, mitochondrial and apicoplast protein synthesis is encoded solely by the nuclear genome: tRNAs are transcribed in the nucleus, exported to the cytoplasm, and later a subset of cytoplasmic tRNAs is actively imported into the mitochondrion or the apicoplast. However, translation of tryptophan codons represents a potential problem because, as in many other eukaryotes, in the mitochondrial genome, the canonical UGG tryptophan codon is often replaced by UGA, which is a stop codon in cytoplasmic translation. This led to the question of how organisms with a single tRNATrp with the anticodon CCA could decode UGG as tryptophan and UGA as a stop codon in the cytoplasm and UGA as tryptophan upon import into the mitochondrion. Beyond C to U editing, this tRNA was found to be thiolated at position33 (to form 2-thiouridine,s2U33) of the anticodon loop, a position that was presumably never modified in tRNAs of any organism. Even more unusual was the fact that this modification only occurred in the edited tRNA, prompting a model by which thiolation at U33 was required for editing. The new map shows the different pathways that lead to the nucleotide exchange. http://sites.huji.ac.il/malaria/maps/tRNAthiol.html
S-adenosylmethionine (AdoMet) is a methyl donor used by a wide variety of methyltransferases, and it is also used as the source of an ?-amino-?-carboxypropyl (‘‘acp’’) group by several enzymes. tRNA-yW synthesizing enzyme-2 (TYW2) is involved in the biogenesis of a hypermodified nucleotide, wybutosine (yW), and it catalyzes the transfer of the ‘‘acp’’ group from AdoMet to the C7 position of the imG-14 base, a yW precursor. This modified nucleoside yW is exclusively located at position 37 of eukaryotic tRNAPhe, and it ensures the anticodon-codon pairing on the ribosomal decoding site. The chemical structure of yW is characterized by the tricyclic 1H-imidazo[1,2-?]purine core with a large side chain. Its bulky, hydrophobic structure is considered to stabilize not only the conformation of the anticodon loop itself but also the codon-anticodon pairing on the ribosome, thereby preventing a potential -1 frame shift at a phenylalanine codon. Since all involved enzymes were found ot have encoding genes in P. falciparum, a map depicting the biosynthesis of wybutosine has been contstructed. http://sites.huji.ac.il/malaria/maps/tRNA-wybut.html
Recent proteomic studies reveal that 5–10% of mammalian and bacterial proteins undergo lysine acetylation, a post-translational modification that adds an acetyl group to the e-amino group of lysine residues. Many of these proteins are not canonical targets, such as histones and transcription factors, suggesting that this modification plays a much wider role than previously appreciated. These studies also suggest that lysine acetylomes are at least comparable with (if not larger than) phosphoproteomes. Although many of the newly identified acetylation events still require validation, they constitute an important framework for further research and the development of new drugs useful in treating a variety of pathologies. In the new map the various modes of histone acetylations that could be reconstructed from genes found in the P. falciparum genome are shown. http://sites.huji.ac.il/malaria/maps/acet_recog_mod.html
Most eukaryotic mRNA precursors (premRNAs) must undergo extensive processing, including cleavage and polyadenylation at the 3’-end. Processing at the 3’-end is controlled by sequence elements in the pre-mRNA (cis elements) as well as protein factors. Despite the seeming biochemical simplicity of the processing reactions, more than 14 proteins have been identified for the mammalian complex, more than 20 proteins for the yeast complex and 18 for P. falciparum. The 3’-end processing machinery also has important roles in transcription and splicing. The n machinery contains several sub-complexes, including cleavage and polyadenylation specificity factor, cleavage stimulation factor, cleavage factor IA, cleavage factor IB, and cleavage factor II. Additional protein factors include poly(A) polymerase, poly(A)-binding protein, symplekin, the Cterminal domain of RNA polymerase II largest subunit, cleavage factor IA, cleavage factor IB, and cleavage and polyadenylation factor. http://sites.huji.ac.il/malaria/maps/mRNA_processing.html
Rab GTPases are tightly and rather specifically involved in intracellular traffic and in the export of proteins. Although they are already represented in several maps of MPMP, it was thought useful to collect them into a table which also characterizes their role in yeast. Not less important, the same table shows all Rab GTPases for which an encoding gene has not been found in the genome of P. falciparum http://sites.huji.ac.il/malaria/maps/rab_prots.html
Microtubule dynamics is controlled and amplified in vivo by complex sets of regulators. Among these regulatory proteins, molecular motors from the kinesin superfamily are taking an increasing importance. A map has been constructed to show how microtubule disassembly or assembly into interphase microtubules and the mitotic spindle may involve kinesins and how protein kinases may participate in these kinesin-dependent regulations. http://sites.huji.ac.il/malaria/maps/spindle_kinesin.html
After invasion of erythrocytes, the human malaria parasite Plasmodium falciparum resides within a parasitophorous vacuole (PV) which forms an interface between the host cell cytosol and the parasite surface. This vacuole protects the parasite from potentially harmful substances, but allows access of essential nutrients to the parasite. Furthermore, the vacuole acts as a transit compartment for parasite proteins en route to the host cell cytoplasm. Recently we developed a strategy to biotin label soluble proteins of the PV. Here, we have paired this strategy with a high-throughput MALDI-TOF-MS analysis to identify 27 vacuolar proteins. These proteins fall into the following main classes: chaperones, proteases, and metabolic enzymes, consistent with the expected functions of the vacuole. These proteins are likely to be involved in several processes including nutrient acquisition from the host cytosol, protein sorting within the vacuole, and release of parasites at the end of the intraerythrocytic cycle. http://sites.huji.ac.il/malaria/maps/protPV.html
The ubiquitin-independent protein quality control of matrix proteins of the mitochondrion is well characterized and until recently the mitochondrion was considered a ‘ubiquitination-free’ organelle. However, a number of studies now indicate multiple roles of the ubiquitin–proteasome pathway in the regulation and maintenance of mitochondrial integrity. Of particular interest is the finding of a mitochondrial ubiquitin-dependent protein quality control and that this pathway may share similarity to the endoplasmic reticulum-associated degradation (ERAD) pathway that acts to eliminate misfolded proteins from the lumen of the endoplasmic reticulum. http://sites.huji.ac.il/malaria/maps/chaperone.html
Kinesins are a superfamily of MT-based motor proteins that convert the chemical energy of ATP hydrolysis into mechanical energy to perform various transport needs. These motor proteins have been classified into 14 subfamilies and share a very similar motor domain, containing nucleotide- and MT-binding sites. Depending on the location of their motor domain, kinesins may walk towards either MT ends. In general, kinesins with an amino-terminal motor domain (N-kinesins) move towards MT plus ends, whereas kinesins containing a carboxy-terminal motor domain (C-kinesins) move in the opposite direction, and kinesins with a central motor domain (M-kinesins) often destabilize MTs rather than move along their surface. Although they bear very similar motor domains, kinesins exhibit an important variety of activities, and it has been found that several kinesin subfamilies are important regulators of MT dynamics, many of them playing pivotal roles in mitosis.
Ribosome biogenesis is a fundamental process that provides cells with the molecular factories for cellular protein production. The process of ribosome assembly comprises the processing and folding of the pre-rRNA and its concomitant assembly with the ribosomal proteins. Eukaryotic ribosome biogenesis relies on a large number (> 200) of non-ribosomal factors, which confer directionality and accuracy to this process. Many of these non-ribosomal factors fall into different families of energy-consuming enzymes, notably including ATP-dependent RNA helicases, AAA-ATPases, GTPases, and kinases. Ribosome biogenesis is highly conserved within eukaryotic organisms; however, due to the combination of powerful genetic and biochemical methods, it is best studied in the yeast Saccharomyces cerevisiae which served as a template to study genes involved in this process in Plasmodium falciparum.
Apicomplexan parasites possess specialized secretory organelles (rhoptries and micronemes) that release their contents during host cell invasion. Although the rhoptries were once thought to be merely a bulbous 'protein reservoir' connected to an anterior neck region, the localization of a protein specifically to the neck suggested that this region was more than just a duct. Recent studies have shown that the rhoptry neck sub-compartment possesses a distinct protein repertoire. Some of these proteins share common features, including conservation across the phylum and involvement in tight-junction formation. A sub-group of rhoptry neck proteins, the RONs, their association with the microneme protein apical membrane antigen AMA1, and their involvement in invasion shown. http://sites.huji.ac.il/malaria/maps/rhop_neck.html
Sirtuins comprise a family of enzymes found in all organisms, where they play a role in diverse processes including transcriptional silencing, aging, regulation of transcription, and metabolism. The predominant reaction catalyzed by these enzymes is NAD+-dependent lysine deacetylation, although some sirtuins exhibit a weaker ADP-ribosyltransferase activity. Although the Sir2 deacetylation mechanism is well established, much less is known about the Sir2 ADP-ribosylation reaction. In bacteria the ADP-ribosylation activity of a sirtuin preferentially ADP-ribosylates some acetuylated proteins. http://sites.huji.ac.il/malaria/maps/sir_deac_adp.html
Central to cell function are RNA-containing complexes involved in gene expression, such as the ribosome, the spliceosome, snoRNAs, RNase P, and telomerase, among others. A deep bioinformatic and experimental analysis of these complexes has allowed the alignment the P. falciparum RNA gene sequence to the other Plasmodium sequences to obtain comparative information about the primary sequence and secondary structure of the RNA. In some cases this allowed reasonable secondary structure prediction.
Damaging environment, certain intracellular defects or heterologous expression of pro-apoptotic genes induce death in yeast cells exhibiting typical markers of apoptosis. In mammals, apoptosis can be directed by the activation of groups of proteases, called caspases, that cleave specific substrates and trigger cell death. In addition, in plants, fungi, Dictyostelium and metazoa, paracaspases and metacaspases have been identified that share some homologies with caspases but showing different substrate specificity. Metacaspases are not biochemical, but sequence and functional homologes of caspases, as deletion of them rescues entirely different death scenarios. The orthologs of apoptosis-related genes were compiled from the genome of P. falciparum and presented in a functional scheme. http://sites.huji.ac.il/malaria/maps/apoptosis.html
Kinesins are a family of molecular motors that use the energy of ATP hydrolysis to move along the surface of, or destabilize, microtubule filaments. Much progress has been made in understanding the mechanics and functions of the kinesin motors that play important parts in cell division, cell motility, intracellular trafficking and ciliary function. How kinesins are regulated in cells to ensure the temporal and spatial fidelity of their microtubule-based activities is less well understood. Recent work has revealed molecular mechanisms that control kinesin autoinhibition and subsequent activation, binding to cargos and microtubule tracks, and localization at specific sites of action. http://sites.huji.ac.il/malaria/maps/dif_kines.html
Post-transcriptional processes have a central role in the regulation of eukaryotic gene expression. Although it has been known for a long time that these processes are functionally linked, often by the use of common protein factors, it has only recently become apparent that many of these processes are also physically connected. Indeed, proteins that are involved in mRNA degradation, translational repression, mRNA surveillance and RNA-mediated gene silencing, together with their mRNA targets, colocalize within discrete cytoplasmic domains known as P bodies. The available evidence indicates that P bodies are sites where mRNAs that are not being translated accumulate, the information carried by associated proteins and regulatory RNAs is integrated, and their fate — either translation, silencing or decay — is decided.
Although P-bodies were not described in malaria, their possible constituents have been compiled from the genome.
Proliferating Cell Nuclear Antigen (PCNA) ubiquitylation plays a crucial role in maintaining genomic stability during DNA replication. DNA damage stalling the DNA replication fork induces PCNA ubiquitylation that activates DNA damage bypass to prevent the collapse of DNA replication forks that could potentially produce double-strand breaks and chromosomal rearrangements. PCNA ubiquitylation dictates the mode of bypass depending on the level of ubiquitylation; monoubiquitylation and polyubiquitylation activate error-prone translesion synthesis and error-free template switching, respectively. Due to the error-prone nature of DNA damage bypass, PCNA ubiquitylation needs to be tightly regulated. Here, we review the molecular mechanisms to remove ubiquitin from PCNA including the emerging role of USP1 and ELG1 in this fascinating process. http://sites.huji.ac.il/malaria/maps/PCNA_UB.html
Replicative DNA polymerases are blocked at DNA lesions. Synthesis past DNA damage requires the replacement of the replicative polymerase by one of a group of specialised translesion synthesis (TLS) polymerases, most of which belong to the Y-family. Each of these has different substrate specificities for different types of damage. In eukaryotes monoubiquitination of PCNA plays a crucial role in the switch fromreplicative to TLS polymerases at stalled forks. All the Y-family polymerases have ubiquitin binding sites that increase their binding affinity for ubiquitinated PCNA at the sites of stalled forks. http://sites.huji.ac.il/malaria/maps/DNA_transles.html
The packaging of chromosomal DNA by nucleosomes condenses and organizes the genome, but occludes many regulatory DNA elements. However, this constraint also allows nucleosomes and other chromatin components to actively participate in the regulation of transcription, chromosome segregation, DNA replication, and DNA repair. To enable dynamic access to packaged DNA and to tailor nucleosome composition in chromosomal regions, cells have evolved a set of specialized chromatin remodeling complexes (remodelers). Remodelers use the energy of ATP hydrolysis to move, destabilize, eject, or restructure nucleosomes. The P. falciparum ortologs of elements involved in chromatin remodeling were compiled in a table. http://sites.huji.ac.il/malaria/maps/chrom_remod.html
Thioesters, amides, and esters are common chemical building blocks in a wide array of natural products. The formation of these bonds can be catalyzed in a variety of ways. For chemists, the use of an activating group is a common strategy and adenylate enzymes are exemplars of this approach. Adenylating enzymes activate the otherwise unreactive carboxylic acid by transforming the normal hydroxyl leaving group into adenosine monophosphate. The stable post-translational modification of proteins by adenylylation is a mechanism to regulate the activity of enzymes. Although many other processes involving the covalent transfer of an AMP residue to an amino acid side chain have been identified since then, these are transient adenylylation events that essentially use the free energy of ATP hydrolysis to activate specific processes. The processes of adenylylation and ADP-ribosylation are shown in details. http://sites.huji.ac.il/malaria/maps/adenylyl.html
Adaptor protein (AP) complexes are cytosolic heterotetramers that mediate the sorting of membrane proteins in the secretory and endocytic pathways. AP complexes are involved in the formation of clathrin-coated vesicles by recruiting the scaffold protein, clathrin. AP complexes also play a pivotal role in the cargo selection by recognizing the sorting signals within the cytoplasmic tail of integral membrane proteins. Six distinct AP complexes have been identified. AP-2 mediates endocytosis from the plasma membrane, while AP-1, AP-3 and AP-4 play a role in the endosomal/lysosomal sorting pathways.