Functions and therapeutic potential of protein phosphatase 1: Insights from mouse genetics.

Protein phosphatase 1 (PP1) catalyzes more than half of all phosphoserine/threonine dephosphorylation reactions in mammalian cells. In vivo PP1 does not exist as a free catalytic subunit but is always associated with at least one regulatory PP1-interacting protein (PIP) to generate a large set of distinct holoenzymes. Each PP1 complex controls the dephosphorylation of only a small subset of PP1 substrates. We screened the literature for genetically engineered mouse models and identified models for all PP1 isoforms and 104 PIPs. PP1 itself and at least 49 PIPs were connected to human disease-associated phenotypes. Additionally, phenotypes related to 17 PIPs were clearly linked to altered PP1 function, while such information was lacking for 32 other PIPs. We propose structural reverse genetics, which combines structural characterization of proteins with mouse genetics, to identify new PP1-related therapeutic targets. The available mouse models confirm the pleiotropic action of PP1 in health and diseases.

Evolutionary insights into Trm112-methyltransferase holoenzymes involved in translation between archaea and eukaryotes.

Protein synthesis is a complex and highly coordinated process requiring many different protein factors as well as various types of nucleic acids. All translation machinery components require multiple maturation events to be functional. These include post-transcriptional and post-translational modification steps and methylations are the most frequent among these events. In eukaryotes, Trm112, a small protein (COG2835) conserved in all three domains of life, interacts and activates four methyltransferases (Bud23, Trm9, Trm11 and Mtq2) that target different components of the translation machinery (rRNA, tRNAs, release factors). To clarify the function of Trm112 in archaea, we have characterized functionally and structurally its interaction network using Haloferax volcanii as model system. This led us to unravel that methyltransferases are also privileged Trm112 partners in archaea and that this Trm112 network is much more complex than anticipated from eukaryotic studies. Interestingly, among the identified enzymes, some are functionally orthologous to eukaryotic Trm112 partners, emphasizing again the similarity between eukaryotic and archaeal translation machineries. Other partners display some similarities with bacterial methyltransferases, suggesting that Trm112 is a general partner for methyltransferases in all living organisms.

AMPK: a contextual oncogene or tumor suppressor?

The AMP-activated protein kinase (AMPK) functions to monitor and maintain energy homeostasis at the cellular and organism level. AMPK was perceived historically primarily as a component of the LKB1/STK11 tumor suppressor (LKB1 mutations cause the Peutz-Jegher cancer predisposition syndrome) cascade upstream of the TSC1/2/mTOR pathway and thus likely to be a tumor suppressor. However, AMPK has recently been shown to promote cancer cell survival in the face of extrinsic and intrinsic stressors including bioenergetic, growth factor, and oncogene stress compatible with studies showing that AMPK is required for oncogenic transformation. Thus, whether AMPK acts as a bona fide tumor suppressor or a contextual oncogene and, of particular importance, whether AMPK should be targeted for activation or inhibition during cancer therapy, is controversial and requires clarification. We aim to initiate discussions of these critical questions by reviewing the role of AMPK with an emphasis on cancer cell adaptation to microenvironment stress and therapeutic intervention.

PP2A holoenzymes negatively and positively regulate cell cycle progression by dephosphorylating pocket proteins and multiple CDK substrates.

Cell cycle progression is negatively regulated by the retinoblastoma family of pocket proteins and CDK inhibitors (CKIs). In contrast, CDKs promote progression through multiple phases of the cell cycle. One prominent way by which CDKs promote cell cycle progression is by inactivation of pocket proteins via hyperphosphorylation. Reactivation of pocket proteins to halt cell cycle progression requires dephosphorylation of multiple CDK-phosphorylated sites and is accomplished by PP2A and PP1 serine/threonine protein phosphatases. The same phosphatases are also implicated in dephosphorylation of multiple CDK substrates as cells exit mitosis and reenter the G1 phase of the cell cycle. This review is primarily focused on the role of PP2A and PP1 in the activation of pocket proteins during the cell cycle and in response to signaling cues that trigger cell cycle exit. Other functions of PP2A during the cell cycle will be discussed in brief, as comprehensive reviews on this topic have been published recently (De Wulf et al., 2009; Wurzenberger and Gerlich, 2011).

Targeting bacterial RNA polymerase: promises for future antisense antibiotics development.

The progress of transcription is synthesized by complex molecules, among which DNA-dependent RNA polymerase (RNAP) is the central enzyme. The prokaryotic RNAP is a large protein composed of core subunits (α2, ß and ß') and a σ factor that is required for specific recognition of the promoter site and the initiation of transcription. Despite its ubiquity, structural and functional similarities, bacterial RNAPs do not share extensive sequence homology with eukaryotic RNAPs. Bacterial RNAP an attractive target for the development of anti-bacterial drugs as its inactivation would lead to bacterial cell death. This review will present the state of knowledge on the assembly and function of RNAP subunits in bacteria with special focus on insights provided by structural analysis of a key component σ factor. Thorough retrospection has been provided for better understanding of progress and problems in targeting RNAP by traditional chemical compounds. Recent progress using innovative strategies including structural biology and phage based screening, especially the antisense technology, has shed light on developing the first set of macro-molecule RNAP inhibitors. In particular, exploration on targeting RNAP σ70 for realization of broad spectrum antisense bactericidal effect in gram negative bacteria presents the first successful example of PNA-peptide conjugate showing attractive potential as conventional broad-spectrum antibiotics, in which possible way the antisense antibiotics might develop into to meet the range and type of usage in future health care.

The Aurora-A/TPX2 complex: a novel oncogenic holoenzyme?

The Aurora-A kinase regulates cell division by phosphorylating multiple downstream targets in the mitotic apparatus. Aurora-A is frequently overexpressed in tumor cells and it is therefore regarded as a novel candidate target in anti-cancer therapy. Its actual contribution to cell transformation, however, is not entirely clarified; furthermore, its transforming ability has been found to vary broadly depending on the systems and experimental conditions in which it was assayed. This variability suggests that Aurora-A overexpression requires the concomitant deregulation of partner factor(s) to fully elicit its oncogenic potential. Molecular and structural studies indicate that the full activation and correct mitotic localisation of Aurora-A require its interaction with the spindle regulator TPX2. In this review we propose a brief reappraisal of Aurora-A intrinsic oncogenic features. We then present literature screening data indicating that TPX2 is also overexpressed in many tumor types, and, furthermore, that Aurora-A and TPX2 are frequently co-overexpressed. We therefore propose that the association of Aurora-A and TPX2 gives rise to a novel functional unit with oncogenic properties. We also suggest that some of the roles that are conventionally attributed to Aurora-A in cell transformation and tumorigenesis could in fact be a consequence of the oncogenic activation of this unit.

The 5' arm of Kluyveromyces lactis telomerase RNA is critical for telomerase function.

Telomerase is a ribonucleoprotein reverse transcriptase that copies a short template within its integral telomerase RNA moiety (TER) onto eukaryotic chromosome ends, thus compensating for incomplete replication and degradation. The highly divergent yeast TER is structured in three long arms, with a catalytic core at its center. A binding site for the protein Ku80 is conserved within the 5' arm of TER in Saccharomyces but not in Kluyveromyces budding yeast species. Consistently, KU80 deletion in Kluyveromyces lactis does not affect telomere length, while it causes telomere shortening in Saccharomyces cerevisiae. We found elements in the 5' arm of K. lactis TER that are crucial for telomerase activity and stability. However, we found no indication of the association of Ku80 with this arm. Although the overexpression of Ku80 rescues a particular mutation in K. lactis TER1 that phenocopies a telomerase null mutation, this effect is indirect, caused by the repression of the recombination pathway competing for telomere maintenance. Interestingly, the overexpression of Est3, an essential telomerase protein whose function is still unknown, suppresses the phenotypes of mutations in this arm. These results indicate that the 5' arm of K. lactis TER has critical roles in telomerase function, which may be linked to the function of Est3.

Structure of 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) holoenzyme from an orthorhombic crystal form: an insight into the bifunctionality of the enzyme.

Mouse 3(17)alpha-hydroxysteroid dehydrogenase (AKR1C21) is a bifunctional enzyme that catalyses the oxidoreduction of the 3- and 17-hydroxy/keto groups of steroid substrates such as oestrogens, androgens and neurosteroids. The structure of the AKR1C21-NADPH binary complex was determined from an orthorhombic crystal belonging to space group P2(1)2(1)2(1) at a resolution of 1.8 A. In order to identify the factors responsible for the bifunctionality of AKR1C21, three steroid substrates including a 17-keto steroid, a 3-keto steroid and a 3alpha-hydroxysteroid were docked into the substrate-binding cavity. Models of the enzyme-coenzyme-substrate complexes suggest that Lys31, Gly225 and Gly226 are important for ligand recognition and orientation in the active site.

The role of an upstream promoter interaction in initiation of bacterial transcription.

The bacterial RNA polymerase (RNAP) recognizes promoters through sequence-specific contacts of its promoter-specificity components (sigma) with two DNA sequence motifs. Contacts with the upstream ('-35') promoter motif are made by sigma domain 4 attached to the flap domain of the RNAP beta subunit. Bacteriophage T4 late promoters consist solely of an extended downstream ('-10') motif specifically recognized by the T4 gene 55 protein (gp55). Low level basal transcription is sustained by gp55-RNAP holoenzyme. The late transcription coactivator gp33 binds to the beta flap and represses this basal transcription. Gp33 can also repress transcription by Escherichia coli sigma70-RNAP holoenzyme mutated to allow gp33 access to the beta flap. We propose that repression is due to gp33 blocking an upstream sequence-independent DNA-binding site on RNAP (as sigma70 domain 4 does) but, unlike sigma70 domain 4, providing no new DNA interaction. We show that this upstream interaction is essential only at an early step of transcription initiation, and discuss the role of this interaction in promoter recognition and transcriptional regulation.

Mutator mutants of Escherichia coli carrying a defect in the DNA polymerase III tau subunit.

To investigate the possible role of accessory subunits of Escherichia coli DNA polymerase III holoenzyme (HE) in determining chromosomal replication fidelity, we have investigated the role of the dnaX gene. This gene encodes both the tau and gamma subunits of HE, which play a central role in the organization and functioning of HE at the replication fork. We find that a classical, temperature-sensitive dnaX allele, dnaX36, displays a pronounced mutator effect, characterized by an unusual specificity: preferential enhancement of transversions and -1 frameshifts. The latter occur predominantly at non-run sequences. The dnaX36 defect does not affect the gamma subunit, but produces a tau subunit carrying a missense substitution (E601K) in its C-terminal domain (domain V) that is involved in interaction with the Pol III alpha subunit. A search for new mutators in the dnaX region of the chromosome yielded six additional dnaX mutators, all carrying a specific tau subunit defect. The new mutators displayed phenotypes similar to dnaX36: strong enhancement of transversions and frameshifts and only weak enhancement for transitions. The combined findings suggest that the tau subunit of HE plays an important role in determining the fidelity of the chromosomal replication, specifically in the avoidance of transversions and frameshift mutations.

CaMKII tethers to L-type Ca2+ channels, establishing a local and dedicated integrator of Ca2+ signals for facilitation.

Ca2+-dependent facilitation (CDF) of voltage-gated calcium current is a powerful mechanism for up-regulation of Ca2+ influx during repeated membrane depolarization. CDF of L-type Ca2+ channels (Ca(v)1.2) contributes to the positive force-frequency effect in the heart and is believed to involve the activation of Ca2+/calmodulin-dependent kinase II (CaMKII). How CaMKII is activated and what its substrates are have not yet been determined. We show that the pore-forming subunit alpha(1C) (Ca(v)alpha1.2) is a CaMKII substrate and that CaMKII interaction with the COOH terminus of alpha1C is essential for CDF of L-type channels. Ca2+ influx triggers distinct features of CaMKII targeting and activity. After Ca2+-induced targeting to alpha1C, CaMKII becomes tightly tethered to the channel, even after calcium returns to normal levels. In contrast, activity of the tethered CaMKII remains fully Ca2+/CaM dependent, explaining its ability to operate as a calcium spike frequency detector. These findings clarify the molecular basis of CDF and demonstrate a novel enzymatic mechanism by which ion channel gating can be modulated by activity.

Characterization of conserved sequence elements in eukaryotic RNase P RNA reveals roles in holoenzyme assembly and tRNA processing.

RNase P is a ubiquitous endoribonuclease responsible for cleavage of the 5' leader of precursor tRNAs (pre-tRNAs). Although the protein composition of RNase P holoenzymes varies significantly among Bacteria, Archaea, and Eukarya, the holoenzymes have essential RNA subunits with several sequences and structural features that are common to all three kingdoms of life. Additional structural elements of the RNA subunits have been found that are conserved in eukaryotes, but not in bacteria, and might have functions specifically required by the more complex eukaryotic holoenzymes. In this study, we have mutated four eukaryotic-specific conserved regions in Saccharomyces cerevisiae nuclear RNase P RNA and characterized the effects of the mutations on cell growth, enzyme function, and biogenesis of RNase P. RNase P with mutations in each of the four regions tested is sufficiently functional to support life although growth of the resulting yeast strains was compromised to varying extents. Further analysis revealed that mutations in three different regions cause differential defects in holoenzyme assembly, localization, and pre-tRNA processing in vivo and in vitro. These data suggest that most, but not all, eukaryotic-specific conserved regions of RNase P RNA are important for the maturation and function of the holoenzyme.

Interaction between the T4 helicase loading protein (gp59) and the DNA polymerase (gp43): unlocking of the gp59-gp43-DNA complex to initiate assembly of a fully functional replisome.

Single-molecule fluorescence resonance energy transfer and functional assays have been used to study the initiation and regulation of the bacteriophage T4 DNA replication system. Previous work has demonstrated that a complex of the helicase loading protein (gp59) and the DNA polymerase (gp43) on forked DNA totally inhibits the polymerase and exonuclease activities of gp43 by a molecular locking mechanism (Xi, J., Zhuang, Z., Zhang, Z., Selzer, T., Spiering, M. M., Hammes, G. G., and Benkovic, S. J. (2005) Biochemistry 44, 2305-2318). We now show that this complex is "unlocked" by the addition of the helicase (gp41) with restoration of the DNA polymerase activity. Gp59 retains its ability to load the helicase while forming a gp59-gp43 complex at a DNA fork in the presence of the single-stranded DNA binding protein (gp32). Upon the addition of gp41 and MgATP, gp59 dissociates from the complex, and the DNA-bound gp41 is capable of recruiting the primase (gp61) to form a functional primosome and, subsequently, a fully active replisome. Functional assays of leading- and lagging-strand synthesis on an active replication fork show that the absence of gp59 has no effect on the coupling of leading- and lagging-strand synthesis or on the size of the Okazaki DNA fragments. We conclude that gp59 acts in a manner similar to the clamp loader to ensure proper assembly of the replisome and does not remain as a replisome component during active replication.

A telomerase holoenzyme protein enhances telomerase RNA assembly with telomerase reverse transcriptase.

Telomerase maintains the simple sequence repeats at chromosome ends, protecting cells from genomic rearrangement, proliferative senescence and death. The telomerase reverse transcriptase (TERT) and telomerase RNA (TER) alone can assemble into active enzyme in a heterologous cell extract, but the physiological process of telomerase biogenesis is more complex. The endogenous accumulation of Tetrahymena thermophila TERT and TER requires an additional telomerase holoenzyme protein, p65. Here, we reconstitute this cellular pathway for telomerase ribonucleoprotein biogenesis in vitro. We demonstrate that tandem RNA interaction domains in p65 recognize the sequence of the TER 3' stem. Notably, the p65-TER complex recruits TERT much more efficiently than does TER alone. Using bacterially expressed p65 and TERT polypeptides, we show that p65 enhances TERT-TER interaction by a mechanism involving a conserved bulge in the protein-bridging TER molecule. These findings reveal a pathway for telomerase holoenzyme biogenesis that preassembles TER for TERT recruitment.

Differential effects of substrate on type I and type II PKA holoenzyme dissociation.

It has been widely accepted that cAMP activates the protein kinase A (PKA) holoenzyme by dissociating the regulatory and catalytic subunits, thus freeing the catalytic subunit to phosphorylate its targets. However, recent experiments suggest that cAMP does not fully dissociate the holoenzyme. Here, we investigate this mechanism further by using small-angle X-ray scattering to study, at physiological enzyme concentrations, the type Ialpha and type IIbeta holoenzyme structures under equilibrium solution conditions without any labeling of the protein subunits. We observe that while the addition of a molar excess of cAMP to the type Ialpha PKA holoenzyme causes partial dissociation, it is only upon addition of a PKA peptide substrate together with cAMP that full dissociation occurs. Similarly, addition of excess cAMP to the type IIbeta holoenzyme causes only a partial dissociation. However, while the addition of peptide substrate as well as excess cAMP causes somewhat more dissociation, a significant percentage of intact type IIbeta holoenzyme remains. These results confirm that both the type Ialpha and the type IIbeta holoenzymes are more stable in the presence of cAMP than previously thought. They also demonstrate that substrate plays a differential role in the activation of type I versus type II holoenzymes, which could explain some important functional differences between PKA isoforms. On the basis of these data and other recently published data, we propose a structural model of type I holoenzyme activation by cAMP.

Holoenzyme proteins required for the physiological assembly and activity of telomerase.

Many proteins have been implicated in the physiological function of telomerase, but specific roles of telomerase-associated proteins other than telomerase reverse transcriptase (TERT) remain ambiguous. To gain a more comprehensive understanding of catalytically active enzyme composition, we performed affinity purification of epitope-tagged, endogenously assembled Tetrahymena telomerase. We identified and cloned genes encoding four telomerase proteins in addition to TERT. We demonstrate that both of the two new proteins characterized in detail, p65 and p45, have essential roles in the maintenance of telomere length as part of a ciliate telomerase holoenzyme. The p65 subunit contains an La motif characteristic of a family of direct RNA-binding proteins. We find that p65 in cell extract is associated specifically with telomerase RNA, and that genetic depletion of p65 reduces telomerase RNA accumulation in vivo. These findings demonstrate that telomerase holoenzyme proteins other than TERT play critical roles in RNP biogenesis and function.

The theta subunit of Escherichia coli DNA polymerase III: a role in stabilizing the epsilon proofreading subunit.

The function of the theta subunit of Escherichia coli DNA polymerase III holoenzyme is not well established. theta is a tightly bound component of the DNA polymerase III core, which contains the alpha subunit (polymerase), the epsilon subunit (3'-->5' exonuclease), and the theta subunit, in the linear order alpha-epsilon-theta. Previous studies have shown that the theta subunit is not essential, as strains carrying a deletion of the holE gene (which encodes theta) proved fully viable. No significant phenotypic effects of the holE deletion could be detected, as the strain displayed normal cell health, morphology, and mutation rates. On the other hand, in vitro experiments have indicated the efficiency of the 3'-exonuclease activity of epsilon to be modestly enhanced by the presence of theta. Here, we report a series of genetic experiments that suggest that theta has a stabilizing role for the epsilon proofreading subunit. The observations include (i) defined DeltaholE mutator effects in mismatch-repair-defective mutL backgrounds, (ii) strong DeltaholE mutator effects in certain proofreading-impaired dnaQ strains, and (iii) yeast two- and three-hybrid experiments demonstrating enhancement of alpha-epsilon interactions by the presence of theta. theta appears conserved among gram-negative organisms which have an exonuclease subunit that exists as a separate protein (i.e., not part of the polymerase polypeptide), and the presence of theta might be uniquely beneficial in those instances where the proofreading 3'-exonuclease is not part of the polymerase polypeptide.

RNA polymerase holoenzyme: structure, function and biological implications.

The past three years have marked the breakthrough in our understanding of the structural and functional organization of RNA polymerase. The latest major advance was the high-resolution structures of bacterial RNA polymerase holoenzyme and the holoenzyme in complex with promoter DNA. Together with an array of genetic, biochemical and biophysical data accumulated to date, the structures provide a comprehensive view of dynamic interactions between the major components of transcription machinery during the early stages of the transcription cycle. They include the binding of sigma factor to the core enzyme, and the recognition of promoter sequences and DNA melting by holoenzyme, transcription initiation and promoter clearance.

Loss of holocytochrome c-type synthetase causes the male lethality of X-linked dominant microphthalmia with linear skin defects (MLS) syndrome.

Girls with MLS syndrome have microphthalmia with linear skin defects of face and neck, sclerocornea, corpus callosum agenesis and other brain anomalies. This X-linked dominant, male-lethal condition is associated with heterozygous deletions of a critical region in Xp22.31, from the 5' untranslated region of MID1 at the telomeric boundary to the ARHGAP6 gene at the centromeric boundary. HCCS, encoding human holocytochrome c-type synthetase, is the only gene located entirely inside the critical region. Because single gene analysis is not feasible in MLS patients (all have deletions), we generated a deletion of the equivalent region in the mouse to study the molecular basis of this syndrome. This deletion inactivates mouse Hccs, whose homologs in lower organisms (cytochrome c or c1 heme lyases) are essential for function of cytochrome c or c1 in the mitochondrial respiratory chain. Ubiquitous deletions generated in vivo lead to lethality of hemizygous, homozygous and heterozygous embryos early in development. This lethality is rescued by expression of the human HCCS gene from a transgenic BAC, resulting in viable homozygous, heterozygous and hemizygous deleted mice with no apparent phenotype. In the presence of the HCCS transgene, the deletion is easily transmitted to subsequent generations. We did obtain a single heterozygous deleted female that does not express human HCCS, which is analogous to the low prevalence of the heterozygous MLS deletion in humans. Through the study of these genetically engineered mice we demonstrate that loss of HCCS causes the male lethality of MLS syndrome.