C-terminal Oligomerization Domain Research Articles

CryoEM analysis of the essential native UDP-glucose pyrophosphorylase from Aspergillus nidulans reveals key conformations for activity regulation and function.

Invasive aspergillosis is one of the most serious clinical invasive fungal infections, resulting in a high case fatality rate among immunocompromised patients. The disease is caused by saprophytic molds in the genus Aspergillus, including Aspergillus fumigatus, the most significant pathogenic species. The fungal cell wall, an essential structure mainly composed of glucan, chitin, galactomannan, and galactosaminogalactan, represents an important target for the development of antifungal drugs. UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) is a central enzyme in the metabolism of carbohydrates that catalyzes the biosynthesis of UDP-glucose, a key precursor of fungal cell wall polysaccharides. Here, we demonstrate that the function of UGP is vital for Aspergillus nidulans (AnUGP). To understand the molecular basis of AnUGP function, we describe a cryoEM structure (global resolution of 3.5 Å for the locally refined subunit and 4 Å for the octameric complex) of a native AnUGP. The structure reveals an octameric architecture with each subunit comprising an N-terminal α-helical domain, a central catalytic glycosyltransferase A-like (GT-A-like) domain, and a C-terminal (CT) left-handed β-helix oligomerization domain. AnUGP displays unprecedented conformational variability between the CT oligomerization domain and the central GT-A-like catalytic domain. In combination with activity measurements and bioinformatics analysis, we unveil the molecular mechanism of substrate recognition and specificity for AnUGP. Altogether, our study not only contributes to understanding the molecular mechanism of catalysis/regulation of an important class of enzymes but also provides the genetic, biochemical, and structural groundwork for the future exploitation of UGP as a potential antifungal target. IMPORTANCE Fungi cause diverse diseases in humans, ranging from allergic syndromes to life-threatening invasive diseases, together affecting more than a billion people worldwide. Increasing drug resistance in Aspergillus species represents an emerging global health threat, making the design of antifungals with novel mechanisms of action a worldwide priority. The cryoEM structure of UDP (uridine diphosphate)-glucose pyrophosphorylase (UGP) from the filamentous fungus Aspergillus nidulans reveals an octameric architecture displaying unprecedented conformational variability between the C-terminal oligomerization domain and the central glycosyltransferase A-like catalytic domain in the individual protomers. While the active site and oligomerization interfaces are more highly conserved, these dynamic interfaces include motifs restricted to specific clades of filamentous fungi. Functional study of these motifs could lead to the definition of new targets for antifungals inhibiting UGP activity and, thus, the architecture of the cell wall of filamentous fungal pathogens.

Oligomerization of Mutant p53 R273H is not Required for Gain-of-Function Chromatin Associated Activities.

The TP53 gene is often mutated in cancer, with missense mutations found in the central DNA binding domain, and less often in the C-terminal oligomerization domain (OD). These types of mutations are found in patients with the rare inherited cancer predisposition disorder called Li-Fraumeni syndrome. We previously found that mutant p53 (mtp53) R273H associates with replicating DNA and promotes the chromatin association of replication-associated proteins mini-chromosome maintenance 2 (MCM2), and poly ADP-ribose polymerase 1(PARP1). Herein, we created dual mutants in order to test if the oligomerization state of mtp53 R273H played a role in chromatin binding oncogenic gain-of-function (GOF) activities. We used site-directed mutagenesis to introduce point mutations in the OD in wild-type p53 (wtp53), and mtp53 R273H expressing plasmids. The glutaraldehyde crosslinking assay revealed that both wtp53 and mtp53 R273H formed predominantly tetramers, while the single OD mutant A347D, and the dual mtp53 R273H-A347D, formed predominantly dimers. The R337C, L344P, mtp53 R273H-R337C, and mtp53 R273H-L344P proteins formed predominantly monomers. Wtp53 was able to activate the cyclin-dependent kinase gene p21/waf and the p53 feedback regulator MDM2. As expected, the transactivation activity was lost for all the single mutants, as well as the mtp53 R273H-dual mutants. Importantly, mtp53 R273H and the dual oligomerization mutants, R273H-A347D, R273H-R337C, and R273H-L344P were able to interact with chromatin. Additionally, the dual oligomerization mutants, R273H-A347D, R273H-R337C, and R273H-L344P, maintained strong interactions with MCM2 and PARP1. Our findings suggest that while mtp53 R273H can form tetramers, tetramer formation is not required for the GOF associated chromatin interactions.

Frame-shift mediated reduction of gain-of-function p53 R273H and deletion of the R273H C-terminus in breast cancer cells result in replication-stress sensitivity

We recently documented that gain-of-function (GOF) mutant p53 (mtp53) R273H in triple negative breast cancer (TNBC) cells interacts with replicating DNA and PARP1. The missense R273H GOF mtp53 has a mutated central DNA binding domain that renders it unable to bind specifically to DNA, but maintains the capacity to interact tightly with chromatin. Both the C-terminal domain (CTD) and oligomerization domain (OD) of GOF mtp53 proteins are intact and it is unclear whether these regions of mtp53 are responsible for chromatin-based DNA replication activities. We generated MDA-MB-468 cells with CRISPR-Cas9 edited versions of the CTD and OD regions of mtp53 R273H. These included a frame-shift mtp53 R273Hfs387, which depleted mtp53 protein expression; mtp53 R273HΔ381-388, which had a small deletion within the CTD; and mtp53 R273HΔ347-393, which had both the OD and CTD regions truncated. The mtp53 R273HΔ347-393 existed exclusively as monomers and disrupted the chromatin interaction of mtp53 R273H. The CRISPR variants proliferated more slowly than the parental cells and mt53 R273Hfs387 showed the most extreme phenotype. We uncovered that after thymidine-induced G1/S synchronization, but not hydroxyurea or aphidicholin, R273Hfs387 cells displayed impairment of S-phase progression while both R273HΔ347-393 and R273HΔ381-388 displayed only moderate impairment. Moreover, reduced chromatin interaction of MCM2 and PCNA in mtp53 depleted R273Hfs387 cells post thymidine-synchronization revealed delayed kinetics of replisome assembly underscoring the slow S-phase progression. Taken together our findings show that the CTD and OD domains of mtp53 R273H play critical roles in mutant p53 GOF that pertain to processes associated with DNA replication.

Progress on the GntR family transcription regulators in bacteria.

In bacteria, GntR family transcription regulators are the widespread family of transcription factors. Members of this family consist of two functional domains, a conserved N-terminal DNA-binding domain that contains a typical helix-turn-helix (HTH) motif and a C-terminal effector-binding or oligomerization domain. Usually, the amino acid sequences of N-terminal DNA-binding domains are highly conserved, but differ in the C-terminal effector-binding or oligomerization domains. In the past several decades, many GntR family transcription regulators have been characterized in a number of bacteria. These regulators control a variety of cellular processes such as cell motility, glucose metabolism, bacterial resistance, pathogenesis and virulence. In this review, we summarized the discovery, C-terminal domains, biological function and regulation mode of GntR family transcription regulators. This review will help researchers to obtain more knowledge about the functions and mechanisms of the GntR family transcriptional regulatory factors.

Abstract 3429: Targeting gain-of-function of mutant p53 C-terminal and oligomerization domains to disrupt activated DNA replication in breast cancer

Abstract We recently documented that gain-of-function (GOF) mutant p53 (mtp53) R273H in triple negative breast cancer (TNBC) cells interacts with replicating DNA and PARP1 (Xiao et. al., Cancer Research 2019). The missense R273H GOF mtp53, without a functional central DNA binding domain, has the capacity to interact tightly with chromatin but it is unclear what regions of mtp53 are responsible for the chromatin-based DNA replication activities. GOF mtp53 proteins possess an intact C-terminal domain (CTD) and oligomerization domain (OD). In wild-type p53 the CTD and OD domain regulate non-specific DNA binding, post-translational modification, and protein tetramerization. We are targeting the CTD and OD regions of mtp53 to test if their disruption limits oncogenic GOF activity. We generated CRISPR/Cas9 edited R273H CTD and OD in the TNBC cell line MDA-MB-468, and also exogenously expressed such dual mutant proteins in p53 null cancers, to evaluate the roles played by oligomerization and non-specific DNA binding in mtp53 interactions with replicating DNA and PARP1. We determined the oligomerization state of R273H mtp53 using the glutaraldehyde crosslinking assay and observed that GOF mtp53 R273H mtp53 in breast cancer cells exists as a tetramer. Experiments are in progress to evaluate cell cycle progression, replication initiation, and mtp53 chromatin interactions in cells expressing missense mtp53 proteins with co-associated mutated CTDs or ODs. Our preliminary data suggests that disrupting the mtp53 CTD inhibits its interactions with chromatin and progression through the S-phase. Unpublished data on the roles played by the mtp53 CTD and OD in DNA replication will be presented at the 2020 AACR meeting. Acknowledgements: This work was funded by grants from the Breast Cancer Research Foundation BCRF-18-011 and BCRF-19-011 to Jill Bargonetti. Devon Lundine and Gu Xiao also received project support from TUFCCC/HC Regional Comprehensive Cancer Health Disparity Partnership, Award Number U54 CA221704(5) from the National Cancer Institute. Special thanks to Ella Freulich for help with CRISPR/Cas9 work and James J. Manfredi for mutant p53 plasmids to exogenously express protein. Citation Format: Devon Lundine, George Annor, Viola Ellison, Gu Xiao, Carol Prives, Jill Bargonetti. Targeting gain-of-function of mutant p53 C-terminal and oligomerization domains to disrupt activated DNA replication in breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3429.

Phosphorylations of the Abutilon Mosaic Virus Movement Protein Affect Its Self-Interaction, Symptom Development, Viral DNA Accumulation, and Host Range.

The genome of bipartite geminiviruses in the genus Begomovirus comprises two circular DNAs: DNA-A and DNA-B. The DNA-B component encodes a nuclear shuttle protein (NSP) and a movement protein (MP), which cooperate for systemic spread of infectious nucleic acids within host plants and affect pathogenicity. MP mediates multiple functions during intra- and intercellular trafficking, such as binding of viral nucleoprotein complexes, targeting to and modification of plasmodesmata, and release of the cargo after cell-to-cell transfer. For Abutilon mosaic virus (AbMV), phosphorylation of MP expressed in bacteria, yeast, and Nicotiana benthamiana plants, respectively, has been demonstrated in previous studies. Three phosphorylation sites (T221, S223, and S250) were identified in its C-terminal oligomerization domain by mass spectrometry, suggesting a regulation of MP by posttranslational modification. To examine the influence of the three sites on the self-interaction in more detail, MP mutants were tested for their interaction in yeast by two-hybrid assays, or by Förster resonance energy transfer (FRET) techniques in planta. Expression constructs with point mutations leading to simultaneous (triple) exchange of T221, S223, and S250 to either uncharged alanine (MPAAA), or phosphorylation charge-mimicking aspartate residues (MPDDD) were compared. MPDDD interfered with MP-MP binding in contrast to MPAAA. The roles of the phosphorylation sites for the viral life cycle were studied further, using plant-infectious AbMV DNA-B variants with the same triple mutants each. When co-inoculated with wild-type DNA-A, both mutants infected N. benthamiana plants systemically, but were unable to do so for some other plant species of the families Solanaceae or Malvaceae. Systemically infected plants developed symptoms and viral DNA levels different from those of wild-type AbMV for most virus-plant combinations. The results indicate a regulation of diverse MP functions by posttranslational modifications and underscore their biological relevance for a complex host plant-geminivirus interaction.

Cotranslational protein assembly imposes evolutionary constraints on homomeric proteins.

Cotranslational protein folding can facilitate rapid formation of functional structures. However, it can also cause premature assembly of protein complexes, if two interacting nascent chains are in close proximity. By analyzing known protein structures, we show that homomeric protein contacts are enriched toward the C termini of polypeptide chains across diverse proteomes. We hypothesize that this is the result of evolutionary constraints for folding to occur before assembly. Using high-throughput imaging of protein homomers in Escherichia coli and engineered protein constructs with N- and C-terminal oligomerization domains, we show that, indeed, proteins with C-terminal homomeric interface residues consistently assemble more efficiently than those with N-terminal interface residues. Using in vivo, in vitro and in silico experiments, we identify features that govern successful assembly of homomers, which have implications for protein design and expression optimization.

Study of DNA binding and bending by Bacillus subtilis GabR, a PLP-dependent transcription factor

BackgroundGabR is a transcriptional regulator belonging to the MocR/GabR family, characterized by a N-terminal wHTH DNA-binding domain and a C-terminal effector binding and/or oligomerization domain, structurally homologous to aminotransferases (ATs). In the presence of γ-aminobutyrate (GABA) and pyridoxal 5′-phosphate (PLP), GabR activates the transcription of gabT and gabD genes involved in GABA metabolism. MethodsHere we report a biochemical and atomic force microscopy characterization of Bacillus subtilis GabR in complex with DNA. Complexes were assembled in vitro to study their stoichiometry, stability and conformation. ResultsThe fractional occupancy of the GabR cognate site suggests that GabR binds as a dimer with Kd of 10nM. Upon binding GabR bends the DNA by 80° as measured by anomalous electrophoretic mobility. With GABA we observed a decrease in affinity and conformational rearrangements compatible with a less compact nucleo-protein complex but no changes of the DNA bending angle. By employing promoter and GabR mutants we found that basic residues of the positively charged groove on the surface of the AT domain affect DNA affinity. ConclusionsThe present data extend current understanding of the GabR-DNA interaction and the effect of GABA and PLP. A model for the GabR-DNA complex, corroborated by a docking simulation, is proposed. General SignificanceCharacterization of the GabR DNA binding mode highlights the key role of DNA bending and interactions with bases outside the canonical direct repeats, and might be of general relevance for the action mechanism of MocR transcription factors.

Structural and dynamics studies of a truncated variant of CI repressor from bacteriophage TP901-1.

The CI repressor from the temperate bacteriophage TP901-1 consists of two folded domains, an N-terminal helix-turn-helix DNA-binding domain (NTD) and a C-terminal oligomerization domain (CTD), which we here suggest to be further divided into CTD1 and CTD2. Full-length CI is a hexameric protein, whereas a truncated version, CI∆58, forms dimers. We identify the dimerization region of CI∆58 as CTD1 and determine its secondary structure to be helical both within the context of CI∆58 and in isolation. To our knowledge this is the first time that a helical dimerization domain has been found in a phage repressor. We also precisely determine the length of the flexible linker connecting the NTD to the CTD. Using electrophoretic mobility shift assays and native mass spectrometry, we show that CI∆58 interacts with the OL operator site as one dimer bound to both half-sites, and with much higher affinity than the isolated NTD domain thus demonstrating cooperativity between the two DNA binding domains. Finally, using small angle X-ray scattering data and state-of-the-art ensemble selection techniques, we delineate the conformational space sampled by CI∆58 in solution, and we discuss the possible role that the dynamics play in CI-repressor function.

An Apoptosis-Inducing Peptidic Heptad That Efficiently Clusters Death Receptor 5.

Multivalent ligands of death receptors hold particular promise as tumor cell-specific therapeutic agents because they induce an apoptotic cascade in cancerous cells. Herein, we present a modular approach to generate death receptor 5 (DR5) binding constructs comprising multiple copies of DR5 targeting peptide (DR5TP) covalently bound to biomolecular scaffolds of peptidic nature. This strategy allows for efficient oligomerization of synthetic DR5TP-derived peptides in different spatial orientations using a set of enzyme-promoted conjugations or recombinant production. Heptameric constructs based on a short (60-75 residues) scaffold of a C-terminal oligomerization domain of human C4b binding protein showed remarkable proapoptotic activity (EC50=3 nm) when DR5TP was ligated to its carboxy terminus. Our data support the notion that inter-ligand distance, relative spatial orientation and copy number of receptor-binding modules are key prerequisites for receptor activation and cell killing.

Phosphorylation of Bacillus subtilis gene regulator AbrB modulates its DNA‐binding properties

AbrB is a global gene regulator involved in transition phase phenomena in Bacillus subtilis. It participates in a complex regulatory network governing the expression of stationary-phase functions. AbrB was previously found to be phosphorylated on serine 86 located close to its C-terminal oligomerization domain. Here we report that AbrB can be phosphorylated by three B. subtilis serine/threonine kinases expressed during the transition and stationary phase: PrkC, PrkD and YabT. Our in vitro findings suggest that AbrB phosphorylation impedes its DNA binding and abolishes binding cooperativity. In vivo we established that a phospho-mimetic mutation abrB S86D leads to a significant loss of AbrB control over several key target functions: exoprotease production, competence development and sporulation. A wider transcriptome analysis of abrB S86D and S86A mutant strains revealed deregulation of a large number of target genes. We therefore propose that AbrB phosphorylation serves as an additional input for fine-tuning the activity of this ambiactive gene regulator.

Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1

RasGRP1 and SOS are Ras-specific nucleotide exchange factors that have distinct roles in lymphocyte development. RasGRP1 is important in some cancers and autoimmune diseases but, in contrast to SOS, its regulatory mechanisms are poorly understood. Activating signals lead to the membrane recruitment of RasGRP1 and Ras engagement, but it is unclear how interactions between RasGRP1 and Ras are suppressed in the absence of such signals. We present a crystal structure of a fragment of RasGRP1 in which the Ras-binding site is blocked by an interdomain linker and the membrane-interaction surface of RasGRP1 is hidden within a dimerization interface that may be stabilized by the C-terminal oligomerization domain. NMR data demonstrate that calcium binding to the regulatory module generates substantial conformational changes that are incompatible with the inactive assembly. These features allow RasGRP1 to be maintained in an inactive state that is poised for activation by calcium and membrane-localization signals. DOI:http://dx.doi.org/10.7554/eLife.00813.001.

P73 – constitutively open for business

p73 – constitutively open for business

Reducing the Number of Subunits in the CAMKIIα Holoenzyme Alters Catalytic Domain Pairing, Diffusion Time, and T286 Autophosphorylation

Calcium/Calmodulin (CaM) dependent protein kinase (CaMKII) is an important regulator of learning and memory, and it is thought to play a key role in sensing neuronal calcium oscillations. In cells, CaMKIIα primarily forms dodecamers comprised of two stacked rings of 6 subunits, each of which has 3 domains: an N-terminal catalytic domain, a C-terminal oligomerization domain, and between them, a regulatory domain. This regulatory domain activates the enzyme upon binding CaM and harbors a T286 autophosphorylation site that is associated with calcium independent persistent activation. To form the holoenzyme, the oligomerization domain of each subunit interacts with three other oligomerization domains: two laterally to form a ring of association domains, and one transversely to stabilize the double ring structure. Projecting outwards from the rings are catalytic domains, which form pairs, as indicated by homo-FRET analysis. Assemblies comprised of 8-14 subunits are also thought to exist, but it is not known if the number of subunits in the holoenzyme alters the activity or behavior of the catalytic domain pairs. To study the impact of subunit stoichiometry on catalytic domain pairing and T286 autophosphorylation, we generated mutants by altering oligomerization domain residues at the lateral interaction sites. Using fluorescence polarization and fluctuation analysis (FPFA) we simultaneously monitored homo-FRET between Venus-tagged catalytic domains, lateral diffusion of the holoenzyme, and the number of subunits present in these altered assemblies. We find that when the holoenzyme has fewer than 8 subunits, the holoenzyme diffusion time changes and homo-FRET between catalytic domains begins to attenuate. Finally, T286 autophosphorylation in a dimeric mutant holoenzyme is approximately half of that observed in the native holoenzyme. The structural implication of these changes will be discussed.

CAMKIIα Catalytic Domain Dimerization is Regulated through T-Site Interactions

The calcium-calmodulin (CaM) dependent protein Kinase-II (CaMKII) regulates various cellular process and has been implicated in illness including schizophrenia, heart failure, arthritis, and even certain types of cancers. CaMKII subunits assemble to form a large dodecameric holoenzyme. The structure of a kinase subunit is organized into 3 domains, a catalytic domain, a regulatory domain, and an oligomerization domain. Each N-terminal catalytic domain harbors the kinase catalytic site, its ATP binding site, as well as the so-called ‘T-site’, thought to interact with the T286 autophosphorylation site in the regulatory domain. The T-site is responsible for mediating the interaction of the holoenzyme with other proteins, such as NMDA receptors, in response to activation. The catalytic domain is attached to an autoinhibitory regulatory domain that contains the T286 autophosphorylation site, and a CaM-binding site. In turn: the regulatory domain is itself tethered to a C-terminal oligomerization domain that organizes the enzyme into ring-shaped oligomers. We have previously shown in living cells using FRET analysis that catalytic domains form pairs in the intact auto-inhibited holoenzyme. In Hippocampal neurons, these pairs separate in response to calcium influx through NMDA-receptors, but it is not known if this is a direct response to CaM binding, or a down stream affect of kinase activation. Here using fluorescence polarization and fluctuation analysis (FPFA), a hybrid method that simultaneously measures homo-FRET and fluorescence correlation spectroscopy (FCS), we show that CaMKIINtide, a T-site ligand, disrupts catalytic domain pairing in the intact holoenzyme. This reaction is dose dependent but was only observed upon co-incubation with CaM to activate the enzyme. The holoenzyme can be activated without catalytic domain pair separation. We also show that T-site specific mutations at I205, stabilize catalytic domain pairing and counteract CaMKIINtide's ability to disrupt catalytic domain pairing.

Arranged Sevenfold: Structural Insights into the C-Terminal Oligomerization Domain of Human C4b-Binding Protein

The complement system as a major part of innate immunity is the first line of defense against invading microorganisms. Orchestrated by more than 60 proteins, its major task is to discriminate between host cells and pathogens and to initiate immune response. Additional recognition of necrotic or apoptotic cells demands a fine-tune regulation of this powerful system. C4b-binding protein (C4BP) is the major inhibitor of the classical complement and lectin pathway. The crystal structure of the human C4BP oligomerization domain in its 7α isoform and molecular simulations provide first structural insights of C4BP oligomerization. The heptameric core structure is stabilized by intermolecular disulfide bonds. In addition, thermal shift assays indicate that layers of electrostatic interactions mainly contribute to the extraordinary thermodynamic stability of the complex. These findings make C4BP a promising scaffold for multivalent ligand display with applications in immunology and biological chemistry.

Solid-state NMR sequential assignments of the C-terminal oligomerization domain of human C4b-binding protein

The complement 4 binding protein (C4bp) plays a crucial role in the inhibition of the complement cascade. It has an extraordinary seven-arm octopus-like structure with 7 tentacle-like identical chains, held together at their C-terminal end. The C-terminal domain does oligomerize in isolation, and is necessary and sufficient to oligomerize full-length C4bp. It is predicted to form a seven-helix coiled coil, and its multimerization properties make it a promising vaccine adjuvant, probably by enhancing the structural stability and binding affinity of the presented antigen. Here, we present the solid-state NMR resonance assignment of the human C4bp C-terminal oligomerization Domain, hC4pbOD, and the corresponding secondary chemical shifts.

GntR family regulators of the pathogen of fish tuberculosis Mycobacterium marinum

Mycobacterium marinum is a slow-growing pathogenic mycobacterium. It was first isolated by Aronson in 1926 from fish, fish mycobacteriosis or called fish tuberculosis is the common causative agent of bacterial disease in many species of freshwater and marine fish. M. marinum can infect wild fish, aquaculture and ornamental fish, and it has a close relative of the causative agent of human tuberculosis, Mycobacterium tuberculosis. The recently sequenced genome of M. marinum has been shown to contain several putative GntR regulators. This family named after gluconate regulator has a helix-turn-helix structure. Characterization of transcription regulators and their network is an important step towards the complete understanding of cellular physiology. The regulator of this family shares a similar and conserved N-terminal DNA-binding domain, but has a highly diverse C-terminal effector-binding and oligomerization domain. According to the heterogeneity, we classify the M. marinum GntR family to four subfamilies: FadR, HutC, MocR, and YtrA, and these regulators are encoded by 8, 3, 1 and 1 genes, respectively. Thus this study extends the annotation of M. marinum GntR family proteins, and can help to understand the pathogenic role of this family in M. marinum and facilitate future drug design against this pathogen.

Divergence in enzyme regulation between Caenorhabditis elegans and human tyrosine hydroxylase, the key enzyme in the synthesis of dopamine

TH (tyrosine hydroxylase) is the rate-limiting enzyme in the synthesis of catecholamines. The cat-2 gene of the nematode Caenorhabditis elegans is expressed in mechanosensory dopaminergic neurons and has been proposed to encode a putative TH. In the present paper, we report the cloning of C. elegans full-length cat-2 cDNA and a detailed biochemical characterization of the encoded CAT-2 protein. Similar to other THs, C. elegans CAT-2 is composed of an N-terminal regulatory domain followed by a catalytic domain and a C-terminal oligomerization domain and shows high substrate specificity for L-tyrosine. Like hTH (human TH), CAT-2 is tetrameric and is phosphorylated at Ser35 (equivalent to Ser40 in hTH) by PKA (cAMP-dependent protein kinase). However, CAT-2 is devoid of characteristic regulatory mechanisms present in hTH, such as negative co-operativity for the cofactor, substrate inhibition or feedback inhibition exerted by catecholamines, end-products of the pathway. Thus TH activity in C. elegans displays a weaker regulation in comparison with the human orthologue, resembling a constitutively active enzyme. Overall, our data suggest that the intricate regulation characteristic of mammalian TH might have evolved from more simple models to adjust to the increasing complexity of the higher eukaryotes neuroendocrine systems.

Abstract 2915: p53 isoforms Δ133p53 and p53β are endogenous regulators of replicative cellular senescence

Abstract The finite proliferative potential of normal human cells leads to replicative cellular senescence, which is a critical barrier to tumor progression in vivo. The p53 signaling pathway plays central roles in the regulation of cellular senescence. Humans, as well as Drosophila and zebrafish, have p53 isoforms; however, their regulation and function are poorly understood. We here examine the expression profiles of two human p53 isoforms, p53β (lacking the C-terminal oligomerization domain due to an alternative mRNA splicing) and Δ133p53 (lacking the N-terminal transactivation and proline-rich domains due to the transcription from an alternative promoter in intron 4), during in vitro and in vivo cellular senescence and their biological activities in regulating cellular senescence. Induced p53β and diminished Δ133p53 were associated with replicative senescence, but not oncogene-induced senescence, in normal human fibroblasts. The replicatively senescent fibroblasts also expressed increased levels of miR-34a, a p53-induced microRNA, the antisense inhibition of which delayed the onset of replicative senescence. The siRNA-mediated knockdown of endogenous Δ133p53 induced cellular senescence, which was attributed to the regulation of p21WAF1 and other p53 transcriptional target genes. In overexpression experiments, while p53β cooperated with full-length p53 to accelerate cellular senescence, Δ133p53 repressed miR-34a expression and extended cellular replicative lifespan, providing a functional connection of this microRNA to the p53 isoform-mediated regulation of senescence. The senescence-associated signature of p53 isoform expression (i.e., elevated p53β and reduced Δ133p53) was observed in vivo in colon adenomas with senescent phenotypes. The decreased p53β and increased Δ133p53 expression found in colon carcinomas might signal an escape from the senescence barrier during the progression from premalignant to malignant tumors in vivo. This study shows that natural p53 isoforms constitute an endogenous regulatory mechanism for p53-mediated replicative senescence and may open up a new p53-based, senescence-mediated strategy to manipulate carcinogenesis and aging. The molecular details of the senescence-associated Δ133p53 repression and p53β induction are currently under investigation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2915.