Biomedical subjects

Sibylle Mittnacht

Publications and source records attributed to Sibylle Mittnacht.

11 recordsLinked to original sources

PubMed2007

Attenuation of disease phenotype through alternative translation initiation in low-penetrance retinoblastoma.

Hereditary predisposition to retinoblastoma (RB) is caused by germline mutations in the retinoblastoma 1 (RB1) gene and transmits as an autosomal dominant trait. In the majority of cases disease develops in greater than 90% of carriers. However, reduced penetrance with a large portion of disease-free carrier is seen in some families. Unambiguous identification of the predisposing mutation in these families is important for accurate risk prediction in relatives and their genetic counseling but also provides conceptual information regarding the relationship between the RB1 genotype and the disease phenotype. In this study we report a novel mutation detected in 10 individuals of an extended family, only three of whom are affected by RB disease. The mutation comprises a 23-basepair (bp) duplication in the first exon of RB1 (c.43_65dup) producing a frameshift in exon 1 and premature chain termination in exon 2. Mutations resulting in premature chain termination classically are associated with high penetrance disease, as message translation may not generate functional product and nonsense mediated RNA decay (NMD) frequently eliminates the mutant transcript. However, appreciable NMD does not follow from the mutation described here and transcript expression in tissue culture cells and translation in vitro reveals that alternative in-frame translation start sites involving Met113 and possibly Met233 are used to generate truncated RB1 products (pRB94 and pRB80), known and suspected to exhibit tumor suppressor activity. These results strongly suggest that modulation of disease penetrance in this family is achieved by internal translation initiation. Our observations provide the first example for rescue of a chain-terminating mutation in RB1 through alternative translation initiation.

Alternative Splicing↗

PubMed2006

Trans-activation of the DNA-damage signalling protein kinase Chk2 by T-loop exchange.

The protein kinase Chk2 (checkpoint kinase 2) is a major effector of the replication checkpoint. Chk2 activation is initiated by phosphorylation of Thr68, in the serine-glutamine/threonine-glutamine cluster domain (SCD), by ATM. The phosphorylated SCD-segment binds to the FHA domain of a second Chk2 molecule, promoting dimerisation of the protein and triggering phosphorylation of the activation segment/T-loop in the kinase domain. We have now determined the structure of the kinase domain of human Chk2 in complexes with ADP and a small-molecule inhibitor debromohymenialdisine. The structure reveals a remarkable dimeric arrangement in which T-loops are exchanged between protomers, to form an active kinase conformation in trans. Biochemical data suggest that this dimer is the biologically active state promoted by ATM-phosphorylation, and also suggests a mechanism for dimerisation-driven activation of Chk2 by trans-phosphorylation.

Adenosine Diphosphate↗

PubMed2006

Direct interaction between the catalytic subunit of Protein Phosphatase 1 and pRb.

BACKGROUND: The product of the retinoblastoma-susceptibility gene (pRb) is a substrate for Protein Phosphatase 1 (PP1). At mitotic exit, all three PP1 isoforms, alpha, gamma1 and delta, bind to pRb and dephosphorylate its Ser/Thr sites in a sequential and site-specific way. The pRb-C terminal has been reported to be necessary and sufficient for PP1alpha binding. The present study investigated whether the three PP1 isoforms from mitotic or asynchronous HeLa cells associate differentially with wild-type and pRb mutants, as well as the holoenzyme composition of the pRb-directed PP1. RESULTS: The requirement for the entire pRb molecule to achieve optimal PP1-binding was indicated by the fact that full-length pRb displayed the highest affinity for all three PP1 isoforms. Ser/Thr-to-Ala substitution for up to 14 pRb sites did not affect the ability of pRb to bind the PP1 isoforms derived from mitotic or asynchronous HeLa cells, thus suggesting that the phosphate-accepting residues on pRb do not regulate the interaction with PP1. To probe for the presence of PP1 targeting subunits in the pRb-directed PP1 complex, PP1 from mitotic or asynchronous HeLa cells was isolated by affinity chromatography on GST-Rb (either full-length or its deletion mutants Rb-big pocket or Rb-C-terminal). The PP1 was always obtained as free catalytic subunit, displaying all three isoforms, thus suggesting direct interaction between pRb and PP1. The direct association was confirmed by the ability of pRb to pull-down purified PP1 catalytic subunits and by in vitro reconstitution of a complex between PP1 catalytic subunit and the pRb-C-terminal. CONCLUSION: The work indicated that the full length of the pRb molecule is required for optimal interaction with the PP1 isoforms and that the association between pRb and PP1 isoforms is direct.

Journal Article↗

PubMed2005

Regulation of microfilament organization by Kaposi sarcoma-associated herpes virus-cyclin.CDK6 phosphorylation of caldesmon.

Kaposi sarcoma-associated herpes virus (KSHV) encodes a D-like cyclin (K-cyclin) that is thought to contribute to the viral oncogenicity. K-cyclin activates cellular cyclin-dependent kinases (CDK) 4 and 6, generating enzymes with a substrate selectivity deviant from CDK4 and CDK6 activated by D-type cyclins, suggesting different biochemical and biological functions. Here we report the identification of the actin- and calmodulin-binding protein caldesmon (CALD1) as a novel K-cyclin.CDK substrate, which is not phosphorylated by D.CDK. CALD1 plays a central role in the regulation of microfilament organization, consequently controlling cell shape, adhesion, cytokinesis and motility. K-cyclin.CDK6 specifically phosphorylates four Ser/Thr sites in the human CALD1 carboxyl terminus, abolishing CALD1 binding to its effector protein, actin, and its regulator protein, calmodulin. CALD1 is hyperphosphorylated in cells following K-cyclin expression and in KSHV-transformed lymphoma cells. Moreover, expression of exogenous K-cyclin results in microfilament loss and changes in cell morphology; both effects are reliant on CDK catalysis and can be reversed by the expression of a phosphorylation defective CALD1. Together, these data strongly suggest that K-cyclin expression modulates the activity of caldesmon and through this the microfilament functions in cells. These results establish a novel link between KSHV infection and the regulation of the actin cytoskeleton.

Actins↗

PubMed2005

Selective ablation of retinoblastoma protein function by the RET finger protein.

The retinoblastoma tumor suppressor protein (Rb) affects gene transcription both negatively and positively and through this regulates distinct cellular responses. Although cell cycle regulation requires gene repression, Rb's ability to promote differentiation and part of its antiproliferative activity appears to rely on the activation of gene transcription. We present evidence here that the RET finger protein (RFP)/tripartite motif protein 27 (TRIM 27) inhibits gene transcription activation by Rb but does not affect gene repression. RFP binds to Rb and prevents the degradation of the EID-1 inhibitor of histone acetylation and differentiation. Furthermore, ablation of RFP in U2OS osteosarcoma cells augments a transcriptional program indicative of lineage-specific differentiation in response to Rb. These findings provide precedent for a regulatory pathway that uncouples different Rb-dependent activities and thus silences specific cellular responses to Rb in a selective way.

Adenovirus E1A Proteins↗

PubMed2005

A novel constitutional mutation affecting splicing of retinoblastoma tumor suppressor gene intron 23 causes partial loss of pRB activity.

Hereditary predisposition to retinoblastoma is caused by germ line mutations in the RB1 gene. Genetic counseling of affected individuals and accurate risk prediction for their families requires identification of the disease causing mutation. Furthermore, the nature of a mutation can determine genetic penetrance, disease presentation and prognosis. We describe, and functionally characterize here, a novel mutant allele of RB1 present in the germ line of a patient with sporadic bilateral retinoblastoma. The mutation generates an operational splice acceptor site resulting in a predicted protein product with loss of 81 amino acids from its carboxy terminus. We demonstrate that the aberrantly spliced transcript is present in substantial amounts in peripheral blood of the patient and present evidence that the predicted protein product displays partial loss of activity reflecting in degree and presentation that of the partially penetrant RB1 missense mutant R661W. This infers that disease with reduced expressivity and incomplete penetrance may arise in individuals that carry the mutation and predicts such presentation for similar mutations with found in sporadic cases in the past.

Adult↗

PubMed2005

The retinoblastoma protein--from bench to bedside.

The retinoblastoma tumour suppressor protein (Rb) has come a long way since its initial discovery in 1986. Encoded by the first candidate tumour suppressor gene it has emerged a versatile and context-dependent modulator of cell behaviour. Its activity is managed by signalling networks sensing intra- and extracellular cues. These cues are relayed to hold or permit inactivation of Rb by phosphorylation. Loss or mutation of the retinoblastoma gene is rare in sporadic cancers but defects in the pathways that license inactivation of Rb are found in the majority of them, suggesting that loss of Rb control is central to tumour development and arguing that its reinstatement might reverse tumour formation. Furthermore, mouse models with engineered defects in the Rb-phosphorylating kinases provide evidence that moderation of Rb inactivation may be a strategy for the prevention of tumour formation. The rationale behind these arguments, their underlying molecular concepts and strategies towards therapeutic application will be discussed.

Animals↗

PubMed2003

High-throughput screening for the identification of small-molecule inhibitors of retinoblastoma protein phosphorylation in cells.

The tumor suppressor protein, pRb, regulates progression through the G1 phase of the cell cycle by its ability to bind to and regulate the activity of a variety of transcription factors. This function of pRb is disabled through its phosphorylation by the cyclin-dependent kinase (CDK) family of serine/threonine kinases. In many human cancers, genetic alteration such as loss of CDK inhibitor function and deregulated G1 cyclin expression leads to inappropriate phosphorylation and hence inactivation of this tumor suppressor. Identification of cell-permeable small molecules that block pRb phosphorylation in these tumors could therefore lead to development of an effective anticancer treatment. As a result, we have developed a high-throughput assay to detect changes in the level of pRb phosphorylation in cells. Signal detection is by a time-resolved fluorescence-based cellular immunosorbant assay on a fixed monolayer of cells. This comprises a mouse monoclonal antibody that recognizes the phosphorylated form of serine 608 on pRb, a known site of CDK phosphorylation, and a Europium-labeled secondary antibody for signal detection. The assay is reproducible and amenable to automation and has been used to screen 2000 compounds in a search for cell-permeable small molecules that will block pRb phosphorylation.

Animals↗

PubMed2003

Crystal structure of the retinoblastoma tumor suppressor protein bound to E2F and the molecular basis of its regulation.

The retinoblastoma tumor suppressor protein (pRb) regulates the cell cycle, facilitates differentiation, and restrains apoptosis. Furthermore, dysfunctional pRb is thought to be involved in the development of most human malignancies. Many of the functions of pRb are mediated by its regulation of the E2F transcription factors. To understand the structural basis for this regulation, we have determined the crystal structure of a fragment of E2F in complex with the pocket domain of the tumor suppressor protein. The pRb pocket, comprising the A and B cyclin-like domains, is the major focus of tumourigenic mutations in the protein. The fragment of E2F used in our structural studies, residues 409-426 of E2F-1, represents the core of the pRb-binding region of the transcription factor. The structure shows that E2F binds at the interface of the A and B domains of the pocket making extensive interactions with conserved residues from both. We show by solution studies that a second site, probably contained within the "marked box" region of E2F, is responsible for additional interactions with the pRb pocket but is insufficient for complex formation on its own. In addition, we show that the interaction of the core binding fragment of E2F with pRb is inhibited by phosphorylation of the tumor suppressor protein by CDK2cyclin DE. Finally, our data reveal that the tight binding of the human papillomavirus E7 oncoprotein to pRb prevents subsequent interactions with the marked box region of E2F but not with its core binding region.

Blotting, Western↗

PubMed2002

RB activation defect in tumor cell lines.

Activation of the retinoblastoma (RB) protein through dephosphorylation arises in cells upon exit from M phase and in response to environmental stresses, including DNA damage. We provide here for the first time evidence that these responses are co-ordinately affected in a subset of tumor derived cell lines. We find that RB dephosphorylation is not apparent in these cells during progression into G(1). Importantly these cells also do not respond with RB activation after DNA damage during S phase. Moreover and as a consequence they display phenotypes classically associated with RB(-) cells, showing accelerated apoptosis after DNA damage and DNA re-replication after spindle-checkpoint activation. A large body of literature provides evidence that controls governing inactivation of RB are lost in tumors. The results presented here indicate that the reverse reaction, namely the activation of RB from an inactive precursor, may also be compromised. Our findings indicate that this type of defect may be coupled with hypersensitivity to DNA damage and an increase in genomic instability in response to spindle-checkpoint activation thus bearing potentially important medical implications.

DNA Damage↗

PubMed2002

p16INK4a loss and sensitivity in KSHV associated primary effusion lymphoma.

The Kaposi's Sarcoma associated Herpes virus (KSHV) encodes two genes with the potential to affect the activity of the retinoblastoma protein (Rb). Open reading frame (orf) 72 encodes a D type cyclin (kcyc) that can elicit p16INK4a resistant cdk activity and orf73 encodes the latency associated nuclear antigen (LNA) that can bind Rb and neutralize E2F regulation. This indicates that, like papilloma and adenovirus associated malignancies, those associated with KSHV are defective with respect to their Rb pathway. To address this we investigated whether KSHV associated primary effusion lymphoma (PEL) derived cell lines are resistant to growth inhibition by p16INK4a. We provide evidence that ectopic expression of p16INK4a in these cells causes an Rb dependent G1 cell cycle block. Importantly, endogenous p16INK4a expression is not detected in six PEL derived cell lines and four primary PEL samples and examination of the p16INK4a locus shows deletion in two out of six and hypermethylation in four out of six PEL lines. Treatment of the latter with the demethylating agent 5'-aza-2' deoxycytidine leads to re-expression of p16INK4a protein. Taken together these results suggest that p16INK4a loss may be a cellular change frequently associated with PEL. They furthermore argue that despite the presence of KSHV DNA and expression of a latent gene program Rb function is intact in PEL.

Adenoviridae↗