PubMed Health⌕ Search

Biomedical subjects

Daimark Bennett

Publications and source records attributed to Daimark Bennett.

11 recordsLinked to original sources
PubMed2024

Dual diagnosis of achondroplasia and mandibulofacial dysostosis with microcephaly.

BACKGROUND: Achondroplasia and mandibulofacial dysostosis with microcephaly (MFDM) are rare monogenic, dominant disorders, caused by gain-of-function fibroblast growth factor receptor 3 (FGFR3) gene variants and loss-of-function elongation factor Tu GTP binding domain-containing 2 (EFTUD2) gene variants, respectively. The coexistence of two distinct Mendelian disorders in a single individual is uncommon and challenges the traditional paradigm of a single genetic disorder explaining a patient's symptoms, opening new avenues for diagnosis and management. CASE PRESENTATION: We present a case of a female patient initially diagnosed with achondroplasia due to a maternally inherited pathogenic FGFR3 variant. She was referred to our genetic department due to her unusually small head circumference and short stature, which were both significantly below the expected range for achondroplasia. Additional features included distinctive facial characteristics, significant speech delay, conductive hearing loss, and epilepsy. Given the complexity of her phenotype, she was recruited to the DDD (Deciphering Developmental Disorders) study and the 100,000 Genomes project for further investigation. Subsequent identification of a complex EFTUD2 intragenic rearrangement confirmed an additional diagnosis of mandibulofacial dysostosis with microcephaly (MFDM). CONCLUSION: This report presents the first case of a dual molecular diagnosis of achondroplasia and mandibulofacial dysostosis with microcephaly in the same patient. This case underscores the complexity of genetic diagnoses and the potential for coexistence of multiple genetic syndromes in a single patient. This case expands our understanding of the molecular basis of dual Mendelian disorders and highlights the importance of considering the possibility of dual molecular diagnoses in patients with phenotypic features that are not fully accounted for by their primary diagnosis.

Humans↗
PubMed2007

Yeast two-hybrid screens to identify Drosophila PP1-binding proteins.

Protein phosphatase type 1 (PP1) is one of the major classes of serine/threonine protein phosphatases and has been found in all eukaryotic cells examined to date. Metazoans from Drosophila to humans have multiple genes encoding catalytic subunits of PP1 (PP1c), which are involved in a wide range of biological processes. Studies in the fruit fly Drosophila melanogaster have revealed some of the essential functions of the PP1c genes. However, the PP1c isoforms have pleiotropic and overlapping functions, making it difficult to characterize their many biological roles and identify their specific in vivo substrates. Regulatory subunits of PP1 provide the key to understanding the role of PP1, as they are responsible for directing PP1c to different intracellular locations and/or affecting their activity or substrate specificity. The existence of isoform-specific PP1 regulatory subunits might also help to explain the unique roles of different PP1 catalytic subunits. Drosophila is an excellent model organism in which to characterize the role of PP1 catalytic and regulatory subunits, because it combines molecular and biochemical approaches with powerful genetics, in a well-characterized animal model. In this chapter, we will describe how the two-hybrid system can be used to identify Drosophila PP1c-interacting proteins and study their interactions with different PP1c isoforms and variants. With the appropriate bait and library constructs, this method should also be equally applicable to identifying binding subunits of related phosphatases or PP1c from other organisms.

Amino Acid Sequence↗
PubMed2006

Towards a comprehensive analysis of the protein phosphatase 1 interactome in Drosophila.

Protein phosphatase type 1 (PP1) is one of the major classes of serine/threonine protein phosphatases, and has been found in all eukaryotic cells examined to date. Metazoans from Drosophila to humans have multiple genes encoding catalytic subunits of PP1 (PP1c), which are involved in a wide range of biological processes. Different PP1c isoforms have pleiotropic and overlapping functions; this has complicated the analysis of their biological roles and the identification of specific in vivo substrates. PP1c isoforms are associated in vivo with regulatory subunits that target them to specific locations and modify their substrate specificity and activity. The PP1c-binding proteins are therefore the key to understanding the role of PP1 in particular biological processes. The existence of isoform specific PP1c-binding subunits may also help to explain the unique roles of different PP1c isoforms. Here we report the identification of 24 genes encoding Drosophila PP1c-binding proteins in the yeast two-hybrid system. Sequence analysis identified a minimal interacting fragment and putative PP1c-binding motif for each protein, delimiting the region involved in binding to PP1c. Further two-hybrid analysis showed that virtually all of the interactors were capable of binding all Drosophila PP1c isoforms. One of the novel interactors, CG1553, was examined further and shown to interact with multiple isoforms by co-immunoprecipitation from Drosophila extracts and functional interaction with PP1c isoforms in vivo. Bioinformatic analyses implicate the putative PP1c-associated subunits in a diverse array of intracellular processes. Our identification of a large number of PP1c-binding proteins with the potential for directing PP1c's specific functions in Drosophila represents a significant step towards a full understanding of the range of PP1 complexes and function in animals.

Amino Acid Sequence↗
PubMed2004

The essential role of PP1beta in Drosophila is to regulate nonmuscle myosin.

Reversible phosphorylation of myosin regulatory light chain (MRLC) is a key regulatory mechanism controlling myosin activity and thus regulating the actin/myosin cytoskeleton. We show that Drosophila PP1beta, a specific isoform of serine/threonine protein phosphatase 1 (PP1), regulates nonmuscle myosin and that this is the essential role of PP1beta. Loss of PP1beta leads to increased levels of phosphorylated nonmuscle MRLC (Sqh) and actin disorganisation; these phenotypes can be suppressed by reducing the amount of active myosin. Drosophila has two nonmuscle myosin targeting subunits, one of which (MYPT-75D) resembles MYPT3, binds specifically to PP1beta, and activates PP1beta's Sqh phosphatase activity. Expression of a mutant form of MYPT-75D that is unable to bind PP1 results in elevation of Sqh phosphorylation in vivo and leads to phenotypes that can also be suppressed by reducing the amount of active myosin. The similarity between fly and human PP1beta and MYPT genes suggests this role may be conserved.

Amino Acid Sequence↗
PubMed2004

PP1beta9C interacts with Trithorax in Drosophila wing development.

Type 1 Ser/Thr protein phosphatase (PP1) has many roles in Drosophila: regulating diverse processes from chromatin condensation to transforming growth factor-beta signaling. The presence of four PP1 genes, PP1alpha87B, PP1beta9C, PP1alpha96A, and PP1alpha13C, encoding very similar proteins complicates analysis of their particular functions. Here, we report that the minor PP1 isoform PP1beta9C binds in vitro and in vivo and genetically interacts with Trithorax (TRX), the archetypal member of the Trx-G family of epigenetic regulators in Drosophila. Direct binding was demonstrated by GST pull-down experiments and PP1beta9C/TRX interaction in vivo was confirmed by coimmune precipitation from Drosophila embryonic extracts. PP1beta9C was found to be present at all TRX sites on the polytene chromosomes. Flies homo- and hemizygous for loss-of-function alleles of PP1beta9C exhibited specific wing defects when combined with various trx mutants, which indicates that PP1beta9C and TRX cooperate in Drosophila wing development.

Animals↗
PubMed2004

Cloning and expression of mars, a novel member of the guanylate kinase associated protein family in Drosophila.

The Membrane-Associated Guanylate Kinase (MAGUK) family of anchor proteins are involved in organising a range of molecules such as cell adhesion molecules, receptors, and intracellular signalling molecules at cell junctions. In mammals, the PSD-95/SAP-90/hDlg class of MAGUK proteins bind to a family of Guanylate Kinase Associated Proteins (GKAPs) that have been found at presumptive synaptic sites in neurons. Here we describe the identification of Mars, a novel Drosophila protein belonging to the GKAP family. RT-PCR analysis reveals that Drosophila mars mRNA and protein are predominantly expressed in embryos and in the adult germline. In embryos, mars is expressed in central nervous system and brain, as determined by RNA in situ hybridisation. In testes, mars is strongly expressed in pre-meiotic germ cells, but is not found in somatic or post-meiotic cells, indicating that in addition to their role in neuronal cells, GKAP proteins are also likely to play a role in germline development.

Amino Acid Sequence↗
PubMed2003

Trithorax interacts with type 1 serine/threonine protein phosphatase in Drosophila.

The catalytic subunit of type 1 serine/threonine protein phosphatase (PP1c) was shown to bind trithorax (TRX) in the yeast two-hybrid system. Interaction between PP1c and TRX was confirmed in vivo by co-immunoprecipitation from Drosophila extracts. An amino-terminal fragment of TRX, containing a putative PP1c-binding motif, was shown to be sufficient for binding to PP1c by in vitro glutathione S-transferase pull-down assays using recombinant protein and fly extracts expressing epitope tagged PP1c. Disruption of the PP1c-binding motif abolished binding, indicating that this motif is necessary for interaction with PP1. On polytene chromosomes, PP1c is found at many discrete bands, which are widely distributed along the chromosomes. Many of the sites that stain strongly for PP1c correspond to sites of TRX, consistent with a physical association of PP1c with chromatin-bound TRX. Homeotic transformations of haltere to wing in flies mutant for trx are dominantly suppressed by PP1c mutants, indicating that PP1c not only binds TRX, but is a physiologically relevant regulator of TRX function in vivo.

Animals↗
PubMed2003

Ectopic expression of inhibitors of protein phosphatase type 1 (PP1) can be used to analyze roles of PP1 in Drosophila development.

We have identified two proteins that bind with high specificity to type 1 serine/threonine protein phosphatase (PP1) and have exploited their inhibitory properties to develop an efficient and flexible strategy for conditional inactivation of PP1 in vivo. We show that modest overexpression of Drosophila homologs of I-2 and NIPP1 (I-2Dm and NIPP1Dm) reduces the level of PP1 activity and phenotypically resembles known PP1 mutants. These phenotypes, which include lethality, abnormal mitotic figures, and defects in muscle development, are suppressed by coexpression of PP1, indicating that the effect is due specifically to loss of PP1 activity. Reactivation of I-2Dm:PP1c complexes suggests that inhibition of PP1 activity in vivo does not result in a compensating increase in synthesis of active PP1. PP1 mutants enhance the wing overgrowth phenotype caused by ectopic expression of the type II TGF beta superfamily signaling receptor Punt. Using I-2Dm, which has a less severe effect than NIPP1Dm, we show that lowering the level of PP1 activity specifically in cells overexpressing Punt is sufficient for wing overgrowth and that the interaction between PP1 and Punt requires the type I receptor Thick-veins (Tkv) but is not strongly sensitive to the level of the ligand, Decapentaplegic (Dpp), nor to that of the other type I receptors. This is consistent with a role for PP1 in antagonizing Punt by preventing phosphorylation of Tkv. These studies demonstrate that inhibitors of PP1 can be used in a tissue- and developmental-specific manner to examine the developmental roles of PP1.

Animals↗
PubMed2002

Functional interaction between nuclear inhibitor of protein phosphatase type 1 (NIPP1) and protein phosphatase type 1 (PP1) in Drosophila: consequences of over-expression of NIPP1 in flies and suppression by co-expression of PP1.

The catalytic subunit of type 1 Ser/Thr protein phosphatases (PP1c) forms complexes with many proteins that target it to particular subcellular locations and regulate its activity towards specific substrates. We report the identification of a Drosophila orthologue of nuclear inhibitor of PP1 (NIPP1Dm) through interaction with PP1c in the yeast two-hybrid system. NIPP1Dm shares many properties with mammalian NIPP1 including inhibition of PP1c in vitro, binding to RNA and PP1c, and localization to nuclear speckles. However, the mechanism controlling interaction of PP1c with NIPP1 is not conserved in Drosophila. NIPP1 can function independently of PP1c as a splicing factor, but the relative importance of this function is unknown. Over-expression of NIPP1Dm in Drosophila is cell-lethal in a range of tissues and developmental stages. The effects of ectopic NIPP1Dm are suppressed by co-expression of PP1c, indicating that the only effect of ectopic NIPP1Dm is to affect PP1c function. Co-expression of NIPP1Dm and PP1c does not have any detectable physiological effect in vivo, suggesting that the NIPP1Dm-PP1c holoenzyme is not normally limiting in Drosophila. These data show that NIPP1Dm and PP1c interact in vivo and suggest that NIPP1's role as a phosphatase regulator is conserved in Drosophila.

Amino Acid Sequence↗
PubMed2002

PP1 binds Sara and negatively regulates Dpp signaling in Drosophila melanogaster.

In signaling involving the transforming growth factor-beta (TGF-beta) superfamily of proteins, ligand binding brings the constitutively active type II receptor kinase into close proximity to its substrate, the type I receptor kinase, which it then activates by phosphorylation. The type I receptor kinase in turn phosphorylates one of the Smad family of transcription factors, which translocates to the nucleus and regulates gene expression. Smads are recruited to the receptor complex by an anchor protein, SARA (Smad anchor for receptor activation). Although several protein kinases in this pathway were known, including the receptors themselves, the relevant phosphatases had not previously been identified. Here we report the isolation of a Drosophila melanogaster homolog of SARA (Sara) in a screen for proteins that bind the catalytic subunit of type 1 serine/threonine protein phosphatase (PP1c). We identified a PP1c-binding motif in Sara, disruption of which reduced the ability of Sara to bind PP1c. Expression of this non-PP1c-binding mutant resulted in hyperphosphorylation of the type I receptor and stimulated expression of a target of TGF-beta signaling. Reducing PP1c activity enhanced the increase in the basal level of expression of genes responsive to Dpp (Decapentaplegic) caused by ectopic expression of the type II receptor Punt. Together these data suggest that PP1c is targeted to Dpp receptor complexes by Sara, where it acts as a negative regulator of Dpp signaling by affecting the phosphorylation state of the type I receptor.

Activin Receptors, Type I↗