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De novo variants of CSNK2B cause a new intellectual disability-craniodigital syndrome by disrupting the canonical Wnt signaling pathway

ABSTRACT: Main purpose of the project is to delineate the consequences of de novo variants identified in patients manifesting intellectual disability-craniodigital syndrome. By employing pulldown assay coupled with mass spectrometry, we investigated the novel binding partners of CK2β and tried to find the impaired interaction due to variant, CK2β:NP_001311.3;p.Asp32His. Furthermore, we also investigated the effects of mutated CK2β by performing phosphoproteome profiling of patient derived LCLs along with the age and gender matched control.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cervical Cell Line

DISEASE(S): Syndromic Intellectual Disability

SUBMITTER: Prerana Wagle

LAB HEAD: Muhammad Sajid Hussain

PROVIDER: PXD029970 | Pride | 2022-05-04

REPOSITORIES: pride

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			Action	DRS
	20170925_Q1_AWH_ColID_422_ProjID_772_Hussain_sample_01.raw	Raw
	20170925_Q1_AWH_ColID_422_ProjID_772_Hussain_sample_02.raw	Raw
	20170925_Q1_AWH_ColID_422_ProjID_772_Hussain_sample_03.raw	Raw
	20170925_Q1_AWH_ColID_422_ProjID_772_Hussain_sample_04.raw	Raw
	20170925_Q1_AWH_ColID_422_ProjID_772_Hussain_sample_05.raw	Raw

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De novo variants of CSNK2B cause a new intellectual disability-craniodigital syndrome by disrupting the canonical Wnt signaling pathway

Project description:Main purpose of the project is to delineate the consequences of de novo variants identified in patients manifesting intellectual disability-craniodigital syndrome. To this end, we investigated the effects of mutated CK2β by performing phosphor proteome profiling of patient derived LCLs along with the age and gender matched control.

2022-05-04 | PXD029983 | Pride

A systematic screening pipeline identifies a new regulator of tyrosinated microtubule dynamics

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An Alternative Splicing Switch Regulates Embryonic Stem Cell Pluripotency and Reprogramming [protein binding microarray]

Project description:Alternative splicing (AS) is a key process underlying the expansion of proteomic diversity and the regulation of gene expression. However, the contribution of AS to the control of embryonic stem cell (ESC) pluripotency is not well understood. Here, we identify an evolutionarily conserved ESC-specific AS event that changes the DNA binding preference of the forkhead family transcription factor FOXP1. We show that the ESC-specific isoform of FOXP1 stimulates the expression of transcription factor genes required for pluripotency including OCT4, NANOG, NR5A2 and GDF3, while concomitantly repressing genes required for ESC differentiation. Remarkably, this isoform also promotes the maintenance of ESC pluripotency and the efficient reprogramming of somatic cells to induced pluripotent stem cells. These results reveal an AS switch that plays a pivotal role in the regulation of pluripotency through the control of critical ESC-specific transcriptional programs. Protein binding microarray (PBM) experiments were performed for two isoforms of the DNA binding domain of the human FOXP1 gene. Briefly, the PBMs involved binding GST-tagged DNA-binding proteins to two double-stranded 4*44K Agilent microarrays, each containing a different DeBruijn sequence design, in order to determine their sequence preferences. The method is described in Berger et al., Nature Biotechnology 2006.

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in vitro kinase assays Identified of STAT1 phosphorylation sites-mediated LATS2

Project description:LATS1/2 are frequently down-regulated or mutated in endometrial cancer (EC), yet their exact role in endometrial tumor progression remains unclear. In this study, we unexpectedly discovered a new function of LATS1/2 in regulating MHC class I expression in EC. Knockout of LATS1/2 in EC cells led to significant down-regulation of multiple genes within the MHC/HLA Class I family, which weakened the killing effects of PBMCs on tumor cells. Importantly, we found that the regulation of MHC/HLA expression by LATS1/2 does not depend on the classical Hippo pathway but rather involves their direct interaction with STAT1. This interaction causes STAT1 to be phosphorylated at Ser727 or Ser532, promoting its accumulation and movement into the nucleus and enhancing the transcriptional activation of IRF1/NLRC5 on MHC-I. Additionally, we demonstrated that the kinase-dead mutant LATS1/2 weakens STAT1 phosphorylation at Ser727, supporting our findings. Our results show a positive correlation between the expression levels of LATS1/2, MHC-I, CD8, and p-STAT1 in tissue samples of EC, providing further evidence to support our discoveries. Overall, our findings reveal novel molecular mechanisms underlying tumor immunogenicity deficiency and suggest that LATS1/2 may serve as an immunotherapy biomarker for targeting EC.

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Affinity purification of in vivo assembled whirlin-associated protein complexes from the zebrafish retina - GST

Project description:Mutations in WHRN lead to Usher syndrome type 2d (USH2d) or to non-syndromic hearing impairment (DFNB31). The WHRN-encoded gene product whirlin directly interacts with the intracellular regions of the other two Usher syndrome type 2 (USH2)-associated proteins, usherin and ADGRV1. Together with PDZD7, these proteins form the transiently present ankle link complex necessary for maturation of the inner ear hair bundle. In photoreceptors, usherin and ADGRV1 are also scaffolded by whirlin and constitute similar fibrous links between the periciliary membrane and the connecting cilium. However, the molecular mechanism of retinal degeneration as a consequence of compromised USH2-associated protein complex formation and function remains elusive. To better understand the function of the USH2-associated proteins in the photoreceptors, we aimed to isolate and characterize whirlin-associated protein complexes from zebrafish photoreceptor cells. We generated transgenic zebrafish that express Strep/FLAG-tagged Whrna, a zebrafish ortholog of human whirlin, under the control of a photoreceptor-specific promoter. Affinity purification of Strep/FLAG-tagged Whrna and associated proteins from adult transgenic zebrafish retinas followed by mass spectrometry analysis identified 19 novel candidate interaction partners. Pull down experiments and dedicated yeast two-hybrid assays confirmed the association of Whrna with 7 of the co-purified proteins. Several of the co-purified proteins are part of the synaptic proteome, which suggest a role for whirlin in the photoreceptor synapse. Future studies will elucidate which of the identified protein-protein interactions contribute to the development of the retinal phenotype observed in USH2d patients.

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